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 .
Claim Status
Claims 1-3, 5, 7-8, 10-14, 16-17, 19-21, 23-26 are under examination (12/8/2023). Applicant previously cancelled claims 4, 6, 9, 15, 18 and 22 (12/8/2023).
Priority
Claims 1-3, 5, 7-8, 10-14, 16-17, 19-21, 23-26 receive the priority date of 2/19/2021, the filing date of United Kingdom Application No. GB2102391.6.
Information Disclosure Statement
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Information Disclosure Statements from 9/27/2023 and 5/21/2025 are considered.
Nucleotide and/or Amino Acid Sequence Disclosures
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Items 1) and 2) provide general guidance related to requirements for sequence disclosures.
37 CFR 1.821(c) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.821(a) must contain a "Sequence Listing," as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.821 - 1.825. This "Sequence Listing" part of the disclosure may be submitted:
In accordance with 37 CFR 1.821(c)(1) via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter "Legal Framework") as an ASCII text file, together with an incorporation-by-reference of the material in the ASCII text file in a separate paragraph of the specification as required by 37 CFR 1.823(b)(1) identifying:
the name of the ASCII text file;
ii) the date of creation; and
iii) the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(1) on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation-by-reference of the material in the ASCII text file according to 37 CFR 1.52(e)(8) and 37 CFR 1.823(b)(1) in a separate paragraph of the specification identifying:
the name of the ASCII text file;
the date of creation; and
the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(2) via the USPTO patent electronic filing system as a PDF file (not recommended); or
In accordance with 37 CFR 1.821(c)(3) on physical sheets of paper (not recommended).
When a “Sequence Listing” has been submitted as a PDF file as in 1(c) above (37 CFR 1.821(c)(2)) or on physical sheets of paper as in 1(d) above (37 CFR 1.821(c)(3)), 37 CFR 1.821(e)(1) requires a computer readable form (CRF) of the “Sequence Listing” in accordance with the requirements of 37 CFR 1.824.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed via the USPTO patent electronic filing system as a PDF, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the PDF copy and the CRF copy (the ASCII text file copy) are identical.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed on paper or read-only optical disc, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the paper or read-only optical disc copy and the CRF are identical.
Specific deficiencies and the required response to this Office Action are as follows:
Specific deficiency - This application fails to comply with the requirements of 37 CFR 1.821 - 1.825 because it does not contain a "Sequence Listing" as a separate part of the disclosure or a CRF of the “Sequence Listing.”.
Required response - Applicant must provide:
A "Sequence Listing" part of the disclosure; together with
An amendment specifically directing its entry into the application in accordance with 37 CFR 1.825(a)(2);
A statement that the "Sequence Listing" includes no new matter as required by 37 CFR 1.821(a)(4); and
A statement that indicates support for the amendment in the application, as filed, as required by 37 CFR 1.825(a)(3).
If the "Sequence Listing" part of the disclosure is submitted according to item 1) a) or b) above, Applicant must also provide:
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required incorporation-by-reference paragraph, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter.
If the "Sequence Listing" part of the disclosure is submitted according to item 1) c) or d) above, applicant must also provide:
A CRF in accordance with 37 CFR 1.821(e)(1) or 1.821(e)(2) as required by 1.825(a)(5); and
A statement according to item 2) a) or b) above.
Specification
The disclosure is objected to because of the following informalities (see MPEP § 608.01):
The use of the terms, “Triton-X” (p. 21,68), “Tween-20” (p. 21), “Biacore System” (p. 32), “Biolayer Interferometry” (p. 32), “Atto550” (p. 48), “Atto647” (p. 48), Atto488” (p. 48), “Sigma Aldrich” (p. 53), “Thermofisher” (p. 53), “Prime BSI Express” (p. 68), “Picasso” (p. 68), “QC-STORM” (p. 68) and “Python” (p. 68) are trade names or marks used in commerce, has been noted in this application. The terms should be accompanied by the generic terminology; furthermore, the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
The spacing of the lines of the specification is such as to make reading difficult. New application papers with lines 1 1/2 or double spaced (see 37 CFR 1.52(b)(2)) on good quality paper are required for p. 68.
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code (p. 22 in reference to Humana Press and p. 38 in reference to Sigma Aldrich). Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
Claim Objections
Claim 8 is objected to because of the following informalities:
Claim 8 at line 4; “derived” should be replaced with “obtained.”
