DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
Applicant's response, filed on 05/01/2026, has been fully considered. The following rejections and/or objections are either reiterated or newly applied. They constitute the complete set presently being applied to the instant application.
Status of claims
Canceled:
15-287
Amended:
1
Pending:
1-14, 288-293
New:
293
Examined:
1-14, 288-293
Independent:
1
Allowable:
none
Drawings
The drawings filed 07/20/2023 are accepted.
Priority
As detailed on the 11/29/2019 filing receipt, this application claims priority to as early as 11/15/2018.
Information Disclosure Statement
The Information Disclosure Statement filed on 05/01/2026 is in compliance with the provisions of 37 CFR 1.97 and have been considered in full. A signed copy of the list of references cited from each IDS is included with this Office Action.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claim(s) 1-14 and 288-293 is/are rejected under 35 U.S.C. 103 as being unpatentable over Callewaert (U.S. Patent No 2020/0231956 A1, published Jul. 23, 2020, Foreign Application Priority Data Sep. 28, 2017; cited on the 03/20/2023 “Notice of References Cited” form 892) in view of Swaminathan (“A theoretical justification for single molecule peptide sequencing.” PLoS computational biology vol. 11,2 e1004080. 25 Feb. 2015; cited on the 12/02/2025 “Notice of References Cited” form 892). Any newly recited portions herein are necessitated by claim amendment.
Regarding claim 1, Callewaert teaches the recited obtaining the data comprises obtaining a plurality of signals including a first plurality of signals, each of the first plurality of signals corresponding to a first type of molecule and being emitted when the first type of molecule is bound to a terminus of the polypeptide and respective ones of the first plurality of signals at least with "FIG. 15. Schematic representation of the Edman degradation mechanism. Edman degradation entails the coupling of phenyl isothiocyanate (PITC) onto the free N-terminus of a protein/peptide (alkaline conditions), followed by the release of the N-terminal amino acid as a phenylthiohydantoin (PTH) derivative (acidic conditions). The released PTH-amino acid is then identified with chromatography" (Para. [0035]). The recited "first type of molecule" corresponds to "phenyl isothiocyanate (PITC)" of Callewaert. The recited "signals" corresponds to "released PTH-amino acid" that is identified by chromatography of Callewaert. Callewaert also teaches "The method of the application can additionally include a step of determining the cleavage of said N-terminal amino acid by measuring an optical, electrical or plasmonical signal of the surface-immobilized polypeptide, wherein a difference in optical, electrical or plasmonical signal is indicative for cleavage of said N-terminal amino acid. The above methods are also provided wherein said surface-immobilized polypeptide is additionally contacted with one or more N-terminal amino acid binding proteins, wherein the kinetics of the binding events of said one or more binding proteins to said N-terminal amino acid identify or is further informative for said N-terminal amino acid. The above methods can also include a first step of polypeptide denaturation or are provided in which polypeptide denaturing conditions are present during one or more of the steps of said methods, wherein said catalytically active aminopeptidase is a thermophilic and/or solvent resistant aminopeptidase and/or wherein said cleavage-inducing agent is isothiocyanate or isothiocyanate analogues. Above methods are also provided wherein N-terminal amino acid is derivatized. The aminopeptidase from above methods can be any of the aminopeptidase disclosed herein." (Para. [0018])
Callewaert teaches the recited discrete binding events of a plurality of binding events that occur between the first type of molecule and the terminus of the polypeptide occurring prior to a cleavage event of the degradation process at least with "FIG. 15. Schematic representation of the Edman degradation mechanism. Edman degradation entails the coupling of phenyl isothiocyanate (PITC) onto the free N-terminus of a protein/peptide (alkaline conditions), followed by the release of the N-terminal amino acid as a phenylthiohydantoin (PTH) derivative (acidic conditions). The released PTH-amino acid is then identified with chromatography" (Para. [0035]). The recited "first type of molecule" corresponds to "phenyl isothiocyanate (PITC)" of Callewaert. It is noted that cleavage is interpreted to be equivalent to the degradation. Callewaert also teaches "...the identity of the N-terminal amino acid derivative is determined by performing, for example, 20 rounds of antibody binding with antibodies specific for each PITC-derivatized N-terminal amino acid, detection, and stripping. The N-terminal amino acid is removed by raising the temperature or lowering pH, and the cycle is repeated to sequence 12-20 amino acids from each peptide on the slide." (para. [0003]).
Callewaert teaches the recited the cleavage event performed by a second type of molecule different than the first type of molecule at least with "Said N-terminal amino acid can be a derivatized N-terminal amino acid and if so said aminopeptidase binds and cleaves said derivatized N-terminal amino acid. Said N-terminal amino acid can be an N-terminal amino acid derivatized with isothiocyanate or isothiocyanate analogues" (Para. [0006]). The recited "second type of molecule" corresponds to "aminopeptidase" of Callewaert.
Callewaert teaches the recited based on the determined portions of data, determining a value for at least one characteristic of the respective determined portions of data, including a first value for the at least one characteristic based on the first portion of the data corresponding to the first amino acid; identifying a portion of a sequence of amino acids of the polypeptide based at least in part on the determined values for the at least one characteristic at least with "...wherein said cleavage-inducing agent is isothiocyanate or isothiocyanate analogues, wherein said residence time is the length of time until said N-terminal amino acid is removed, wherein said N-terminal amino acid is identified by comparing said length of time to a set of reference values for different amino acids." (Para. [0016]) and “…wherein said cleavage-inducing agent binds and cleaves the N-terminal amino acid from said polypeptide; measuring the residence time of said cleavage-inducing agent on said N-terminal amino acid; and comparing said measured residence time to a set of reference residence time values characteristic for said cleavage-inducing agent and a set of N-terminal amino acids to identify or categorize said N-terminal amino acid.” (Para. [0009]). The recited "value" corresponds to "residence time" and "length of time" of Callewaert. Callewaert also teaches (Paragraph [0018]) the method of the application can additionally include a step of determining the cleavage of said N-terminal amino acid by measuring an optical, electrical or plasmonical signal of the surface-immobilized polypeptide, wherein a difference in optical, electrical or plasmonical signal is indicative for cleavage of said N-terminal amino acid (Paragraph [0018]). The identity of the N-terminal amino acid corresponds to the signal value and the characteristic of the signal allows one to determine its identity.
Callewaert further teaches that the N-terminal amino acid of single molecules of peptides are identified (or categorized) using the catalytic properties of an aminopeptidase and the kinetics of the enzymatic reaction (Paragraph [0005]). The method is based on the correlation between the turnover number (kcat) of engineered aminopeptidases and the N-terminal amino acid which it cleaves (Paragraph [0005]). Therefore, by measuring the time the engineered aminopeptidase resides upon addition on the peptide substrate before the N-terminal amino acid is cleaved off, N-terminal amino acid is identified (Paragraph [0005]). This corresponds to the claim limitation of obtaining data during a degradation process of a polypeptide and analyzing the plurality of signals to determine portions of the data corresponding to respective amino acids that are exposed at a terminus of the polypeptide during the degradation process.
Callewaert teaches including a first portion of the data corresponding to a first amino acid exposed at the terminus of the polypeptide during the degradation process when the first plurality of signals are emitted with “One of the additional parts of the methods of the application is that the cleavage of the terminal amino acid is to be detected or confirmed. Hence also provided herein are the methods of current application, additionally including a step of determining the cleavage of said terminal amino acid by measuring an optical, electrical or plasmonical signal of the surface-immobilized polypeptide, wherein a difference in optical, electrical or plasmonical signal is indicative for cleavage of said terminal amino acid. Indeed, immobilized peptides with a free N-terminus have several properties which are utilized to determine when an N-terminal amino acid has been cleaved off by the cleaving-inducing agents of the present invention.” (para. [0115]).
Callewaert teaches identifying a portion of a sequence of amino acids of the polypeptide based at least in part on the determined values for the at least one characteristic; with "...the application provides a method to sequence proteins comprising the following step cycle: the N-terminal derivatization of peptides immobilized through a moiety of the peptide C-terminal to the scissile bond, measuring the time it takes for a cleavage-inducing agent to cleave off the N-terminal amino acid, leading to release of the N-terminal amino acid from the immobilisation surface, setting the system ready for the next cycle (FIG. 1)." (Para. [0005]) and “…wherein said cleavage-inducing agent binds and cleaves the N-terminal amino acid from said polypeptide; measuring the residence time of said cleavage-inducing agent on said N-terminal amino acid; and comparing said measured residence time to a set of reference residence time values characteristic for said cleavage-inducing agent and a set of N-terminal amino acids to identify or categorize said N-terminal amino acid.” (Para. [0009]).
Callewaert teaches the application provides a method to sequence proteins comprising the following step cycle: the N-terminal derivatization of peptides immobilized through a moiety of the peptide C-terminal to the scissile bond, measuring the time it takes for a cleavage-inducing agent to cleave off the N-terminal amino acid, leading to release of the N-terminal amino acid from the immobilization surface, setting the system ready for the next cycle (FIG. 1) (Paragraph [0005]). Callewaert also teaches “The invention discloses means and methods for single molecule protein sequencing and/or amino acid identification using cleavage inducing agent.” (Abstract) This corresponds to the claim limitation of identifying the sequence of amino acids of the polypeptide and outputting the identified sequence of amino acids.
Callewaert teaches identifying, using the first value for the at least one characteristic, the first amino acid exposed at the terminus of the polypeptide during the degradation process when the first plurality of signals are emitted and a second amino acid of the polypeptide downstream of the first amino acid when the first plurality of signals are emitted with “The invention discloses means and methods for single molecule protein sequencing and/or amino acid identification using cleavage-inducing agents. Said cleavage-inducing agents, which are not specific for one particular amino acid, cleave polypeptides step by step from the N-terminus onwards and provide information on the identity of the cleaved amino acids based on the kinetics of the engagement between said cleavage inducing agent with the polypeptide or on the kinetics of said polypeptide cleaving reaction.” (para. [0001]); “d) identifying or categorizing said N-terminal amino acid by comparing said measured residence time to a set of reference residence time values characteristic for said cleavage-inducing agent and a set of N-terminal amino acids;” (para. [0014]) and “e) repeating steps a) through d) one or more times or repeating steps b) through d) one or more times.” (para. [0015]).
Callewaert does not explicitly teach wherein the identifying the first amino acid using the first value for the at least one characteristic is based on an effect of the second amino acid on the first value for the at least one characteristic when the first plurality of signals are emitted that is reflected in the first portion of the data and identifying the sequence of amino acids of the polypeptide based on the identified portion of the sequence of amino acids, wherein a total number of amino acids in the identified sequence of amino acids is greater than a total number of amino acids in the portion of the sequence of amino acids; and the total number of amino acids in the identified sequence of amino acids is greater than a total number of the portions of data corresponding to the amino acids that are exposed at the terminus of the polypeptide during the degradation process of claim 1. However, this limitation is taught by Swaminathan.
Swaminathan teaches wherein the identifying the first amino acid using the first value for the at least one characteristic is based on an effect of the second amino acid on the first value for the at least one characteristic when the first plurality of signals are emitted that is reflected in the first portion of the data with Figure 1 (page 3). Figure 1 depicts a relative fluorescence intensity for the different amino acids of a peptide. The relative fluorescence intensity is highest for the first amino acid and decreases with each degradation cycle. The second amino acid has a lower fluorescence intensity than the first amino acid.
Swaminathan teaches identifying the sequence of amino acids of the polypeptide based on the identified portion of the sequence of amino acids, wherein a total number of amino acids in the identified sequence of amino acids is greater than a total number of amino acids in the portion of the sequence of amino acids; and the total number of amino acids in the identified sequence of amino acids is greater than a total number of the portions of data corresponding to the amino acids that are exposed at the terminus of the polypeptide during the degradation process with “Mapping the partial sequence back to a reference proteome of potential proteins, such as might be derived from a genome sequence, would determine if the fluorosequence uniquely identifies a peptide, and ultimately, its parent protein.” (page 4, para. 3) and “Matching this partial sequence to a reference protein database identifies the peptide.” (Fig. 1 caption, page 3).
It would have been prima facia obvious to combine the teachings of Callewaert and Swaminathan to arrive at the claimed invention. Swaminathan’s method is capable of generating partial peptide sequences in a highly parallel fashion, scalable to entire proteomes (page 3, Paragraph 1). A person of ordinary skill in the art would have been motivated to modify the method of Callewaert to identify the first amino acid and portions of amino acid sequences as taught by Swaminathan for the benefit of utilizing the partial sequence to uniquely identify the peptide from the entire proteomes. Furthermore, there would have been a reasonable expectation of success, since both Callewaert and Swaminathan teach methods that pertain to single molecule peptide sequencing.
With respect to claim 2, Callewaert teaches that the N-terminal amino acid of single molecules of peptides are identified (or categorized) using the catalytic properties of an aminopeptidase and the kinetics of the enzymatic reaction (Paragraph [0005]). The method is based on the correlation between the turnover number (kcat) of engineered aminopeptidases and the N-terminal amino acid which it cleaves (Paragraph [0005]). Therefore, by measuring the time the engineered aminopeptidase resides upon addition on the peptide substrate before the N-terminal amino acid is cleaved off, N-terminal amino acid is identified (Paragraph [0005]). This corresponds to the claim limitation of wherein the data is indicative of amino acid identity at the terminus of the polypeptide during the degradation process.
With respect to claim 3, Callewaert teaches that the N-terminal amino acid of single molecules of peptides are identified (or categorized) using the catalytic properties of an aminopeptidase and the kinetics of the enzymatic reaction (Paragraph [0005]). An aminopeptidase taught by Callewaert is equivalent to amino acid recognition molecule. Therefore, this corresponds to the claim limitation of the data is indicative of signals produced by one or more amino acid recognition molecules binding to different types of terminal amino acids at the terminus during the degradation process.