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-3, 5, 7-8, 10-14, 16-17, 19, 21, 25 and26 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 is rejected. Claim 1 recites the limitation “the sequence of T-oligonucleotide” (part a, line 4). There is insufficient antecedent basis for this limitation in the claim.
Claim 1 is further rejected. Claim 1 recites the limitation “the fluorescent kinetic profile” (part d, line 1). There is insufficient antecedent basis for this limitation in the claim.
Claims 2-3, 5, 7-8, 10-14, 16-17, 19 and 26 are included in this rejection due to their dependency on claim 1.
Claim 2 is further rejected. Claim 2 recites the limitation “the calculation” (line 1). There is insufficient antecedent basis for this limitation in the claim.
Claim 8 is included in this rejection due to its dependency on claim 2.
Claim 12 is further rejected. Claim 12 recites the limitation “the corresponding FRET oligonucleotides” at step (i) at line 3. There is insufficient antecedent basis for this limitation in the claim.
Claims 13-14, and 16 are included in this rejection due to their dependency on claim 12.
Claim 19 is further rejected. Claim 19 recites the limitation “the corresponding FRET-oligonucleotide” at step a at line 2. There is insufficient antecedent basis for this limitation in the claim.
Claim 21 is rejected. Claim 21 recites the limitation “the corresponding FRET-oligonucleotide” at line 9. There is insufficient antecedent basis for this limitation in the claim.
Claim 25 is rejected. Claim 25 recites the limitation “the sequence of T-oligonucleotide” at (line 6). There is insufficient antecedent basis for this limitation in the claim.
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.
Claims 1-3, 5, 7-8, 10-14, 16-17, 19-21, 23-26 are rejected under 35 U.S.C. 101 because the claimed invention is directed to an abstract idea without significantly more.
The claims recite obtaining fluorescence kinetic profile information from a sample, calculating or deriving one or more metrics from the fluorescence kinetic profile (i.e. average time between fluorescence emissions, average duration of fluorescence emission, rate of fluorescence emissions, dissociation constants, and association constants), and assigning an identity to one or more pixels based on the calculated metrics. These limitations recite the evaluation and comparison of information, followed by identification of a result based upon that information.
Such steps constitute a mental process because they involve observation, analysis, and classification of information that could practically be performed in the human mind or with a pen and paper, albeit more efficiently using laboratory equipment and computers. To the extent the claims recite calculating metrics such as dissociation constants, association constants, averages, rates, or other numerical values from observed fluorescence data, the claims additionally recite mathematical concepts.
The additional elements, including contacting a sample with binding agent-T-oligonucleotide conjugates, contacting the sample with FRET-oligonucleotides, illuminating the sample, and observing fluorescence emissions, merely gather data for use in the recited analysis and do not integrate the judicial exception into a practical application. These steps represent routine and conventional laboratory techniques for generating information that is subsequently analyzed according to the claimed mental process.
The integration of the judicial exception into the claims does not render them patent eligible because the claims are written at a high level of generality and merely use well-known, routine, and conventional techniques in the field.
Subject Matter Eligibility Test for Products and Processes
Step 1 - Is the Claim to a Process, Machine, Manufacture or Composition of Matter? YES.
The claims provide for a method comprising:
contacting a fixed sample with a set of binding agent-T-oligonucleotide conjugates to allow the binding agents to bind to binding partners present in the sample, wherein the set comprises a plurality of binding agents having different specificities and wherein the sequence of the T-oligonucleotide is unique to the binding agent to which it is conjugated;
contacting the sample and any bound binding agents resulting from step (a) with a FRET-oligonucleotide
illuminating the sample with a wavelength to cause excitation of the FRET-oligonucleotide emitter molecule; and
observing the fluorescent kinetic profile of the sample at the FRET-oligonucleotide emitter molecule’s emission wavelength at one or more pixels over time; wherein the FRET-oligonucleotide can hybridize to multiple T-oligonucleotides in the set to form multiple pairs, and wherein the disassociation and reassociation between each different pair generates a fluorescent kinetic profile that is unique within that set to that pair.
The claims further recite calculating or deriving one or more metrics from fluorescent kinetic profile and assigning an identity to one or more pixels based upon the calculated metrics.
Thus, the claims are directed to statutory categories (i.e., processes and products).
Step 2A, Prong One — Does the Claim Recite an Abstract Idea, Law of Nature, or Natural Phenomenon? YES.
Abstract ideas have been identified by the courts by way of example, including fundamental economic practices, certain methods of organizing human activities, an idea ‘of itself,’ and mathematical relationships/formulas. The claims recite a judicial exception. The “mental process” of comparing dissociation and association calculated metrics is “an abstraction” (an idea having no particular concrete or tangible form). The mathematical concepts involving statistical measures and threshold comparisons are abstract ideas. Thus, the claimed invention describes a judicial exception, which correspond to abstractions (ideas, having no particular concrete or tangible form) and mathematical relationships.
Step 2A, Prong Two — Does the Claim Recite an Additional Elements that Integrate the Judicial Exception into a Practical Application? NO.
The Supreme Court has long distinguished between principles themselves, which are not patent eligible, and the integration of those principles into practical applications, which are patent eligible. However, absent are any additional elements recited in the claim beyond the judicial exceptions which integrate the exception into a practical application of the exception. The “integration into a practical application” requires an additional element or a combination of additional elements in the claim to apply, rely on, or use the judicial exception in a manner that imposes a meaningful limit on the judicial exception, such that it is more than a drafting effort designed to monopolize the exception.
The claim limitations are considered to be directed to the abstract idea of evaluating and analyzing information. Specifically, the claims recite observing a fluorescent kinetic profile, calculating or deriving one or more metrics from the fluorescent kinetic profile, including metrics such as dissociation constants (Koff), association constants (Kon), average emission duration, average time between emissions, or rate of fluorescence events, and assigning an identity to one or more pixels based on the calculated metrics. These limitations recite observation, evaluation, and analysis of information, followed by identification of a result based upon that information, which constitutes a mental process and, to the extent mathematical relationships are used to derive kinetic parameters, a mathematical concept.
While the claims further recite contacting a sample with binding agent-T-oligonucleotide conjugates, contacting the sample with a FRET-oligonucleotide, illuminating the sample, and observing fluorescence emissions over time, these additional steps do not integrate the judicial exception into a practical application. Rather, these steps are recited as data gathering activities used to obtain information that is subsequently analyzed through the calculation and identification steps.
There are no additional steps which apply either of the identified judicial exceptions into a practical application. Thus, the claims do not provide for any element/step that integrates the law of nature into a practical application. Specifically, the claims do not recite and particular improvement in microscopy technology, fluorescence detection technology, FRET chemistry, image acquisition technology, or laboratory instrumentation. For example, the claims broadly recite the use of binding agent-T-oligonucleotide conjugates, FRET-oligonucleotides, excitation of fluorescent emitters, and observation of fluorescent kinetic profiles, but do not require any specific improvement in the manner by which fluorescence data are generated, detected, or collected. Instead, these elements are used as tools to obtain information that is subsequently evaluated through the claimed metric calculation and pixel identification steps. Instead, the additional elements merely use conventional laboratory activity as tools to collect information that is then analyzed through the abstract comparison and identification steps.
As a result, there are no additional steps that apply the identified judicial exception in a manner that imposes a meaningful limit on the exception. Thus, the claims do not integrate the abstract idea into a practical application.
Step 2B - Does the Claim Recite Additional Elements that Amount to Significantly More than the Judicial Exception? NO.
The Supreme Court has identified a number of considerations for determining whether a claim with additional elements amounts to “significantly more” than the judicial exception(s) itself. The claims as a whole are analyzed to determine whether any additional element/step, or combination of additional elements/steps, in addition to the identified judicial exception(s) is sufficient to ensure that the claim amounts to “significantly more” than the exception(s).
However, the additional elements of the instant application, individually and in combination, do not amount to “significantly more.” Under the Step 2B analysis, the “physical” elements/steps of, contacting a sample with binding agent-T-oligonucleotide conjugates, contacting the sample with a FRET-oligonucleotide, illuminating the sample with an excitation wavelength, observing fluorescence emissions over time, and generating a fluorescent kinetic profile are merely physical steps used to obtain information that is subsequently evaluated through the abstract comparison, calculation, and identification steps.
For example, Hohng et al., (WO 2019/177345 A1, published 9/19/2019, from IDS 9/27/2023), teaches attaching biomarker-specific probes, combining fluorescent donor and acceptor strands, and detecting fluorescence signals for multiplex biomarker detection (Abstract). Thus, fluorescence-based probe hybridization and signal detection were well-understood, routine and conventional activities in the art.