With respect to claim 4, Callewaert teaches “Surface” as used herein is a synonym for carrier or layer (Paragraph [0077]). The surface or layer of current application is suitable to use in the detection of molecular labels, electrochemical signals, electromagnetic signals, plasmon related events (Paragraph [0077]). Said molecular label can be an optical (comprising but not limited to luminescent and fluorescent labels) or electrical (comprising but not limited to potentiometric, voltametric, coulometric labels) label (Paragraph [0077]).
Callewaert also teaches that in order to detect and measure the “on-time” values or the residence time of the aminopeptidase of the application on the N-terminal amino acid of an immobilized polypeptide or until the N-terminal amino acid of an immobilized polypeptide is cleaved off (see above), said aminopeptidase needs to be detected (Paragraph [0080]). The aminopeptidase can interact cleavage-productively or cleavage non-productively with the substrate within the measured residence time until the N-terminal amino acid is cleaved off (Paragraph [0080]). Of both interaction types, their length, sum of length and average lengths can be part of the measured residence time relevant to the present invention, as these parameters all are part of the measurement that provides information on how long it takes until the aminopeptidase cleaves off the N-terminal amino acid (Paragraph [0080]). The nature of detection is not vital to the invention, as long as the enzyme “on-time” or the residence time of the aminopeptidase can be detected (Paragraph [0080]). In certain embodiments of the application, the “on-time” of the aminopeptidase is detected optically, electrically or plasmonicall(Paragraph [0080]). One way of detecting the aminopeptidases of the application is by fusing it to a molecular label and subsequent detection of the molecular label (Paragraph [0080]). Similar to the above, aminopeptidases can be labelled optically, electrically or plasmonically (Paragraph [0080]). Therefore, Callewaert teaches that the molecular label could be luminescent or fluorescent labels that allows for the “on-time” of the aminopeptidase to be detected optically. This corresponds to the claim limitation of wherein the signals comprise of a luminescent signal generated during the degradation process.
With respect to claim 5, Callewaert teaches to detect the “on-time” values or residence time, two labelling options can be selected (Paragraph [0079]). First, the polypeptides to be sequenced can be labelled for example through their N-terminal amino acids. Alternatively or additionally, internal amino acids can be labelled for example as shown in FIG. 14 (Paragraph [0079]). The labelling of polypeptides can be done using fluorescent probes, such as but not limited to fluorescamine, o-phthalaldehyde, dansyl chloride and coumarinyl isothiocyanate (CITC) (Paragraph [0079]). In particular embodiments, the N-terminal amino acid of the immobilized polypeptide of the application is CITC-derivatized or alternatively phrased labeled with CITC (Paragraph [0079]). The polypeptides can also be electrically labeled (Paragraph [0079]). Electroanalytical methods are a class of techniques by which the presence of an analyte, peptide, enzyme, . . . can be determined by measuring the potential (volts) and/or current (amperes) of the electrical label on the analyte, peptide, enzyme . . . These methods can be broken down into several categories depending on the label (Paragraph [0079]). The three main categories are potentiometry (the difference in electrode potentials is measured), coulometry (the current is measured over time), and voltammetry (the current is measured while the potential is actively altered) (Paragraph [0079]). There are two basic categories of coulometric techniques (Paragraph [0079]). Potentiostatic coulometry involves holding the electric potential constant during the reaction using a potentiostat (Paragraph [0079]). The other, called coulometric titration or amperostatic coulometry, keeps the current (measured in amperes) constant using an amperostat (Paragraph [0079]). A non-limiting example of an electrical label is sulfophenyl isothiocyanate (SPITC) (Paragraph [0079]). SPITC is a negatively charged variant of the phenyl isothiocyanate (PITC) probe that is used in MS de novo peptide sequencing for neutralizing N-terminal fragment ions (Samyn et al. 2004 J Am Soc Mass Spectrom 15:1838-1852) (Paragraph [0079]). In particular embodiments, electrically labeled can be potentiometrically, amperometrically or voltametrically labeled (Paragraph [0079]). This corresponds to the claim limitation of wherein the signals comprise an electrical signal generated during the degradation process.
With respect to claim 6, Callewaert teaches that the present invention relates to the field of biochemistry, more particularly to proteomics, more particularly to protein sequencing, even more particularly to single molecule peptide sequencing (Abstract). The invention discloses means and methods for single molecule protein sequencing and/or amino acid identification using cleavage inducing agent (Abstract). Said cleavage inducing agents which are not specific for one particular amino acid, cleave polypeptides step by step from the N-terminus onwards and provide information on the identity of the cleaved amino acids based on the kinetics of said reaction (Abstract). It is noted that the process of cleaving polypeptides step by step taught by Callewaert is equivalent to series of cleavage events and successive cleavage events. The process of identifying the cleaved amino acids based on the kinetics of said reaction is equivalent to the claim limitation of detecting cleavage events including the cleavage event and determining the portions of the data between successive cleavage events.
With respect to claim 7, Callewaert teaches in claim 17 a method of obtaining sequence information of a polypeptide immobilized on a surface via its C-terminus, the method comprising:
a. contacting the surface-immobilized polypeptide with a cleavage-inducing agent, wherein the agent binds and cleaves the N-terminal amino acid from the polypeptide;
b. measuring the residence time of the cleavage-inducing agent on the N-terminal amino acid of the surface-immobilized polypeptide;
c. allowing the cleavage-inducing agent to cleave off said the N-terminal amino acid; and
d. wherein comparing the measured residence time to a set of reference residence time values characteristic for the cleavage-inducing agent and a set of N-terminal amino acids allows determining the identity of the N-terminal amino acid;
e. repeating the measuring and cleaving one or more times.
Each amino acid of a sequence is considered to be equivalent to individual portions. Repetition of the process taught by Callewaert allows for the sequential measuring, cleaving, and identifying of amino acids resulting in a series of amino acids being identified. Therefore, this corresponds to the claim limitation of determining a type of amino acid for each of the portions.
With respect to claim 8, Callewaert teaches after immobilizing the peptide substrates and determining the single molecule substrate locations in the field-of-view, the fluorescently labeled aminopeptidase is added and the consecutive enzyme residence times on the substrate locations is measured (Paragraph [0151]). Considering that both the enzyme-substrate binding kinetics (KM) and the substrate cleavage kinetics (kcat) depend on the identity of the N-terminal amino acid, by measuring the number of enzyme-substrate ‘on-off’ events and the overall time until substrate cleavage, the identity of the N-terminal amino acid is derived or categorized (FIG. 14) (Paragraph [0151]). Verification of substrate cleavage is derived from a measurable change in frequency of enzyme-substrate ‘on-off’ events before and after cleavage (FIG. 14 below) (Paragraph [0151]). When using a thermophilic aminopeptidase (e.g. T. aquaticus aminopeptidase T) at a far-below optimal temperature, cleave kinetics are significantly reduced leading to an increase in the number of enzyme-substrate ‘on-off’ events (Paragraph [0151]). Callewaert also teaches in Figure 14 the pulse pattern that is observed to be the number of enzyme-substrate ‘on-off’ events and the overall time until substrate cleavage that corresponds to N-terminal amino acid identity. Therefore, this corresponds to the claim limitation of wherein each of the individual portions comprises a pulse pattern, and analyzing the data further comprises determining a type of amino acid for one or more of the portions based on its respective pulse pattern.
With respect to claim 9, Callewaert teaches in Figure 14 the pulse pattern that is observed to be the number of enzyme-substrate ‘on-off’ events and the overall time until substrate cleavage that corresponds to N-terminal amino acid identity. It is interpreted that the event when a pulse is detected corresponds to the time when threshold value is reached or is above threshold. From the data depicted in Figure 4, the amount of time within the portion could be compared with the duration of the portion. Therefore, this corresponds to the claim limitation of determining the type of amino acid further comprises identifying an amount of time within a portion when the data is above a threshold value and comparing the amount of time to a duration of time for the portion.
With respect to claim 10, Callewaert teaches that both the enzyme-substrate binding kinetics (KM) and the substrate cleavage kinetics (kcat) depend on the identity of the N-terminal amino acid, by measuring the number of enzyme-substrate ‘on-off’ events and the overall time until substrate cleavage, the identity of the N-terminal amino acid is derived or categorized (FIG. 14) (Paragraph [0151]). Callewaert also teaches in Figure 14 the pulse pattern that is observed to be the number of enzyme-substrate ‘on-off’ events and the overall time until substrate cleavage that corresponds to N-terminal amino acid identity. Therefore, this corresponds to the claim limitation of wherein the at least one characteristic comprises pulse duration and determining the type of amino acid further comprises identifying at least one pulse duration for each of the one or more portions.
With respect to claim 11, Callewaert teaches that both the enzyme-substrate binding kinetics (KM) and the substrate cleavage kinetics (kcat) depend on the identity of the N-terminal amino acid, by measuring the number of enzyme-substrate ‘on-off’ events and the overall time until substrate cleavage, the identity of the N-terminal amino acid is derived or categorized (FIG. 14) (Paragraph [0151]). Callewaert also teaches in Figure 14 the pulse pattern that is observed to be the number of enzyme-substrate ‘on-off’ events and the overall time until substrate cleavage that corresponds to N-terminal amino acid identity. The off events are interpreted to be equivalent to the interpulse. Therefore, this corresponds to the claim limitation of wherein the at least one characteristic comprises interpulse duration and determining the type of amino acid further comprises identifying at least one interpulse duration for each of the one or more portions.
With respect to claim 12, Callewaert teaches in claim 17 a method of obtaining sequence information of a polypeptide immobilized on a surface via its C-terminus, the method comprising:
a. contacting the surface-immobilized polypeptide with a cleavage-inducing agent, wherein the agent binds and cleaves the N-terminal amino acid from the polypeptide;
b. measuring the residence time of the cleavage-inducing agent on the N-terminal amino acid of the surface-immobilized polypeptide;
c. allowing the cleavage-inducing agent to cleave off said the N-terminal amino acid; and
d. wherein comparing the measured residence time to a set of reference residence time values characteristic for the cleavage-inducing agent and a set of N-terminal amino acids allows determining the identity of the N-terminal amino acid;
e. repeating the measuring and cleaving one or more times.
Each amino acid of a sequence is considered to be equivalent to individual portions. Repetition of the process taught by Callewaert allows for the sequential measuring, cleaving, and identifying of amino acids resulting in a series of amino acids being identified. Therefore, this corresponds to the claim limitation of wherein the sequence of amino acids includes a series of amino acids corresponding to the portions.
Regarding claim 288, Callewaert teaches the recited wherein the first type of molecule is an amino acid recognition molecule, and the second type of molecule is an aminopeptidase at least with "FIG. 15. Schematic representation of the Edman degradation mechanism. Edman degradation entails the coupling of phenyl isothiocyanate (PITC) onto the free N-terminus of a protein/peptide (alkaline conditions), followed by the release of the N-terminal amino acid as a phenylthiohydantoin (PTH) derivative (acidic conditions). The released PTH-amino acid is then identified with chromatography" (Para. [0035]) and with "Said N-terminal amino acid can be a derivatized N-terminal amino acid and if so said aminopeptidase binds and cleaves said derivatized N-terminal amino acid. Said N-terminal amino acid can be an N-terminal amino acid derivatized with isothiocyanate or isothiocyanate analogues" (Para. [0006]). The recited "first type of molecule" corresponds to "phenyl isothiocyanate (PITC)" of Callewaert. The recited "second type of molecule" corresponds to "aminopeptidase" of Callewaert.
Regarding claim 289, Callewaert teaches the recited plurality of signals further comprise a second plurality of signals different from the first plurality of signals; the first plurality of signals correspond to binding events between a first type of amino acid recognition molecule and a first type of amino acid at the terminus of the polypeptide at least with "FIG. 15. Schematic representation of the Edman degradation mechanism. Edman degradation entails the coupling of phenyl isothiocyanate (PITC) onto the free N-terminus of a protein/peptide (alkaline conditions), followed by the release of the N-terminal amino acid as a phenylthiohydantoin (PTH) derivative (acidic conditions). The released PTH-amino acid is then identified with chromatography" (Para. [0035]). The recited "first type of molecule" corresponds to "phenyl isothiocyanate (PITC)" of Callewaert. The recited "first type of amino acid" corresponds to "free N-terminus of a protein/peptide" of Callewaert.
Callewaert teaches the recited the second plurality of signals correspond to binding events between a second type of amino acid recognition molecule and a second type of amino acid at the terminus of the polypeptide at least with "Said N-terminal amino acid can be a derivatized N-terminal amino acid and if so said aminopeptidase binds and cleaves said derivatized N-terminal amino acid. Said N-terminal amino acid can be an N-terminal amino acid derivatized with isothiocyanate or isothiocyanate analogues" (Para. [0006]). The recited "second type of molecule" corresponds to "aminopeptidase" of Callewaert. The recited "second type of amino acid" corresponds to "derivatized N-terminal amino acid" of Callewaert.
Regarding claim 290, Callewaert teaches the recited wherein the plurality of signals corresponds to discrete binding events between one or more amino acid recognition molecules and amino acids that are exposed at the terminus of the polypeptide during the degradation process at least with "Therefore, by measuring the time the engineered aminopeptidase resides upon addition on the peptide substrate before the N-terminal amino acid is cleaved off, N-terminal amino acid is identified. Said aminopeptidase can also be replaced by a chemical cleavage-inducing agent. Similar to what is observed using aminopeptidases, the residence time of chemical cleavage-inducing agents is a read-out for the identity of the N-terminal amino acid to which it binds." (Para. [0005]).