Further, Pereira et al. (“Super-Beacons: open-source probes with spontaneous tunable blinking compatible with live-cell super-resolution microscopy”, BioRxiv, 2020; from IDS 9/27/2023) discloses, that fluorescence microscopy techniques in which fluorescence blinking behavior is observed over time and characterized using kinetic parameters including fluorescence on-times, off-times, blinking frequencies, and transitions between fluorescent and non-fluorescent states (Abstract; Introduction: Paragraphs 1-2; Figure 1). Thus, collection and characterization of fluorescence kinetic information from fluorescent probes was well-understood, routine and conventional in the art.
Further, Nieves et al. (“DNA-Based Super-Resolution Microscopy: DNA-PAINT”, MDPI Genes, published 2018, from IDS 5/21/2025) discloses super-resolution microscopy techniques based on transient hybridization between complementary oligonucleotides, stochastic fluorescence blinking, repeated observation of fluorescence events over time, and analysis of intensity-versus-time behavior associated with binding and dissociation events (Abstract; Introduction: Paragraphs 1-2; PAINT: Paragraphs 1-2; Figure 1). Thus, fluorescence-based observation of transient binding kinetics and collection of kinetic profile information were well-understood, routine, and conventional activities in the art.
Therefore, preparing the nucleic acid constructs, hybridizing complementary oligonucleotides, detecting fluorescence emission events, collecting fluorescence intensity information over time, and generating fluorescence kinetic profiles were routine and conventional before the effective filing date of the claimed invention.
Simply appending routine and conventional activities previously known to the industry specified at a high level of generality to the judicial exception and/or generally linking the use of the judicial exception(s) to a particular technological environment or field of use, are not found to be enough to qualify as “significantly more.” Nothing is added by identifying the techniques to be used (i.e., specific fluorescence detection techniques) because those techniques were well-understood, routine, and conventional techniques that a practitioner would have thought of when instructed to obtain fluorescence measurements associated with nucleic acid binding events.
In context with the other recited claim limitations, the language requiring determination of a fluorescence kinetic profile, calculation of kinetic parameters associated with the profile, and identification of a target nucleic acid based on the calculated kinetic information merely indicates whether a relationship exists between the measured fluorescence behavior and the identified target nucleic acid.
This information simply tells a practitioner about the relevant informational relationship between the measured fluorescence kinetic profile and the identified target nucleic acid, and does not recite any technological improvement in fluorescence microscopy, fluorescence detection, image acquisition, nucleic acid hybridization, or kinetic measurement technology. Thus, when viewed both individually and as an ordered combination, the claimed elements/steps in addition to the identified judicial exception are found insufficient to supply an inventive concept because the elements/steps are considered conventional and specified at a high level of generality. The claim limitations do not transform the abstract idea that they recite into patent-eligible subject matter because “the claims simply instruct the practitioner to implement the abstract idea with routine, conventional activity.”
Accordingly, the claims do not qualify as patent-eligible subject matter.
Claim Rejections - 35 USC § 103
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.