Regarding claim 291, Callewaert teaches the recited wherein the degradation process comprises terminal amino acid cleavage performed by one or more aminopeptidases during the degradation process at least with "Said N-terminal amino acid can be a derivatized N-terminal amino acid and if so said aminopeptidase binds and cleaves said derivatized N-terminal amino acid. Said N-terminal amino acid can be an N-terminal amino acid derivatized with isothiocyanate or isothiocyanate analogues" (Para. [0006]).
Regarding claim 292, Callewaert teaches the recited wherein the first type of molecule comprises an amino acid recognition molecule comprising a fluorophore at least with "In yet another embodiment, the N-terminal amino acid is derivatized (e.g. with biotin, for example using a biotinylated isothiocyanate) such that a binding agent (e.g. an avidin such as streptavidin or neutravidin) that carries a spectroscopically distinguishable label (e.g. a fluorophore) can bind the derivatized N-terminal amino acid" (Para. [0118]).
Regarding claim 293, Callewaert teaches wherein the second amino acid is a penultimate amino acid of the polypeptide when the first amino acid is exposed at the terminus of the polypeptide with Figure 1; “In a first aspect, an engineered, catalytically active aminopeptidase acting on a polypeptide is provided, wherein said polypeptide is immobilized on a surface via its C-terminus or via a peptide moiety C-terminal to the first peptide bond of said polypeptide, wherein said aminopeptidase cleaves the N-terminal amino acid of said polypeptide, and wherein the residence time of said aminopeptidase until cleavage of said N-terminal amino acid identifies or categorizes said N-terminal amino acid. Said N-terminal amino acid can be a derivatized N-terminal amino acid and if so said aminopeptidase binds and cleaves said derivatized N-terminal amino acid. Said N-terminal amino acid can be an N-terminal amino acid derivatized with isothiocyanate or isothiocyanate analogues.” (para. [0006]) and “Said aminopeptidase of current application can also have at least 80% sequence identity to SEQ ID No. 7, wherein a cysteine residue is inserted between the methionine residue at position 1 and the alanine residue at position 2.” (para. [0007]). Figure 1 depicts the first amino acid being released exposing the second amino acid. The recited “penultimate amino acid” corresponds to the second amino acid after the N-terminal amino acid is cleaved off as taught by Callewaert. The recited “penultimate amino acid” also corresponds to the “wherein a cysteine residue is inserted between the methionine residue at position 1 and the alanine residue at position 2” as taught by Callewaert.
Regarding claims 13 and 14, Callewaert does not explicitly teach a system comprising: at least one hardware processor; and at least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by the at least one hardware processor, cause the at least one hardware processor to perform the method of claim 1 in claim 13 and at least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by at least one hardware processor, cause the at least one hardware processor to perform the method of claim 1 in claim 14. However, Callewaert teaches making use of computer programs that are available in the art for sequence alignment (Paragraph [0073]). Callewaert teaches Determining the percentage of sequence homology can be done manually, or by making use of computer programs that are available in the art. Examples of useful algorithms are PILEUP (Higgins & Sharp, CABIOS 5:151 (1989), BLAST and BLAST 2.0 (Altschul et al. J. Mol. Biol. 215: 403 (1990) (Paragraph [0073]). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) (Paragraph [0073]). It is well known that computer programs and software have to be executed on a computer. It is also well known that a generic computer system includes hardware processor and a non-transitory computer-readable storage medium. Therefore, Callewaert’s teachings meet the claim limitations of claims 13 and 14. It would have been obvious to one of ordinary skill in the art at the time of the invention to use the generic computer system and hardware of claims 13 and 14 to automate the data analysis of amino acid sequences to save time on sequence comparison.
Response to 35 USC §103 (05/01/2026, Pages 7-9 of remarks)
It is noted that Applicants arguments are based on amended claim 1.
Applicant argues that the cited portions of Callewaert and Swaminathan do not teach the limitations of amended claim 1.
In response, Applicant's arguments are based on amended claim 1 and are not persuasive. As discussed above Callewaert and Swaminathan was found to teach the limitations of amended claim 1. For instance, Callewaert teaches identifying, using the first value for the at least one characteristic, the first amino acid exposed at the terminus of the polypeptide during the degradation process when the first plurality of signals are emitted and a second amino acid of the polypeptide downstream of the first amino acid when the first plurality of signals are emitted with “The invention discloses means and methods for single molecule protein sequencing and/or amino acid identification using cleavage-inducing agents. Said cleavage-inducing agents, which are not specific for one particular amino acid, cleave polypeptides step by step from the N-terminus onwards and provide information on the identity of the cleaved amino acids based on the kinetics of the engagement between said cleavage inducing agent with the polypeptide or on the kinetics of said polypeptide cleaving reaction.” (para. [0001]); “d) identifying or categorizing said N-terminal amino acid by comparing said measured residence time to a set of reference residence time values characteristic for said cleavage-inducing agent and a set of N-terminal amino acids;” (para. [0014]) and “e) repeating steps a) through d) one or more times or repeating steps b) through d) one or more times.” (para. [0015]).
Swaminathan teaches wherein the identifying the first amino acid using the first value for the at least one characteristic is based on an effect of the second amino acid on the first value for the at least one characteristic when the first plurality of signals are emitted that is reflected in the first portion of the data with Figure 1 (page 3). Figure 1 depicts a relative fluorescence intensity for the different amino acids of a peptide. The relative fluorescence intensity is highest for the first amino acid and decreases with each degradation cycle. The second amino acid has a lower fluorescence intensity than the first amino acid.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Judicial exceptions (JEs) to 101 patentability
Claims 1-14 and 288-293 are rejected under 35 USC 101 because the claimed inventions are not directed to patent eligible subject matter. After consideration of relevant factors with respect to each claim as a whole, each claim is directed to one or more judicially-recognized exceptions to patentability (JEs), i.e. an abstract idea, a natural phenomenon, a law of nature and/or a product of nature, as identified below. Below, it is not clear that any element or combination of elements in addition to the JE(s), i.e. and "additional elements," either integrate the identified JE(s) into a practical application and/or is a non-conventional additional element, such that it is not clear that any claim is directed to significantly more than the identified JE(s).
MPEP 2106 organizes JE analysis into Steps 1, 2A (1st prong & 2nd prong) and 2B as analyzed below. MPEP 2106 and the following USPTO website provide further explanation and case law citations: www.uspto.gov/patent/laws-and-regulations/examination-policy/examination-guidance-and-training-materials.
Analysis of claims 1-14 and 288-293
Step 1: Are the claims directed to a 101 process, machine, manufacture, or composition of matter (MPEP 2106.03)?
Independent claim 1 is directed to a 101 process, here a "method comprising: obtaining data during a degradation process of a polypeptide …," with process steps such as " analyzing … and determining…"
[Step 1: claims 1-14 and 288-293: YES]
Step 2A, 1st prong: Do the claims recite a judicially-recognized exception (JE), e.g. a law of nature, a natural phenomenon or product, or an abstract idea (MPEP 2106.04.II.A.1 & .04(a))?
The MPEP at 2106.I, 2nd para. explains that JEs have been court-recognized as: abstract ideas, laws of nature and natural phenomena (including natural products).
MPEP § 2106.04(a)(2) further explains that abstract ideas may be grouped as:
• mathematical concepts (mathematical formulas or equations, mathematical relationships
and mathematical calculations);
• certain methods of organizing human activity (fundamental economic practices or
principles, managing personal behavior or relationships or interactions between people);
and/or
• mental processes (procedures for observing, evaluating, analyzing/ judging and
organizing information).
Regarding the instant claims and with respect to Step 2A, 1st prong, at least preliminarily these claims recite JEs in the form of abstract ideas (claims 1, 6-11 and 13-14) as follows.
Mental processes recited include:
Claims 1, 13, 14 recite: “…analyzing plurality of signals to determine portions to determine portions of the data corresponding to respective amino acids that are sequentially exposed at the terminus of the polypeptide during the degradation process…; …determining a value for at least one characteristic of the first plurality of signals produced by the discrete binding events between the first molecule and the terminus of the polypeptide …; determining the probabilistic assessment and identifying a portion of a sequence of amino acids of the polypeptide based on at least in part on the analyzing determined values for the at least one characteristic; identifying, using the first value for the at least one characteristic, the first amino acid exposed at the terminus of the polypeptide during the degradation process when the first plurality of signals are emitted and a second amino acid of the polypeptide downstream of the first amino acid when the first plurality of signals are emitted, wherein the identifying the first amino acid using the first value for the at least one characteristic is based on an effect of the second amino acid on the first value for the at least one characteristic when the first plurality of signals are emitted that is reflected in the first portion of the data; identifying the sequence of amino acids of the polypeptide based on the identified portion of the sequence of amino acids, wherein a total number of amino acids in the identified sequence of amino acids is greater than a total number of amino acids in the portion of the sequence of amino acids; and the total number of amino acids in the identified sequence of amino acids is greater than a total number of the portions of data corresponding to the amino acids that are exposed at the terminus of the polypeptide during the degradation process." The claims recite the process of analyzing, determining, identifying and comparing which are data analytics steps and are acts of analyzing, evaluating, organizing and judging information that could be practically performed in the human mind and/or with pen and paper. Acts of evaluating and analyzing data could be practically performed in the human mind and/or with pen and paper because they merely require making observations, evaluations, judgments, and opinions (See MPEP 2106.04(a)(2) subsection III).
Claim 6 recites: “…wherein analyzing the data further comprises detecting cleavage events... and determining the portions of the data between successive cleavage events.” The process of analyzing and determining are data analytics steps that could be performed with the human mind or with pen and paper.
Claim 7 recites: “…wherein analyzing the data further comprises determining a type of amino acid for each of the portions.” The process of analyzing is a data analytics step that could be performed with the human mind or with pen and paper.
Claim 8 recites: “…wherein each of the portions comprises a pulse pattern, and analyzing the data further comprises determining a type of amino acid for one or more of the portions based on its respective pulse pattern.” The process of analyzing and determining are data analytics steps that could be performed with the human mind or with pen and paper.
Claim 9 recites: “…wherein determining the type of amino acid further comprises identifying an amount of time within a portion when the data is above a threshold value and comparing the amount of time to a duration of time for the portion.” The process of determining and identifying are data analytics steps that could be performed with the human mind or with pen and paper.
Claim 10 recites: “…wherein the at least one characteristic comprises pulse duration and determining the type of amino acid further comprises identifying at least one pulse duration for each of the one or more portions.” The process of determining and identifying are data analytics steps that could be performed with the human mind or with pen and paper.
Claim 11 recites: “…wherein the at least one characteristic comprises interpulse duration and determining the type of amino acid further comprises identifying at least one interpulse duration for each of the one or more portions.” The process of determining is a data analytics step could be performed with the human mind or with pen and paper.
At this step of the analysis, elements of claims 1, 6-11 and 13-14 are interpreted as directed to the abstract idea of mental processes including the particularly recited JE steps/elements of " analyzing...," "…determining...," and "identifying...," each of which, including all recitation within each listed element, in at least some embodiments within a BRI, involves only manipulation of data. The above-identified steps/elements are interpreted as directed to the abstract ideas identified below.
BRIs of the claims are analogous to the JE of an abstract idea in the form of a mental process, including obtaining and comparing intangible data. Instant examples of mental process that cause the claim(s) to be directed to the above-identified JE(s) are identified above for claims 1, 6-11 and 13-14. In a BRI, it is not clear that the claim embodiments are limited so as to require complexity precluding analogy to a mental process. Case law, establishing the mental process JE and to which the instant claims are analogized, is presented in MPEP 2106.04(a)(2).III, including examples of analogous mental process JEs.
[Step 2A, 1st prong: claims 1-14 and 288-293: YES]
Step 2A, 2nd prong: Are the above-identified JEs integrated into a practical application (MPEP 2106.04.II.A.2 & .04(d))?
Generally regarding Step 2A, 2nd prong
MPEP 2106.04(d).I lists the following considerations for evaluating whether additional elements integrate a judicial exception into a practical application:
An improvement in the functioning of a computer, or an improvement to other technology or technical field, as discussed in MPEP §§ 2106.04(d)(1) and 2106.05(a);
Applying or using a judicial exception to affect a particular treatment or prophylaxis for a disease or medical condition, as discussed in MPEP § 2106.04(d)(2);
Implementing a judicial exception with, or using a judicial exception in conjunction with, a particular machine or manufacture that is integral to the claim, as discussed in MPEP § 2106.05(b);
Effecting a transformation or reduction of a particular article to a different state or thing, as discussed in MPEP § 2106.05(c); and
Applying or using the judicial exception in some other meaningful way beyond generally linking the use of the judicial exception to a particular technological environment, such that the claim as a whole is more than a drafting effort designed to monopolize the exception, as discussed in MPEP § 2106.05(e).
Additionally, the courts have also identified limitations that did not integrate a judicial exception into a practical application:
Merely reciting a phrase such as "apply it" (or an equivalent) along with the judicial exception, or merely including instructions to implement an abstract idea on a computer, or merely using a computer as a tool to perform an abstract idea, as discussed in MPEP 2106.05(f);
Adding insignificant extra-solution activity to the judicial exception, as discussed in MPEP 2106.05(g); and
Generally linking the use of a judicial exception to a particular technological environment or field of use, as discussed in MPEP 2106.05(h).
At this point in examination, it is not clear that the identified JEs are integrated into a practical application according to any of the "considerations" exemplified in MPEP 2106.04(d).
For example, according to the first consideration at MPEP 2106.04(d)(1), it is not yet clear in the record that application of the above-identified JEs results in an improvement to the technology field notwithstanding the specification at paragraph [0064].
[Step 2A, 2nd prong: claims 1-14 and 288-293: NO]
Step 2B: Do the claims recite a non-conventional arrangement of additional elements (i.e. elements in addition to any identified JE) (MPEP 2106.05)?
All elements of claims 1, 6-11 and 13-14 are part of one or more identified JEs (as described above), except for elements identified here as conventional elements in addition to the above JEs:
The recited " … obtaining data during a degradation process of a polypeptide, wherein obtaining the data comprises obtaining a plurality of signals including a first plurality of signals, each of the first plurality of signals corresponding to a first type of molecule and being emitted when the first type of molecule is bound to a terminus of the polypeptide and respective ones of the first plurality of signals correspond to respective discrete binding events between the first type of molecule and the terminus of the polypeptide occurring prior to a cleavage event of the degradation process performed by a second molecule and outputting the identified sequence of amino acid." steps/elements (claims 1, 13 and 14); “a system comprising: at least one hardware processor; and at least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by the at least one hardware processor, cause the at least one hardware processor to perform the method of claim 1.” steps/elements (claim 13) and “At least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by at least one hardware processor, cause the at least one hardware processor to perform the method of claim 1.” steps/elements (claim 14) are conventional elements of a laboratory and/or computing environment, conventional data gathering/input elements, and/or conventional post-processing or output elements, as exemplified by the following evidence.
The use of a computer or other machinery in its ordinary capacity for economic or other tasks (e.g., to receive, store, or transmit data) or simply adding a general purpose computer or computer components after the fact to an abstract idea (e.g., a fundamental economic practice or mathematical equation) does not integrate a judicial exception into a practical application or provide significantly more as identified by the courts in Affinity Labs v. DirecTV, 838 F.3d 1253, 1262, 120 USPQ2d 1201, 1207 (Fed. Cir. 2016) (cellular telephone); TLI Communications LLC v. AV Auto, LLC, 823 F.3d 607, 613, 118 USPQ2d 1744, 1748 (Fed. Cir. 2016) (computer server and telephone unit). Also, data gathering steps are not an abstract idea, they are extra-solution activity, as they collect the data needed to carry out the abstract idea. Data gathering does not impose any meaningful limitation on the abstract idea, or how the abstract idea is performed. (MPEP 2106.05(g)). For instance, mere data gathering in conjunction with a law of nature or abstract idea such as a step of obtaining information about credit card transactions so that the information can be analyzed by an abstract mental process, as discussed in CyberSource v. Retail Decisions, Inc., 654 F.3d 1366, 1375, 99 USPQ2d 1690, 1694 (Fed. Cir. 2011) (see MPEP § 2106.05(g)) is considered by the courts to be insignificant extra-solution activity. The additional limitation must have more than a nominal or insignificant relationship to the identified judicial exception. (MPEP 2106.04(d)(2))
[Step 2B: claims 1-14 and 288-293: NO]
Summary and conclusion regarding claims 1, 6-11 and 13-14
Summing up the above 101 JE analysis of claims 1, 6-11 and 13-14, each viewed as a whole and considering all elements individually and in combination, no claim recites limitations that transform the claim, finally interpreted as directed to the above-identified JE(s), into patent eligible subject matter.
Remaining claims
Claims 2-5, 12 and 288-293 are providing information on what the data represents and do not change the character of the data obtaining step beyond mere data gathering activity.
The claims have all been examined to identify the presence of one or more judicial exceptions. Each additional element in the claims has been addressed, alone and in combination, to determine whether the additional elements integrate the judicial exception into a practical application. Each additional limitation in the claims has been addressed, alone and in combination, to determine whether those additional limitations provide an inventive concept which provides significantly more than those exceptions. Individually, the limitations of the claims and the claims as a whole have been found to be patent ineligible under 35 U.S.C. 101.
Response to 35 USC § 101 Remarks received 05/01/2026 (pages 9-12)
Applicant amended claim 1.
In Applicant's remarks for Claim Rejections under 35 U.S.C. §101, see pages 9-12, Applicant states the pending claims do not recite any abstract ideas of mental processes. According to Applicant, the supposed abstract ideas of claim 1 are patent-eligible under Prong 2 of Step 2A because the claims recite an improvement to polypeptide sequencing technology by enabling high-accuracy polypeptide sequencing based on partial sequence information. Applicant provides references to paragraphs 63-65 and 266 of the specification that describes the claimed improvement. Applicant states that the paragraph 0064 of the specification describes that the techniques disclosed in the present application present an advancement in polypeptide sequencing that allows for the analysis of polypeptides by amino acid detection throughout an ongoing degradation reaction in real time. Applicant further states that the claimed techniques do not require polypeptide labeling and/or harsh chemical reagents used in certain conventional polypeptide sequencing approaches, thereby increasing throughput and/or accuracy of sequence information obtained from a sample. Applicant also states that the claim techniques improve upon conventional protein identification systems by identifying a sequence of amino acids of a polypeptide without requiring individually identifying each amino acid in the sequence. Applicant asserts that the claimed techniques facilitate increased throughout and/or accuracy of sequence information based on partial sequence information, enabling protein sequencing based on partial polypeptide data, which is an improvement to polypeptide sequencing technology. Applicant states that the claims reflect the described improvements.
In response, Applicant’s arguments under Step 2A Prong 2 regarding improvement have been fully considered and are not persuasive. Applicant’s argument of improvement in increased throughout and/or accuracy of sequence information is not persuasive because the argument is a bare assertion of an improvement without the detail necessary to be apparent to a person of ordinary skill in the art. As stated in the MPEP 2106.05(a) and MPEP 2106.04(d), if the specification explicitly sets forth an improvement but in a conclusory manner (i.e., a bare assertion of an improvement without the detail necessary to be apparent to a person of ordinary skill in the art), the examiner should not determine the claim improves technology. (see MPEP 2106.05(a) and MPEP 2106.04(d)).
Double Patenting Rejections
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/process/file/efs/guidance/eTD-info-I.jsp.
Any newly recited portions herein are necessitated by claim amendment.
Claims 1-8, 10-11, 13-14 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 68-70, 73, 77, 80, 106, 107, 118 and 119 of copending Application No. 16/708,989 (reference application) in view of Callewaert and Swaminathan. Although the claims at issue are not identical, they are not patentably distinct from each other because both sets of claims recite a method of sequencing a polypeptide and identifying amino acids in a polypeptide during the degradation process. Both groups of claims are also involved with detecting pulses for amino acid identification. Callewaert also teaches these elements.
See table below for similarities between the claims.
Instant Application
Reference Application 16/708,989
Claim 2
Claim 77
2. (Original) The method of claim 1, wherein the data is indicative of amino acid identity at the terminus of the polypeptide during the degradation process.
77. The method of claim 69, wherein the characteristic pattern is indicative of the amino acid exposed at the terminus of the single polypeptide molecule and an amino acid at a contiguous position.
Claim 3
Claim 106
3. (Original) The method of claim 2, wherein the data is indicative of a signal produced by one or more amino acid recognition molecules binding to different types of terminal amino acids at the terminus during the degradation process.
106. The method of claim 68, wherein each of the one or more terminal amino acid recognition molecules comprises a detectable label.
Claim 4
Claim 107
4. (Original) The method of claim 1, wherein the data is indicative of a luminescent signal generated during the degradation process.
107. The method of claim 106, wherein the detectable label is a luminescent label or a conductivity label.
Claim 5
Claim 107
5. (Original) The method of claim 1, wherein the data is indicative of an electrical signal generated during the degradation process.
107. The method of claim 106, wherein the detectable label is a luminescent label or a conductivity label.
Claim 6
Claim 80
6. (Original) The method of claim 1, wherein analyzing the data further comprises detecting a series of cleavage events and determining the portions of the data between successive cleavage events.
80. The method of claim 68, wherein sequencing comprises identifying at least a portion of all types of successive amino acids exposed at the terminus of the single polypeptide while the single polypeptide is being degraded by the cleaving reagent.
Claim 7
Claim 77
7. (Original) The method of claim 1, wherein analyzing the data further comprises determining a type of amino acid for each of the individual portions.
77. (Previously Presented) The method of claim 69, wherein the characteristic pattern is
indicative of the amino acid exposed at the terminus of the single polypeptide molecule and an
amino acid at a contiguous position.
Claim 8
Claims 68 and 69
8. (Original) The method of claim 1, wherein each of the individual portions comprises a pulse pattern, and analyzing the data further comprises determining a type of amino acid for one or more of the portions based on its respective pulse pattern.
68. A method of sequencing a polypeptide, the method comprising:
contacting a single polypeptide molecule in a reaction mixture with a composition
comprising one or more terminal amino acid recognition molecules and a cleaving reagent, wherein
the single polypeptide molecule is immobilized to a surface through a linker comprising an
oligonucleotide; and
detecting a series of signal pulses indicative of association of the one or more terminal
amino acid recognition molecules with a terminus of the single polypeptide molecule in the
presence of the cleaving reagent, wherein the series of signal pulses is indicative of a series of
amino acids exposed at the terminus over time as a result of terminal amino acid cleavage by the
cleaving reagent.
69. (Original) The method of claim 68, wherein association of the one or more terminal amino
acid recognition molecules with each type of amino acid exposed at the terminus produces a
characteristic pattern in the series of signal pulses that is different from other types of amino acids exposed at the terminus.
Claim 10
Claims 70 and 77
10. (Original) The method of claim 8, wherein determining the type of amino acid further comprises identifying at least one pulse duration for each of the one or more portions.
70. The method of claim 69, wherein the characteristic pattern comprises a portion of the series of signal pulses.
77. The method of claim 69, wherein the characteristic pattern is indicative of the amino acid exposed at the terminus of the single polypeptide molecule and an amino acid at a contiguous position.
Claim 11
Claim 73
11. (Original) The method of claim 8, wherein determining the type of amino acid further comprises identifying at least one interpulse duration for each of the one or more portions.
73. The method of claim 72, wherein each signal pulse of the characteristic pattern is separated from another by an interpulse duration that is characteristic of an association rate of terminal amino acid recognition molecule binding.
Claim 13
Claim 118
13. (Currently Amended) A system comprising: at least one hardware processor; and at least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by the at least one hardware processor, cause the at least one hardware processor to perform the method of claim 1.
118. A system comprising:
at least one hardware processor; and
at least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by the at least one hardware processor, cause the at least one hardware processor to perform the method of claim 68.
Claim 14
Claim 119
14. (Currently Amended) At least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by at least one hardware processor, cause the at least one hardware processor to perform the method of claim 1
119. At least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by at least one hardware processor, cause the at least one hardware processor to perform the method of claim 68.
Reference Application No. 16/708,989 does not teach obtaining data during the degradation process of a polypeptide, wherein obtaining the data comprises obtaining a plurality of signals including a first plurality of signals, each of the first plurality of signals corresponding to a first type of molecule and being emitted when the first type of molecule is bound to a terminus of the polypeptide and respective ones of the first plurality of signals correspond to respective discrete binding events of a plurality of binding events that occur between the first type of molecule and the terminus of the polypeptide occurring in, prior to a cleavage event of the degradation process, the cleavage event performed by a second type of molecule different than the first type of molecule;
analyzing the plurality of signals to determine portions of the data corresponding to respective amino acids that are exposed at the terminus of the polypeptide during the degradation process, including a first portion of the data corresponding to a first amino acid exposed at the terminus of the polypeptide during the degradation process when the first plurality of signals are emitted; based on the determined portions of data, determining a value for at least one characteristic of the respective determined portions of data; including a first value for the at least one characteristic based on the first portion of the data corresponding to the first amino acid; identifying a portion of a sequence of amino acids of the polypeptide based at least in part on the determined values for the at least one characteristic at least in part by: identifying, using the first value for the at least one characteristic, the first amino acid exposed at the terminus of the polypeptide during the degradation process when the first plurality of signals are emitted and a second amino acid of the polypeptide downstream of the first amino acid when the first plurality of signals are emitted,; and outputting the identified sequence of amino acids of claim 1. However, these limitations are taught by Callewaert.
Application No. 16/708,989 also does not teach wherein the identifying the first amino acid using the first value for the at least one characteristic is based on an effect of the second amino acid on the first value for the at least one characteristic when the first plurality of signals are emitted that is reflected in the first portion of the data; identifying the sequence of amino acids of the polypeptide based on the identified portion of the sequence of amino acids, wherein a total number of amino acids in the identified sequence of amino acids is greater than a total number of amino acids in the portion of the sequence of amino acids; and the total number of amino acids in the identified sequence of amino acids is greater than a total number of the portions of data corresponding to the amino acids that are exposed at the terminus of the polypeptide during the degradation process; based on the determined portions of data of claim 1. However, this limitation is taught by Swaminathan.