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 text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-3, 5, 7-8, 10-14, 16-17, 19-21, 23-26 are rejected under 35 U.S.C. 103 as being unpatentable over Jungmann et al., (USPGPub 2016/0161472, published 6/9/2016) and Dai et al. (US 10006917, published 6/26/2018), in further view of Saetrom et al. (US 2018/0305689 A1; published 10/20/2017) and Williams et al. (US 2021/0199660 A1; filed 11/20/2020),
Regarding claims 1-2, Jungmann teaches that far-field fluorescence microscopy has seen major advances since the advent of methods that circumvent the classical diffraction limit, e.g., super-resolution microscopy and most implementations switch molecules between fluorescent ON- and OFF-states to allow consecutive localization of individual molecules (Paragraph 3, lines 1-5). Specifically, Jungmann teaches that in some embodiments, a method may be carried out involving a plurality of docking strand and imager strand pairs where such a method can be used to detect a plurality of targets (e.g., with each docking strand-imager strand pair corresponding to one target) and the docking strand-imager strand pairs in the plurality must share an approximately equal probability of hybridizing under a single environment or condition (as defined for example by temperature, salt concentration, strand molarity, etc.), such that if there is an observed difference between the level of binding (and thus the detection) of a population of imager strands, an end user can conclude that such difference is a function of the amount of docking strand and thus ultimately the amount of target (Paragraph 8, lines 1-5). Further, Jungmann teaches that with this range of thermal stability, it is possible to select at least 200 orthogonal (e.g., different) sequences to be used in these multiplexing methods and in some embodiments, a protein is an antibody such as a primary antibody or a secondary antibody, an antigen-binding antibody fragment, or a peptide aptamer (Paragraph 8, lines 10-15). Jungmann also teaches that by linking a short docking strand to a binding partner (e.g., a protein-binding moiety or a nucleic acid-binding moiety, whether primary or secondary), such as an antibody including a primary and a secondary antibody, different species of targets (e.g., biomolecules, optionally in a cellular environment) can be labeled and subsequently detected by introducing fluorescently-labeled imager strands that are complementary to and bind to the docking strands through transient Watson-Crick interactions (Paragraph 5, lines 1-5). Also, Jungmann teaches that the methods, compositions and kits of the present disclosure take advantage of repetitive, transient binding of short, labeled (e.g., fluorescently labeled) oligonucleotides (e.g., DNA oligonucleotides), or “imager” strands, to complementary “docking” strands, which are attached to targets of interest, in some embodiments, through an intermediate molecule such as an antibody such as a primary or a secondary antibody, to obtain stochastic switching between fluorescent ON- and OFF-states (FIGS. 1A and 1B; Paragraph 4, lines 1-10).
Further, Jungmann teaches in FIG. 11A which shows that in the traditional method of detection, where a single fluorophore is stably attached to the imaging surface (see FIG. 11B(1)), a limited number of photons per “switching” event is emitted (top panel), that extraction of all photons from “replenishable” imager strands (see FIG. 11B(2)) leads to higher localization accuracy per switching event (middle panel), and that a DNA metafluorophore (see FIG. 11B(3) and FIG. 11B(4)) yields a significantly larger number of photons per switching event than the single fluorophore in FIG. 11B(2) (bottom) (Paragraph 141, lines 1-5) at a corresponding pixel size (Paragraph 317, lines 1-5).
Regarding claim 3, Jungmann teaches that upon binding and immobilization of an imager strand, fluorescence emission is detected using, for example, total internal reflection (TIR) or highly inclined and laminated optical sheet (HILO) microscopy (ref. 9) and this is considered an “ON” state where in general, the methods, compositions and kits as provided herein increase the imaging resolution and thus the sensitivity of detection and in some aspects, they also increase the specificity as well as the number of utilizable fluorophores available for detecting targets of interest including but not limited to, e.g., naturally-occurring biomolecules (Paragraph 4, lines 5-10).
Regarding claim 5, Jungmann teaches that in some embodiments, a complementary labeled, optionally fluorescently labeled, imager strand is about 4 to about 30 nucleotides, or about 8 to about 10 nucleotides, in length. In some embodiments, a complementary labeled imager strand is longer than 30 nucleotides (Paragraph 12, lines 1-5).
Regarding claim 7, Jungmann teaches that with this range of thermal stability, it is possible to select at least 200 orthogonal (e.g., different) sequences to be used in these multiplexing methods and in some embodiments, a protein is an antibody such as a primary antibody or a secondary antibody, an antigen-binding antibody fragment, or a peptide aptamer (Paragraph 8, lines 10-15).
Regarding claim 8, Jungmann teaches that in FIG. 20B shows a bulk fluorescence measurement of the Spinach-DFHBI before (bottom line) and after (top line) addition of the aptamer shows that the DFHBI binding activity is well maintained after the addition an extension to Spinach required for immobilizing to the glass surface in FIG. 20A (Paragraph 151, lines 1-5).
Regarding claim 10, Jungmann teaches in FIG. 11A which shows that in the traditional method of detection, where a single fluorophore is stably attached to the imaging surface (see FIG. 11B(1)), a limited number of photons per “switching” event is emitted (top panel), that extraction of all photons from “replenishable” imager strands (see FIG. 11B(2)) leads to higher localization accuracy per switching event (middle panel), and that a DNA metafluorophore (see FIG. 11B(3) and FIG. 11B(4)) yields a significantly larger number of photons per switching event than the single fluorophore in FIG. 11B(2) (bottom) (Paragraph 141, lines 1-5) at a corresponding pixel size (Paragraph 317, lines 1-5).