Regarding claim 1, Callewaert teaches the recited obtaining the data comprises obtaining a plurality of signals including a first plurality of signals, each of the first plurality of signals corresponding to a first type of molecule and being emitted when the first type of molecule is bound to a terminus of the polypeptide and respective ones of the first plurality of signals at least with "FIG. 15. Schematic representation of the Edman degradation mechanism. Edman degradation entails the coupling of phenyl isothiocyanate (PITC) onto the free N-terminus of a protein/peptide (alkaline conditions), followed by the release of the N-terminal amino acid as a phenylthiohydantoin (PTH) derivative (acidic conditions). The released PTH-amino acid is then identified with chromatography" (Para. [0035]). The recited "first type of molecule" corresponds to "phenyl isothiocyanate (PITC)" of Callewaert. The recited "signals" corresponds to "released PTH-amino acid" that is identified by chromatography of Callewaert. Callewaert also teaches "The method of the application can additionally include a step of determining the cleavage of said N-terminal amino acid by measuring an optical, electrical or plasmonical signal of the surface-immobilized polypeptide, wherein a difference in optical, electrical or plasmonical signal is indicative for cleavage of said N-terminal amino acid. The above methods are also provided wherein said surface-immobilized polypeptide is additionally contacted with one or more N-terminal amino acid binding proteins, wherein the kinetics of the binding events of said one or more binding proteins to said N-terminal amino acid identify or is further informative for said N-terminal amino acid. The above methods can also include a first step of polypeptide denaturation or are provided in which polypeptide denaturing conditions are present during one or more of the steps of said methods, wherein said catalytically active aminopeptidase is a thermophilic and/or solvent resistant aminopeptidase and/or wherein said cleavage-inducing agent is isothiocyanate or isothiocyanate analogues. Above methods are also provided wherein N-terminal amino acid is derivatized. The aminopeptidase from above methods can be any of the aminopeptidase disclosed herein." (Para. [0018])
Callewaert teaches the recited discrete binding events of a plurality of binding events that occur between the first type of molecule and the terminus of the polypeptide occurring prior to a cleavage event of the degradation process at least with "FIG. 15. Schematic representation of the Edman degradation mechanism. Edman degradation entails the coupling of phenyl isothiocyanate (PITC) onto the free N-terminus of a protein/peptide (alkaline conditions), followed by the release of the N-terminal amino acid as a phenylthiohydantoin (PTH) derivative (acidic conditions). The released PTH-amino acid is then identified with chromatography" (Para. [0035]). The recited "first type of molecule" corresponds to "phenyl isothiocyanate (PITC)" of Callewaert. It is noted that cleavage is interpreted to be equivalent to the degradation. Callewaert also teaches "...the identity of the N-terminal amino acid derivative is determined by performing, for example, 20 rounds of antibody binding with antibodies specific for each PITC-derivatized N-terminal amino acid, detection, and stripping. The N-terminal amino acid is removed by raising the temperature or lowering pH, and the cycle is repeated to sequence 12-20 amino acids from each peptide on the slide." (para. [0003]).
Callewaert teaches the recited the cleavage event performed by a second type of molecule different than the first type of molecule at least with "Said N-terminal amino acid can be a derivatized N-terminal amino acid and if so said aminopeptidase binds and cleaves said derivatized N-terminal amino acid. Said N-terminal amino acid can be an N-terminal amino acid derivatized with isothiocyanate or isothiocyanate analogues" (Para. [0006]). The recited "second type of molecule" corresponds to "aminopeptidase" of Callewaert.
Callewaert teaches the recited based on the determined portions of data, determining a value for at least one characteristic of the respective determined portions of data, including a first value for the at least one characteristic based on the first portion of the data corresponding to the first amino acid; identifying a portion of a sequence of amino acids of the polypeptide based at least in part on the determined values for the at least one characteristic at least with "...wherein said cleavage-inducing agent is isothiocyanate or isothiocyanate analogues, wherein said residence time is the length of time until said N-terminal amino acid is removed, wherein said N-terminal amino acid is identified by comparing said length of time to a set of reference values for different amino acids." (Para. [0016]) and “…wherein said cleavage-inducing agent binds and cleaves the N-terminal amino acid from said polypeptide; measuring the residence time of said cleavage-inducing agent on said N-terminal amino acid; and comparing said measured residence time to a set of reference residence time values characteristic for said cleavage-inducing agent and a set of N-terminal amino acids to identify or categorize said N-terminal amino acid.” (Para. [0009]). The recited "value" corresponds to "residence time" and "length of time" of Callewaert. Callewaert also teaches (Paragraph [0018]) the method of the application can additionally include a step of determining the cleavage of said N-terminal amino acid by measuring an optical, electrical or plasmonical signal of the surface-immobilized polypeptide, wherein a difference in optical, electrical or plasmonical signal is indicative for cleavage of said N-terminal amino acid (Paragraph [0018]). The identity of the N-terminal amino acid corresponds to the signal value and the characteristic of the signal allows one to determine its identity.
Callewaert further teaches that the N-terminal amino acid of single molecules of peptides are identified (or categorized) using the catalytic properties of an aminopeptidase and the kinetics of the enzymatic reaction (Paragraph [0005]). The method is based on the correlation between the turnover number (kcat) of engineered aminopeptidases and the N-terminal amino acid which it cleaves (Paragraph [0005]). Therefore, by measuring the time the engineered aminopeptidase resides upon addition on the peptide substrate before the N-terminal amino acid is cleaved off, N-terminal amino acid is identified (Paragraph [0005]). This corresponds to the claim limitation of obtaining data during a degradation process of a polypeptide and analyzing the plurality of signals to determine portions of the data corresponding to respective amino acids that are exposed at a terminus of the polypeptide during the degradation process.
Callewaert teaches including a first portion of the data corresponding to a first amino acid exposed at the terminus of the polypeptide during the degradation process when the first plurality of signals are emitted with “One of the additional parts of the methods of the application is that the cleavage of the terminal amino acid is to be detected or confirmed. Hence also provided herein are the methods of current application, additionally including a step of determining the cleavage of said terminal amino acid by measuring an optical, electrical or plasmonical signal of the surface-immobilized polypeptide, wherein a difference in optical, electrical or plasmonical signal is indicative for cleavage of said terminal amino acid. Indeed, immobilized peptides with a free N-terminus have several properties which are utilized to determine when an N-terminal amino acid has been cleaved off by the cleaving-inducing agents of the present invention.” (para. [0115]).
Callewaert teaches identifying a portion of a sequence of amino acids of the polypeptide based at least in part on the determined values for the at least one characteristic; with "...the application provides a method to sequence proteins comprising the following step cycle: the N-terminal derivatization of peptides immobilized through a moiety of the peptide C-terminal to the scissile bond, measuring the time it takes for a cleavage-inducing agent to cleave off the N-terminal amino acid, leading to release of the N-terminal amino acid from the immobilisation surface, setting the system ready for the next cycle (FIG. 1)." (Para. [0005]) and “…wherein said cleavage-inducing agent binds and cleaves the N-terminal amino acid from said polypeptide; measuring the residence time of said cleavage-inducing agent on said N-terminal amino acid; and comparing said measured residence time to a set of reference residence time values characteristic for said cleavage-inducing agent and a set of N-terminal amino acids to identify or categorize said N-terminal amino acid.” (Para. [0009]).
Callewaert teaches the application provides a method to sequence proteins comprising the following step cycle: the N-terminal derivatization of peptides immobilized through a moiety of the peptide C-terminal to the scissile bond, measuring the time it takes for a cleavage-inducing agent to cleave off the N-terminal amino acid, leading to release of the N-terminal amino acid from the immobilization surface, setting the system ready for the next cycle (FIG. 1) (Paragraph [0005]). Callewaert also teaches “The invention discloses means and methods for single molecule protein sequencing and/or amino acid identification using cleavage inducing agent.” (Abstract) This corresponds to the claim limitation of identifying the sequence of amino acids of the polypeptide and outputting the identified sequence of amino acids.
Callewaert teaches identifying, using the first value for the at least one characteristic, the first amino acid exposed at the terminus of the polypeptide during the degradation process when the first plurality of signals are emitted and a second amino acid of the polypeptide downstream of the first amino acid when the first plurality of signals are emitted with “The invention discloses means and methods for single molecule protein sequencing and/or amino acid identification using cleavage-inducing agents. Said cleavage-inducing agents, which are not specific for one particular amino acid, cleave polypeptides step by step from the N-terminus onwards and provide information on the identity of the cleaved amino acids based on the kinetics of the engagement between said cleavage inducing agent with the polypeptide or on the kinetics of said polypeptide cleaving reaction.” (para. [0001]); “d) identifying or categorizing said N-terminal amino acid by comparing said measured residence time to a set of reference residence time values characteristic for said cleavage-inducing agent and a set of N-terminal amino acids;” (para. [0014]) and “e) repeating steps a) through d) one or more times or repeating steps b) through d) one or more times.” (para. [0015]).
Callewaert does not explicitly teach wherein the identifying the first amino acid using the first value for the at least one characteristic is based on an effect of the second amino acid on the first value for the at least one characteristic when the first plurality of signals are emitted that is reflected in the first portion of the data and identifying the sequence of amino acids of the polypeptide based on the identified portion of the sequence of amino acids, wherein a total number of amino acids in the identified sequence of amino acids is greater than a total number of amino acids in the portion of the sequence of amino acids; and the total number of amino acids in the identified sequence of amino acids is greater than a total number of the portions of data corresponding to the amino acids that are exposed at the terminus of the polypeptide during the degradation process of claim 1. However, this limitation is taught by Swaminathan.
Swaminathan teaches wherein the identifying the first amino acid using the first value for the at least one characteristic is based on an effect of the second amino acid on the first value for the at least one characteristic when the first plurality of signals are emitted that is reflected in the first portion of the data with Figure 1 (page 3). Figure 1 depicts a relative fluorescence intensity for the different amino acids of a peptide. The relative fluorescence intensity is highest for the first amino acid and decreases with each degradation cycle. The second amino acid has a lower fluorescence intensity than the first amino acid.
Swaminathan teaches identifying the sequence of amino acids of the polypeptide based on the identified portion of the sequence of amino acids, wherein a total number of amino acids in the identified sequence of amino acids is greater than a total number of amino acids in the portion of the sequence of amino acids; and the total number of amino acids in the identified sequence of amino acids is greater than a total number of the portions of data corresponding to the amino acids that are exposed at the terminus of the polypeptide during the degradation process with “Mapping the partial sequence back to a reference proteome of potential proteins, such as might be derived from a genome sequence, would determine if the fluorosequence uniquely identifies a peptide, and ultimately, its parent protein.” (page 4, para. 3) and “Matching this partial sequence to a reference protein database identifies the peptide.” (Fig. 1 caption, page 3).
A person of ordinary skill in the art would have been motivated to combine the method of Callewaert with Application No. 16/708,989 (reference application) to identify N-terminal amino acids. Furthermore, there would have been a reasonable expectation of success, since both Callewaert and reference application teach methods that pertain to determining amino acid sequences.
A person of ordinary skill in the art would have also been motivated to modify the method of Application No. 16/708,989 to identify portions of amino acid sequences as taught by Swaminathan for the benefit of utilizing the partial sequence to uniquely identify the peptide from the entire proteomes. Furthermore, there would have been a reasonable expectation of success, since both Application No. 16/708,989 and Swaminathan teach methods that pertain to single molecule peptide sequencing.
Claims 1-2, 4-5 and 7-14 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 152, 154, 156, 160, 161, 190 and 205-206 of copending Application No. 16/709,052 (reference application) in view of Callewaert and Swaminathan. Although the claims at issue are not identical, they are not patentably distinct from each other because both sets of claims recite a method of sequencing a polypeptide and identifying amino acids in a polypeptide during the degradation process. Both groups of claims are also involved with detecting pulses for amino acid identification. Callewaert also teaches these elements.
See table below for similarities between the claims.
Instant Application
Reference Application 16/709,052, 12/06/2023
Claim 2
Claim 152
2. (Original) The method of claim 1, wherein the data is indicative of amino acid identity at the terminus of the polypeptide during the degradation process.
152. A method of identifying an amino acid of a polypeptide, the method
comprising:
contacting a single polypeptide molecule with one or more amino acid recognition
molecules that bind to the single polypeptide molecule, wherein at least one amino acid recognition
molecule comprises an N-end rule pathway protein;
detecting a series of signal pulses indicative of association of the one or more amino acid
recognition molecules with the single polypeptide molecule under polypeptide degradation
conditions in a reaction mixture comprising two or more aminopeptidases, wherein at least two aminopeptidases in the reaction mixture each comprise an amino acid sequence that is at least 80% identical to a different sequence selected from the group consisting of SEQ ID NOs: 48, 59, and 69; and
identifying a first type of amino acid in the single polypeptide molecule based on a first
characteristic pattern in the series of signal pulses.
Claim 4
Claim 190
4. (Original) The method of claim 1, wherein the data is indicative of a luminescent signal generated during the degradation process.
190. The method of claim 189, wherein the detectable label is a luminescent label or a conductivity label.
Claim 5
Claim 190
5. (Original) The method of claim 1, wherein the data is indicative of an electrical signal generated during the degradation process.
190. The method of claim 189, wherein the detectable label is a luminescent label or a conductivity label.
Claim 7
Claim 160
7. (Original) The method of claim 1, wherein analyzing the data further comprises determining a type of amino acid for each of the portions.
160. The method of claim 152, wherein the single polypeptide molecule comprises the first type of amino acid at a terminal position of the single polypeptide molecule
Claim 8
Claim 154
8. (Original) The method of claim 1, wherein each of the individual portions comprises a pulse pattern, and analyzing the data further comprises determining a type of amino acid for one or more of the portions based on its respective pulse pattern.
154. The method of claim 153, wherein a signal pulse of the first characteristic pattern corresponds to an individual association event between an amino acid recognition molecule and the first type of amino acid.
Claim 10
Claim 161
10. (Original) The method of claim 8, wherein determining the type of amino acid further comprises identifying at least one pulse duration for each of the one or more portions.
161. The method of claim 160, wherein the first characteristic pattern is indicative of the first type of amino acid at the terminal position and an amino acid type at a contiguous position.
Claim 11
Claim 156
11. (Original) The method of claim 8, wherein determining the type of amino acid further comprises identifying at least one interpulse duration for each of the one or more portions.
156. The method of claim 155, wherein each signal pulse of the first characteristic pattern is separated from another by an interpulse duration that is characteristic of an association rate of amino acid recognition molecule binding.
Claim 12
Claim 161
12. (Original) The method of claim 1, wherein the sequence of amino acids includes a series of amino acids corresponding to the portions.
161. The method of claim 160, wherein the first characteristic pattern is indicative of the first type of amino acid at the terminal position and an amino acid type at a contiguous position.
Claim 13
Claim 205
13. (Currently Amended) A system comprising: at least one hardware processor; and at least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by the at least one hardware processor, cause the at least one hardware processor to perform the method of claim 1.
205. A system comprising:
at least one hardware processor; and
at least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by the at least one hardware processor, cause the at least one hardware processor to perform the method of claim 152.
Claim 14
Claim 206
14. (Currently Amended) At least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by at least one hardware processor, cause the at least one hardware processor to perform the method of claim 1
206. At least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by at least one hardware processor, cause the at least one hardware processor to perform the method of claim 152.
Copending Application No. 16/709,052 (reference application) does not teach performing sequencing of a polypeptide at least in part by: obtaining data during a degradation process of the polypeptide, wherein obtaining the data comprises obtaining a plurality of signals including a first plurality of signals, each of the first plurality of signals corresponding to a first type of molecule and being emitted when the first type of molecule is bound to a terminus of the polypeptide and respective ones of the first plurality of signals correspond to respective discrete binding events of a plurality of binding events that occur between the first type of molecule and the terminus of the polypeptide occurring prior to a cleavage event of the degradation process, the cleavage event performed by a second type of molecule different than the first type of molecule; analyzing the plurality of signals to determine portions of the data corresponding to respective amino acids that are exposed at the terminus of the polypeptide during the degradation process; based on the determined portions of data, determining a value for at least one characteristic of the respective determined portions of data ; identifying a portion of a sequence of amino acids of the polypeptide based at least in part on the determined values for the at least one characteristic; identifying the sequence of amino acids of the polypeptide based on the identified portion of the sequence of amino acids, wherein a total number of amino acids in the identified sequence of amino acids is greater than a total number of amino acids in the portion of the sequence of amino acids; and the total number of amino acids in the identified sequence of amino acids is greater than a total number of the portions of data corresponding to the amino acids that are exposed at the terminus of the polypeptide during the degradation process; and outputting the identified sequence of amino acids. of claim 1. However, these limitations are taught by Callewaert and Swaminathan.
Regarding claim 1, Callewaert teaches the recited obtaining the data comprises obtaining a plurality of signals including a first plurality of signals, each of the first plurality of signals corresponding to a first type of molecule and being emitted when the first type of molecule is bound to a terminus of the polypeptide and respective ones of the first plurality of signals at least with "FIG. 15. Schematic representation of the Edman degradation mechanism. Edman degradation entails the coupling of phenyl isothiocyanate (PITC) onto the free N-terminus of a protein/peptide (alkaline conditions), followed by the release of the N-terminal amino acid as a phenylthiohydantoin (PTH) derivative (acidic conditions). The released PTH-amino acid is then identified with chromatography" (Para. [0035]). The recited "first type of molecule" corresponds to "phenyl isothiocyanate (PITC)" of Callewaert. The recited "signals" corresponds to "released PTH-amino acid" that is identified by chromatography of Callewaert. Callewaert also teaches "The method of the application can additionally include a step of determining the cleavage of said N-terminal amino acid by measuring an optical, electrical or plasmonical signal of the surface-immobilized polypeptide, wherein a difference in optical, electrical or plasmonical signal is indicative for cleavage of said N-terminal amino acid. The above methods are also provided wherein said surface-immobilized polypeptide is additionally contacted with one or more N-terminal amino acid binding proteins, wherein the kinetics of the binding events of said one or more binding proteins to said N-terminal amino acid identify or is further informative for said N-terminal amino acid. The above methods can also include a first step of polypeptide denaturation or are provided in which polypeptide denaturing conditions are present during one or more of the steps of said methods, wherein said catalytically active aminopeptidase is a thermophilic and/or solvent resistant aminopeptidase and/or wherein said cleavage-inducing agent is isothiocyanate or isothiocyanate analogues. Above methods are also provided wherein N-terminal amino acid is derivatized. The aminopeptidase from above methods can be any of the aminopeptidase disclosed herein." (Para. [0018])
Callewaert teaches the recited discrete binding events of a plurality of binding events that occur between the first type of molecule and the terminus of the polypeptide occurring prior to a cleavage event of the degradation process at least with "FIG. 15. Schematic representation of the Edman degradation mechanism. Edman degradation entails the coupling of phenyl isothiocyanate (PITC) onto the free N-terminus of a protein/peptide (alkaline conditions), followed by the release of the N-terminal amino acid as a phenylthiohydantoin (PTH) derivative (acidic conditions). The released PTH-amino acid is then identified with chromatography" (Para. [0035]). The recited "first type of molecule" corresponds to "phenyl isothiocyanate (PITC)" of Callewaert. It is noted that cleavage is interpreted to be equivalent to the degradation. Callewaert also teaches "...the identity of the N-terminal amino acid derivative is determined by performing, for example, 20 rounds of antibody binding with antibodies specific for each PITC-derivatized N-terminal amino acid, detection, and stripping. The N-terminal amino acid is removed by raising the temperature or lowering pH, and the cycle is repeated to sequence 12-20 amino acids from each peptide on the slide." (para. [0003]).
Callewaert teaches the recited the cleavage event performed by a second type of molecule different than the first type of molecule at least with "Said N-terminal amino acid can be a derivatized N-terminal amino acid and if so said aminopeptidase binds and cleaves said derivatized N-terminal amino acid. Said N-terminal amino acid can be an N-terminal amino acid derivatized with isothiocyanate or isothiocyanate analogues" (Para. [0006]). The recited "second type of molecule" corresponds to "aminopeptidase" of Callewaert.
Callewaert teaches the recited based on the determined portions of data, determining a value for at least one characteristic of the respective determined portions of data, including a first value for the at least one characteristic based on the first portion of the data corresponding to the first amino acid; identifying a portion of a sequence of amino acids of the polypeptide based at least in part on the determined values for the at least one characteristic at least with "...wherein said cleavage-inducing agent is isothiocyanate or isothiocyanate analogues, wherein said residence time is the length of time until said N-terminal amino acid is removed, wherein said N-terminal amino acid is identified by comparing said length of time to a set of reference values for different amino acids." (Para. [0016]) and “…wherein said cleavage-inducing agent binds and cleaves the N-terminal amino acid from said polypeptide; measuring the residence time of said cleavage-inducing agent on said N-terminal amino acid; and comparing said measured residence time to a set of reference residence time values characteristic for said cleavage-inducing agent and a set of N-terminal amino acids to identify or categorize said N-terminal amino acid.” (Para. [0009]). The recited "value" corresponds to "residence time" and "length of time" of Callewaert. Callewaert also teaches (Paragraph [0018]) the method of the application can additionally include a step of determining the cleavage of said N-terminal amino acid by measuring an optical, electrical or plasmonical signal of the surface-immobilized polypeptide, wherein a difference in optical, electrical or plasmonical signal is indicative for cleavage of said N-terminal amino acid (Paragraph [0018]). The identity of the N-terminal amino acid corresponds to the signal value and the characteristic of the signal allows one to determine its identity.
Callewaert further teaches that the N-terminal amino acid of single molecules of peptides are identified (or categorized) using the catalytic properties of an aminopeptidase and the kinetics of the enzymatic reaction (Paragraph [0005]). The method is based on the correlation between the turnover number (kcat) of engineered aminopeptidases and the N-terminal amino acid which it cleaves (Paragraph [0005]). Therefore, by measuring the time the engineered aminopeptidase resides upon addition on the peptide substrate before the N-terminal amino acid is cleaved off, N-terminal amino acid is identified (Paragraph [0005]). This corresponds to the claim limitation of obtaining data during a degradation process of a polypeptide and analyzing the plurality of signals to determine portions of the data corresponding to respective amino acids that are exposed at a terminus of the polypeptide during the degradation process.
Callewaert teaches including a first portion of the data corresponding to a first amino acid exposed at the terminus of the polypeptide during the degradation process when the first plurality of signals are emitted with “One of the additional parts of the methods of the application is that the cleavage of the terminal amino acid is to be detected or confirmed. Hence also provided herein are the methods of current application, additionally including a step of determining the cleavage of said terminal amino acid by measuring an optical, electrical or plasmonical signal of the surface-immobilized polypeptide, wherein a difference in optical, electrical or plasmonical signal is indicative for cleavage of said terminal amino acid. Indeed, immobilized peptides with a free N-terminus have several properties which are utilized to determine when an N-terminal amino acid has been cleaved off by the cleaving-inducing agents of the present invention.” (para. [0115]).
Callewaert teaches identifying a portion of a sequence of amino acids of the polypeptide based at least in part on the determined values for the at least one characteristic; with "...the application provides a method to sequence proteins comprising the following step cycle: the N-terminal derivatization of peptides immobilized through a moiety of the peptide C-terminal to the scissile bond, measuring the time it takes for a cleavage-inducing agent to cleave off the N-terminal amino acid, leading to release of the N-terminal amino acid from the immobilisation surface, setting the system ready for the next cycle (FIG. 1)." (Para. [0005]) and “…wherein said cleavage-inducing agent binds and cleaves the N-terminal amino acid from said polypeptide; measuring the residence time of said cleavage-inducing agent on said N-terminal amino acid; and comparing said measured residence time to a set of reference residence time values characteristic for said cleavage-inducing agent and a set of N-terminal amino acids to identify or categorize said N-terminal amino acid.” (Para. [0009]).
Callewaert teaches the application provides a method to sequence proteins comprising the following step cycle: the N-terminal derivatization of peptides immobilized through a moiety of the peptide C-terminal to the scissile bond, measuring the time it takes for a cleavage-inducing agent to cleave off the N-terminal amino acid, leading to release of the N-terminal amino acid from the immobilization surface, setting the system ready for the next cycle (FIG. 1) (Paragraph [0005]). Callewaert also teaches “The invention discloses means and methods for single molecule protein sequencing and/or amino acid identification using cleavage inducing agent.” (Abstract) This corresponds to the claim limitation of identifying the sequence of amino acids of the polypeptide and outputting the identified sequence of amino acids.
Callewaert teaches identifying, using the first value for the at least one characteristic, the first amino acid exposed at the terminus of the polypeptide during the degradation process when the first plurality of signals are emitted and a second amino acid of the polypeptide downstream of the first amino acid when the first plurality of signals are emitted with “The invention discloses means and methods for single molecule protein sequencing and/or amino acid identification using cleavage-inducing agents. Said cleavage-inducing agents, which are not specific for one particular amino acid, cleave polypeptides step by step from the N-terminus onwards and provide information on the identity of the cleaved amino acids based on the kinetics of the engagement between said cleavage inducing agent with the polypeptide or on the kinetics of said polypeptide cleaving reaction.” (para. [0001]); “d) identifying or categorizing said N-terminal amino acid by comparing said measured residence time to a set of reference residence time values characteristic for said cleavage-inducing agent and a set of N-terminal amino acids;” (para. [0014]) and “e) repeating steps a) through d) one or more times or repeating steps b) through d) one or more times.” (para. [0015]).
Callewaert does not explicitly teach wherein the identifying the first amino acid using the first value for the at least one characteristic is based on an effect of the second amino acid on the first value for the at least one characteristic when the first plurality of signals are emitted that is reflected in the first portion of the data and identifying the sequence of amino acids of the polypeptide based on the identified portion of the sequence of amino acids, wherein a total number of amino acids in the identified sequence of amino acids is greater than a total number of amino acids in the portion of the sequence of amino acids; and the total number of amino acids in the identified sequence of amino acids is greater than a total number of the portions of data corresponding to the amino acids that are exposed at the terminus of the polypeptide during the degradation process of claim 1. However, this limitation is taught by Swaminathan.
Swaminathan teaches wherein the identifying the first amino acid using the first value for the at least one characteristic is based on an effect of the second amino acid on the first value for the at least one characteristic when the first plurality of signals are emitted that is reflected in the first portion of the data with Figure 1 (page 3). Figure 1 depicts a relative fluorescence intensity for the different amino acids of a peptide. The relative fluorescence intensity is highest for the first amino acid and decreases with each degradation cycle. The second amino acid has a lower fluorescence intensity than the first amino acid.
Swaminathan teaches identifying the sequence of amino acids of the polypeptide based on the identified portion of the sequence of amino acids, wherein a total number of amino acids in the identified sequence of amino acids is greater than a total number of amino acids in the portion of the sequence of amino acids; and the total number of amino acids in the identified sequence of amino acids is greater than a total number of the portions of data corresponding to the amino acids that are exposed at the terminus of the polypeptide during the degradation process with “Mapping the partial sequence back to a reference proteome of potential proteins, such as might be derived from a genome sequence, would determine if the fluorosequence uniquely identifies a peptide, and ultimately, its parent protein.” (page 4, para. 3) and “Matching this partial sequence to a reference protein database identifies the peptide.” (Fig. 1 caption, page 3).
A person of ordinary skill in the art would have been motivated to combine the method of Callewaert with Application No. 16/709,052 (reference application) to identify N-terminal amino acids. Furthermore, there would have been a reasonable expectation of success, since both Callewaert and reference application teach methods that pertain to determining amino acid sequences.
A person of ordinary skill in the art would have also been motivated to modify the method of Application No. 16/709,052 to identify portions of amino acid sequences as taught by Swaminathan for the benefit of utilizing the partial sequence to uniquely identify the peptide from the entire proteomes. Furthermore, there would have been a reasonable expectation of success, since both Application No. 16/709,052 and Swaminathan teach methods that pertain to single molecule peptide sequencing.
Claims 1-4, 8, 10 and 13-14 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 120, 125, 138 and 150-151 of copending Application No. 16/709,024 (reference application) in view of Callewaert and Swaminathan. . Although the claims at issue are not identical, they are not patentably distinct from each other because both sets of claims recite a method of sequencing a polypeptide and identifying amino acids in a polypeptide during the degradation process. Both groups of claims are also involved with detecting pulses for amino acid identification. Callewaert also teaches these elements.
See table below for a mapping of the claims of the reference application and the claims of the instant application.
Instant Application
Reference Application 16/709,024, 02/11/2025
Claim 1
Claim 120
1. (Currently amended) A method comprising: performing sequencing of a polypeptide at least in part by: obtaining data during a degradation process of the polypeptide, wherein obtaining the data comprises obtaining a plurality of signals including a first plurality of signals, each of the first plurality of signals corresponding to a first type of molecule and being emitted when the first type of molecule is bound to a terminus of the polypeptide and respective ones of the first plurality of signals correspond to respective discrete binding events of a plurality of binding events that occur between the first type of molecule and the terminus of the polypeptide occurring prior to a cleavage event of the degradation process, the cleavage event performed by a second type of molecule different than the first type of molecule;
analyzing the plurality of signals to determine portions of the data corresponding to respective amino acids that are exposed at the terminus of the polypeptide during the degradation process, including a first portion of the data corresponding to a first amino acid exposed at the terminus of the polypeptide during the degradation process when the first plurality of signals are emitted; based on the determined portions of data, determining a value for at least one characteristic of the respective determined portions of data, including a first value for the at least one characteristic based on the first portion of the data corresponding to the first amino acid;
identifying a portion of a sequence of amino acids of the polypeptide based at least in part on the determined values for the at least one characteristic at least in part by:
identifying, using the first value for the at least one characteristic, the first amino acid exposed at the terminus of the polypeptide during the degradation process when the first plurality of signals are emitted and a second amino acid of the polypeptide downstream of the first amino acid when the first plurality of signals are emitted,
wherein the identifying the first amino acid using the first value for the at least one characteristic is based on an effect of the second amino acid on the first value for the at least one characteristic when the first plurality of signals are emitted that is reflected in the first portion of the data;
identifying the sequence of amino acids of the polypeptide based on the identified portion of the sequence of amino acids, wherein: a total number of amino acids in the identified sequence of amino acids is greater than [[a]] the total number of amino acids in the portion of the sequence of amino acids; and the total number of amino acids in the identified sequence of amino acids is greater than [[a]] the total number of the portions of data corresponding to the amino acids that are exposed at the terminus of the polypeptide during the degradation process; and outputting the identified sequence of amino acids.
120. (Currently Amended) A method of polypeptide sequencing, the method comprising:(a) detecting a first series of signal pulses corresponding to binding events between one or more amino acid recognition molecules and a first amino acid at a terminus of a single polypeptide molecule; (b) removing the first amino acid from the terminus of the single polypeptide molecule, wherein a second amino acid is exposed at the terminus of the single polypeptide molecule; and (c) identifying the second amino acid based at least in part on a first characteristic pattern in the first series of signal pulses, wherein each signal pulse of the first characteristic pattern is indicative of an individual association event between an amino acid recognition molecule and the first amino acid prior to (b).
Claim 2
Claim 120
2. (Original) The method of claim 1, wherein the data is indicative of amino acid identity at the terminus of the polypeptide during the degradation process.
120. (Currently Amended) A method of polypeptide sequencing, the method comprising:(a) detecting a first series of signal pulses corresponding to binding events between one or more amino acid recognition molecules and a first amino acid at a terminus of a single polypeptide molecule; (b) removing the first amino acid from the terminus of the single polypeptide molecule, wherein a second amino acid is exposed at the terminus of the single polypeptide molecule; and (c) identifying the second amino acid based at least in part on a first characteristic pattern in the first series of signal pulses.
Claim 3
Claim 125
3. (Original) The method of claim 2, wherein the data is indicative of a signal produced by one or more amino acid recognition molecules binding to different types of terminal amino acids at the terminus during the degradation process.
125. The method of claim 120, wherein
the first amino acid is identified based on the
produces a characteristic pattern in the series of signal pulses that is different from the second amino acid.
Claim 4
Claim 138
4. (Original) The method of claim 1, wherein the data is indicative of a luminescent signal generated during the degradation process.
138. The method of claim 137, wherein the detectable label is a luminescent label or a conductivity label.
Claim 8
Claim 125
8. (Original) The method of claim 1, wherein each of the individual portions comprises a pulse pattern, and analyzing the data further comprises determining a type of amino acid for one or more of the portions based on its respective pulse pattern.
125. The method of claim 120, wherein the first amino acid is identified based on the characteristic pattern in the series of signal pulses.
Claim 10
Claim 125
10. (Original) The method of claim 8, wherein determining the type of amino acid further comprises identifying at least one pulse duration for each of the one or more portions.
125. The method of claim 120, wherein the first amino acid is identified based on the characteristic pattern in the series of signal pulses.
Claim 13
Claim 150
13. (Currently Amended) A system comprising: at least one hardware processor; and at least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by the at least one hardware processor, cause the at least one hardware processor to perform the method of claim 1.
150. (Withdrawn) A system comprising:
at least one hardware processor; and
at least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by the at least one hardware processor, cause the at least one hardware processor to perform the method of claim 120.
Claim 14
Claim 151
14. (Currently Amended) At least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by at least one hardware processor, cause the at least one hardware processor to perform the method of claim 1
151. (Withdrawn) At least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by at least one hardware processor, cause the at least one hardware processor to perform the method of claim 120.
Copending Application No. 16/709,024 (reference application) does not teach the cleavage event performed by a second type of molecule different than the first type of molecule; analyzing the plurality of signals to determine portions of the data corresponding to respective amino acids that are sequentially exposed at the terminus of the polypeptide during the degradation process, based on the determined portions of data, determining a value for at least one characteristic of the respective determined portions of data; identifying a portion of a sequence of amino acids of the polypeptide based at least in part on the determined values for the at least one characteristic; and outputting the identified sequence of amino acids. However, these limitations are taught by Callewaert.
Application No. 16/709,024 also does not teach wherein the identifying the first amino acid using the first value for the at least one characteristic is based on an effect of the second amino acid on the first value for the at least one characteristic when the first plurality of signals are emitted that is reflected in the first portion of the data; identifying the sequence of amino acids of the polypeptide based on the identified portion of the sequence of amino acids, wherein a total number of amino acids in the identified sequence of amino acids is greater than a total number of amino acids in the portion of the sequence of amino acids; and the total number of amino acids in the identified sequence of amino acids is greater than a total number of the portions of data corresponding to the amino acids that are exposed at the terminus of the polypeptide during the degradation process of claim 1. However, this limitation is taught by Swaminathan.
Regarding claim 1, Callewaert teaches the recited the cleavage event performed by a second type of molecule different than the first type of molecule at least with "Said N-terminal amino acid can be a derivatized N-terminal amino acid and if so said aminopeptidase binds and cleaves said derivatized N-terminal amino acid. Said N-terminal amino acid can be an N-terminal amino acid derivatized with isothiocyanate or isothiocyanate analogues" (Para. [0006]). The recited "second type of molecule" corresponds to "aminopeptidase" of Callewaert.
Callewaert teaches the recited determining a value for at least one characteristic of the respective determined portions of data at least with "...wherein said cleavage-inducing agent is isothiocyanate or isothiocyanate analogues, wherein said residence time is the length of time until said N-terminal amino acid is removed, wherein said N-terminal amino acid is identified by comparing said length of time to a set of reference values for different amino acids." (Para. [0016]). The recited "value" corresponds to "residence time" and "length of time" of Callewaert. Callewaert also teaches (Paragraph [0018]) the method of the application can additionally include a step of determining the cleavage of said N-terminal amino acid by measuring an optical, electrical or plasmonical signal of the surface-immobilized polypeptide, wherein a difference in optical, electrical or plasmonical signal is indicative for cleavage of said N-terminal amino acid (Paragraph [0018]). The identity of the N-terminal amino acid corresponds to the signal value and the characteristic of the signal allows one to determine its identity.
Callewaert further teaches that the N-terminal amino acid of single molecules of peptides are identified (or categorized) using the catalytic properties of an aminopeptidase and the kinetics of the enzymatic reaction (Paragraph [0005]). The method is based on the correlation between the turnover number (kcat) of engineered aminopeptidases and the N-terminal amino acid which it cleaves (Paragraph [0005]). Therefore, by measuring the time the engineered aminopeptidase resides upon addition on the peptide substrate before the N-terminal amino acid is cleaved off, N-terminal amino acid is identified (Paragraph [0005]). This corresponds to the claim limitation of obtaining data during a degradation process of a polypeptide and analyzing the plurality of signals to determine portions of the data corresponding to respective amino acids that are sequentially exposed at a terminus of the polypeptide during the degradation process.
Callewaert teaches identifying a portion of a sequence of amino acids of the polypeptide based at least in part on the determined values for the at least one characteristic; with "...the application provides a method to sequence proteins comprising the following step cycle: the N-terminal derivatization of peptides immobilized through a moiety of the peptide C-terminal to the scissile bond, measuring the time it takes for a cleavage-inducing agent to cleave off the N-terminal amino acid, leading to release of the N-terminal amino acid from the immobilisation surface, setting the system ready for the next cycle (FIG. 1)." (Para. [0005]) and “…wherein said cleavage-inducing agent binds and cleaves the N-terminal amino acid from said polypeptide; measuring the residence time of said cleavage-inducing agent on said N-terminal amino acid; and comparing said measured residence time to a set of reference residence time values characteristic for said cleavage-inducing agent and a set of N-terminal amino acids to identify or categorize said N-terminal amino acid.” (Para. [0009]).
Callewaert teaches the application provides a method to sequence proteins comprising the following step cycle: the N-terminal derivatization of peptides immobilized through a moiety of the peptide C-terminal to the scissile bond, measuring the time it takes for a cleavage-inducing agent to cleave off the N-terminal amino acid, leading to release of the N-terminal amino acid from the immobilization surface, setting the system ready for the next cycle (FIG. 1) (Paragraph [0005]). Callewaert also teaches “The invention discloses means and methods for single molecule protein sequencing and/or amino acid identification using cleavage inducing agent.” (Abstract) This corresponds to the claim limitation of identifying the sequence of amino acids of the polypeptide and outputting the identified sequence of amino acids.
Swaminathan teaches wherein the identifying the first amino acid using the first value for the at least one characteristic is based on an effect of the second amino acid on the first value for the at least one characteristic when the first plurality of signals are emitted that is reflected in the first portion of the data with Figure 1 (page 3). Figure 1 depicts a relative fluorescence intensity for the different amino acids of a peptide. The relative fluorescence intensity is highest for the first amino acid and decreases with each degradation cycle. The second amino acid has a lower fluorescence intensity than the first amino acid.
Swaminathan teaches identifying the sequence of amino acids of the polypeptide based on the identified portion of the sequence of amino acids, wherein a total number of amino acids in the identified sequence of amino acids is greater than a total number of amino acids in the portion of the sequence of amino acids; and the total number of amino acids in the identified sequence of amino acids is greater than a total number of the portions of data corresponding to the amino acids that are exposed at the terminus of the polypeptide during the degradation process with “Mapping the partial sequence back to a reference proteome of potential proteins, such as might be derived from a genome sequence, would determine if the fluorosequence uniquely identifies a peptide, and ultimately, its parent protein.” (page 4, para. 3) and “Matching this partial sequence to a reference protein database identifies the peptide.” (Fig. 1 caption, page 3).
A person of ordinary skill in the art would have been motivated to combine the method of Callewaert with Application No. 16/709,024 (reference application) to identify N-terminal amino acids. Furthermore, there would have been a reasonable expectation of success, since both Callewaert and reference application teach methods that pertain to determining amino acid sequences.
A person of ordinary skill in the art would have also been motivated to modify the method of Application No. 16/709,024 to identify portions of amino acid sequences as taught by Swaminathan for the benefit of utilizing the partial sequence to uniquely identify the peptide from the entire proteomes. Furthermore, there would have been a reasonable expectation of success, since both Application No. 16/709,024 and Swaminathan teach methods that pertain to single molecule peptide sequencing.
Claim 1-4, 6, 8, 10-11, and 13-14 provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 15, 20, 24, 27, 53-54 and 66-67 of copending Application No. 16/708,956 (reference application)in view of Callewaert and Swaminathan. Although the claims at issue are not identical, they are not patentably distinct from each other because both sets of claims recite a method of sequencing a polypeptide and identifying amino acids in a polypeptide during the degradation process. Both groups of claims are also involved with detecting pulses for amino acid identification. Callewaert also teaches these elements.
See table below for similarities between the claims.
Instant Application
Reference Application 16/708,956
Claim 1
Claims 24 and 27
1. (Currently amended) A method comprising: performing sequencing of a polypeptide at least in part by: obtaining data during a degradation process of the polypeptide, wherein obtaining the data comprises obtaining a plurality of signals including a first plurality of signals, each of the first plurality of signals corresponding to a first type of molecule and being emitted when the first type of molecule is bound to a terminus of the polypeptide and respective ones of the first plurality of signals correspond to respective discrete binding events of a plurality of binding events that occur between the first type of molecule and the terminus of the polypeptide occurring prior to a cleavage event of the degradation process, the cleavage event performed by a second type of molecule different than the first type of molecule;
analyzing the plurality of signals to determine portions of the data corresponding to respective amino acids that are exposed at the terminus of the polypeptide during the degradation process, including a first portion of the data corresponding to a first amino acid exposed at the terminus of the polypeptide during the degradation process when the first plurality of signals are emitted; based on the determined portions of data, determining a value for at least one characteristic of the respective determined portions of data, including a first value for the at least one characteristic based on the first portion of the data corresponding to the first amino acid;
identifying a portion of a sequence of amino acids of the polypeptide based at least in part on the determined values for the at least one characteristic at least in part by:
identifying, using the first value for the at least one characteristic, the first amino acid exposed at the terminus of the polypeptide during the degradation process when the first plurality of signals are emitted and a second amino acid of the polypeptide downstream of the first amino acid when the first plurality of signals are emitted,
wherein the identifying the first amino acid using the first value for the at least one characteristic is based on an effect of the second amino acid on the first value for the at least one characteristic when the first plurality of signals are emitted that is reflected in the first portion of the data;
identifying the sequence of amino acids of the polypeptide based on the identified portion of the sequence of amino acids, wherein: a total number of amino acids in the identified sequence of amino acids is greater than [[a]] the total number of amino acids in the portion of the sequence of amino acids; and the total number of amino acids in the identified sequence of amino acids is greater than [[a]] the total number of the portions of data corresponding to the amino acids that are exposed at the terminus of the polypeptide during the degradation process; and outputting the identified sequence of amino acids.
24. (Currently Amended) The method of claim 15, wherein the characteristic pattern is
indicative of the amino acid exposed at the terminus of the single polypeptide molecule and an amino acid at a contiguous position.
27. (Previously Presented) The method of claim 15, wherein sequencing comprises identifying
at least a portion of all types of successive amino acids exposed at the terminus of the single
polypeptide while the single polypeptide is being degraded.
Claim 2
Claim 27
2. (Original) The method of claim 1, wherein the data is indicative of amino acid identity at the terminus of the polypeptide during the degradation process.
27. (Previously Presented) The method of claim 15, wherein sequencing comprises identifying
at least a portion of all types of successive amino acids exposed at the terminus of the single
polypeptide while the single polypeptide is being degraded.
Claim 3
Claim 53
3. (Original) The method of claim 2, wherein the data is indicative of a signal produced by one or more amino acid recognition molecules binding to different types of terminal amino acids at the terminus during the degradation process.
53. (Previously Presented) The method of claim 15, wherein each of the one or more terminal
amino acid recognition molecules comprises a detectable label.
Claim 4
Claim 54
4. (Original) The method of claim 1, wherein the data is indicative of a luminescent signal generated during the degradation process.
54. (Original) The method of claim 53, wherein the detectable label is a luminescent label or a
conductivity label.
Claim 6
Claim 15
6. (Original) The method of claim 1, wherein analyzing the data further comprises detecting cleavage events including the cleavage event and determining the portions of the data between successive cleavage events.
15. (Currently Amended) A method of polypeptide sequencing, the method comprising: contacting a single polypeptide molecule with one or more terminal amino acid recognition molecules that is contacted with the SH2 domain-containing protein; and detecting a series of signal pulses indicative of association of the one or more terminal amino acid recognition molecules with successive amino acids exposed at a terminus of the single polypeptide while the single polypeptide is being degraded, thereby sequencing the single polypeptide molecule, wherein sequencing comprises identifying that an amino acid of the single polypeptide molecule comprises a phosphorylated side chain based at least in part on a characteristic pattern in the series of signal pulses.
Claim 8
Claim 15
8. (Original) The method of claim 1, wherein each of the individual portions comprises a pulse pattern, and analyzing the data further comprises determining a type of amino acid for one or more of the portions based on its respective pulse pattern.
15. (Currently Amended) A method of polypeptide sequencing, the method comprising: contacting a single polypeptide molecule with one or more terminal amino acid recognition molecules, wherein at least one of the one or more terminal amino acid recognition molecules is an SH2 domain-containing protein; and detecting a series of signal pulses indicative of association of the one or more terminal amino acid recognition molecules with successive amino acids exposed at a terminus of the single polypeptide while the single polypeptide is being degraded, thereby sequencing the single polypeptide molecule, wherein sequencing comprises identifying that an amino acid of the single polypeptide molecule comprises a phosphorylated side chain
Claim 10
Claim 24
10. (Original) The method of claim 8, wherein determining the type of amino acid further comprises identifying at least one pulse duration for each of the one or more portions.
24. (Currently Amended) The method of claim 15, wherein the characteristic pattern is
indicative of the amino acid exposed at the terminus of the single polypeptide molecule and an amino acid at a contiguous position.
Claim 11
Claim 20
11. (Original) The method of claim 8, wherein determining the type of amino acid further comprises identifying at least one interpulse duration for each of the one or more portions.
20. (Previously Presented) The method of claim 19, wherein each signal pulse of the
characteristic pattern is separated from another by an interpulse duration that is characteristic of an
association rate of binding between the terminal amino acid recognition molecule and the amino
acid exposed at the terminus.
Claim 13
Claim 66
13. (Currently Amended) A system comprising: at least one hardware processor; and at least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by the at least one hardware processor, cause the at least one hardware processor to perform the method of claim 1.
66. (Previously Presented) A system comprising:
at least one hardware processor; and
at least one non-transitory computer-readable storage medium storing processor-executable
instructions that, when executed by the at least one hardware processor, cause the at least one
hardware processor to perform the method of claim 15.
Claim 14
Claim 67
14. (Currently Amended) At least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by at least one hardware processor, cause the at least one hardware processor to perform the method of claim 1
67. (Previously Presented) At least one non-transitory computer-readable storage medium
storing processor-executable instructions that, when executed by at least one hardware processor,
cause the at least one hardware processor to perform the method of claim 15.
Copending Application No. 16/708,956 (reference application) does not teach based on the determined portions of data, determining a value for at least one characteristic of the respective determined portions of data; identifying a portion of a sequence of amino acids of the polypeptide based at least in part on the determined values for the at least one characteristic;; and outputting the identified sequence of amino acids of claim 1. However, these limitations are taught by Callewaert.
Application No. 16/708,956 also does not teach identifying the sequence of amino acids of the polypeptide based on the identified portion of the sequence of amino acids, wherein a total number of amino acids in the identified sequence of amino acids is greater than a total number of amino acids in the portion of the sequence of amino acids; and the total number of amino acids in the identified sequence of amino acids is greater than a total number of the portions of data corresponding to the amino acids that are exposed at the terminus of the polypeptide during the degradation process; based on the determined portions of data, of claim 1. However, this limitation is taught by Swaminathan.
Regarding claim 1, Callewaert teaches the recited determining a value for at least one characteristic of the respective determined portions of data at least with "...wherein said cleavage-inducing agent is isothiocyanate or isothiocyanate analogues, wherein said residence time is the length of time until said N-terminal amino acid is removed, wherein said N-terminal amino acid is identified by comparing said length of time to a set of reference values for different amino acids." (Para. [0016]). The recited "value" corresponds to "residence time" and "length of time" of Callewaert. Callewaert also teaches (Paragraph [0018]) the method of the application can additionally include a step of determining the cleavage of said N-terminal amino acid by measuring an optical, electrical or plasmonical signal of the surface-immobilized polypeptide, wherein a difference in optical, electrical or plasmonical signal is indicative for cleavage of said N-terminal amino acid (Paragraph [0018]). The identity of the N-terminal amino acid corresponds to the signal value and the characteristic of the signal allows one to determine its identity.
Callewaert further teaches that the N-terminal amino acid of single molecules of peptides are identified (or categorized) using the catalytic properties of an aminopeptidase and the kinetics of the enzymatic reaction (Paragraph [0005]). The method is based on the correlation between the turnover number (kcat) of engineered aminopeptidases and the N-terminal amino acid which it cleaves (Paragraph [0005]). Therefore, by measuring the time the engineered aminopeptidase resides upon addition on the peptide substrate before the N-terminal amino acid is cleaved off, N-terminal amino acid is identified (Paragraph [0005]). This corresponds to the claim limitation of obtaining data during a degradation process of a polypeptide and analyzing the plurality of signals to determine portions of the data corresponding to respective amino acids that are exposed at a terminus of the polypeptide during the degradation process.
Callewaert teaches identifying a portion of a sequence of amino acids of the polypeptide based at least in part on the determined values for the at least one characteristic; with "...the application provides a method to sequence proteins comprising the following step cycle: the N-terminal derivatization of peptides immobilized through a moiety of the peptide C-terminal to the scissile bond, measuring the time it takes for a cleavage-inducing agent to cleave off the N-terminal amino acid, leading to release of the N-terminal amino acid from the immobilisation surface, setting the system ready for the next cycle (FIG. 1)." (Para. [0005]) and “…wherein said cleavage-inducing agent binds and cleaves the N-terminal amino acid from said polypeptide; measuring the residence time of said cleavage-inducing agent on said N-terminal amino acid; and comparing said measured residence time to a set of reference residence time values characteristic for said cleavage-inducing agent and a set of N-terminal amino acids to identify or categorize said N-terminal amino acid.” (Para. [0009]).
Callewaert teaches the application provides a method to sequence proteins comprising the following step cycle: the N-terminal derivatization of peptides immobilized through a moiety of the peptide C-terminal to the scissile bond, measuring the time it takes for a cleavage-inducing agent to cleave off the N-terminal amino acid, leading to release of the N-terminal amino acid from the immobilization surface, setting the system ready for the next cycle (FIG. 1) (Paragraph [0005]). Callewaert also teaches “The invention discloses means and methods for single molecule protein sequencing and/or amino acid identification using cleavage inducing agent.” (Abstract) This corresponds to the claim limitation of identifying the sequence of amino acids of the polypeptide and outputting the identified sequence of amino acids.
Callewaert teaches identifying, using the first value for the at least one characteristic, the first amino acid exposed at the terminus of the polypeptide during the degradation process when the first plurality of signals are emitted and a second amino acid of the polypeptide downstream of the first amino acid when the first plurality of signals are emitted with “The invention discloses means and methods for single molecule protein sequencing and/or amino acid identification using cleavage-inducing agents. Said cleavage-inducing agents, which are not specific for one particular amino acid, cleave polypeptides step by step from the N-terminus onwards and provide information on the identity of the cleaved amino acids based on the kinetics of the engagement between said cleavage inducing agent with the polypeptide or on the kinetics of said polypeptide cleaving reaction.” (para. [0001]); “d) identifying or categorizing said N-terminal amino acid by comparing said measured residence time to a set of reference residence time values characteristic for said cleavage-inducing agent and a set of N-terminal amino acids;” (para. [0014]) and “e) repeating steps a) through d) one or more times or repeating steps b) through d) one or more times.” (para. [0015]).
Swaminathan teaches wherein the identifying the first amino acid using the first value for the at least one characteristic is based on an effect of the second amino acid on the first value for the at least one characteristic when the first plurality of signals are emitted that is reflected in the first portion of the data with Figure 1 (page 3). Figure 1 depicts a relative fluorescence intensity for the different amino acids of a peptide. The relative fluorescence intensity is highest for the first amino acid and decreases with each degradation cycle. The second amino acid has a lower fluorescence intensity than the first amino acid.
Swaminathan teaches identifying the sequence of amino acids of the polypeptide based on the identified portion of the sequence of amino acids, wherein a total number of amino acids in the identified sequence of amino acids is greater than a total number of amino acids in the portion of the sequence of amino acids; and the total number of amino acids in the identified sequence of amino acids is greater than a total number of the portions of data corresponding to the amino acids that are exposed at the terminus of the polypeptide during the degradation process with “Mapping the partial sequence back to a reference proteome of potential proteins, such as might be derived from a genome sequence, would determine if the fluorosequence uniquely identifies a peptide, and ultimately, its parent protein.” (page 4, para. 3) and “Matching this partial sequence to a reference protein database identifies the peptide.” (Fig. 1 caption, page 3).
A person of ordinary skill in the art would have been motivated to combine the method of Callewaert with Application No. 16/708,956 (reference application) to identify N-terminal amino acids. Furthermore, there would have been a reasonable expectation of success, since both Callewaert and reference application teach methods that pertain to determining amino acid sequences.
A person of ordinary skill in the art would have also been motivated to modify the method of Application No. 16/708,956 to identify portions of amino acid sequences as taught by Swaminathan for the benefit of utilizing the partial sequence to uniquely identify the peptide from the entire proteomes. Furthermore, there would have been a reasonable expectation of success, since both Application No. 16/708,956 and Swaminathan teach methods that pertain to single molecule peptide sequencing.
Response to Double Patenting Remarks
Applicant’s arguments, see page 13, filed 05/01/2026, with respect to the double patenting rejection(s) of claim(s) 1-14 and 288-293 have been fully considered and are not persuasive. Although the instant claims are amended to differ from the claims of the reference applications 16/708,989, 16/709,052, 16/709,024 and 16/708,956, both the instant claims and the claims of the reference applications are not patentably distinct from each other because both sets of claims are involved with a method of sequencing a polypeptide, identifying amino acids in a polypeptide during the degradation process and detecting pulses for amino acid identification. Callewaert and Swaminathan also teaches the claim limitations that the reference applications do not teach. Therefore, the claims of the reference applications in view of Callewaert and Swaminathan teaches the instant claims as discussed above in the Double Patenting Rejections section.
Conclusion
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/K.K./Examiner, Art Unit 1686
/LARRY D RIGGS II/Supervisory Patent Examiner, Art Unit 1686
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