Regarding claims 11-13, Jungmann teaches that far-field fluorescence microscopy has seen major advances since the advent of methods that circumvent the classical diffraction limit, e.g., super-resolution microscopy and most implementations switch molecules between fluorescent ON- and OFF-states to allow consecutive localization of individual molecules (Paragraph 3, lines 1-5). Specifically, Jungmann teaches that in some embodiments, a method may be carried out involving a plurality of docking strand and imager strand pairs where such a method can be used to detect a plurality of targets (e.g., with each docking strand-imager strand pair corresponding to one target) and the docking strand-imager strand pairs in the plurality must share an approximately equal probability of hybridizing under a single environment or condition (as defined for example by temperature, salt concentration, strand molarity, etc.), such that if there is an observed difference between the level of binding (and thus the detection) of a population of imager strands, an end user can conclude that such difference is a function of the amount of docking strand and thus ultimately the amount of target (Paragraph 8, lines 1-5). Further, Jungmann teaches that with this range of thermal stability, it is possible to select at least 200 orthogonal (e.g., different) sequences to be used in these multiplexing methods and in some embodiments, a protein is an antibody such as a primary antibody or a secondary antibody, an antigen-binding antibody fragment, or a peptide aptamer (Paragraph 8, lines 10-15). Jungmann also teaches that by linking a short docking strand to a binding partner (e.g., a protein-binding moiety or a nucleic acid-binding moiety, whether primary or secondary), such as an antibody including a primary and a secondary antibody, different species of targets (e.g., biomolecules, optionally in a cellular environment) can be labeled and subsequently detected by introducing fluorescently-labeled imager strands that are complementary to and bind to the docking strands through transient Watson-Crick interactions (Paragraph 5, lines 1-5). Also, Jungmann teaches that the methods, compositions and kits of the present disclosure take advantage of repetitive, transient binding of short, labeled (e.g., fluorescently labeled) oligonucleotides (e.g., DNA oligonucleotides), or “imager” strands, to complementary “docking” strands, which are attached to targets of interest, in some embodiments, through an intermediate molecule such as an antibody such as a primary or a secondary antibody, to obtain stochastic switching between fluorescent ON- and OFF-states (FIGS. 1A and 1B; Paragraph 4, lines 1-10).
Regarding claim 14, Jungmann teaches that, “spectrally indistinct” molecules refer to molecules with labels having the same spectral signal or wavelength—that is, the emission wavelength of the labels cannot be used to distinguish between two spectrally indistinct fluorescently labeled molecules (e.g., because the wavelengths are the same or close together) (Paragraph 167, lines 5-10).
Regarding claim 16, Jungmann teaches that far-field fluorescence microscopy has seen major advances since the advent of methods that circumvent the classical diffraction limit, e.g., super-resolution microscopy and most implementations switch molecules between fluorescent ON- and OFF-states to allow consecutive localization of individual molecules (Paragraph 3, lines 1-5). Specifically, Jungmann teaches that in some embodiments, a method may be carried out involving a plurality of docking strand and imager strand pairs where such a method can be used to detect a plurality of targets (e.g., with each docking strand-imager strand pair corresponding to one target) and the docking strand-imager strand pairs in the plurality must share an approximately equal probability of hybridizing under a single environment or condition (as defined for example by temperature, salt concentration, strand molarity, etc.), such that if there is an observed difference between the level of binding (and thus the detection) of a population of imager strands, an end user can conclude that such difference is a function of the amount of docking strand and thus ultimately the amount of target (Paragraph 8, lines 1-5). Further, Jungmann teaches that with this range of thermal stability, it is possible to select at least 200 orthogonal (e.g., different) sequences to be used in these multiplexing methods and in some embodiments, a protein is an antibody such as a primary antibody or a secondary antibody, an antigen-binding antibody fragment, or a peptide aptamer (Paragraph 8, lines 10-15). Jungmann also teaches that by linking a short docking strand to a binding partner (e.g., a protein-binding moiety or a nucleic acid-binding moiety, whether primary or secondary), such as an antibody including a primary and a secondary antibody, different species of targets (e.g., biomolecules, optionally in a cellular environment) can be labeled and subsequently detected by introducing fluorescently-labeled imager strands that are complementary to and bind to the docking strands through transient Watson-Crick interactions (Paragraph 5, lines 1-5). Also, Jungmann teaches that the methods, compositions and kits of the present disclosure take advantage of repetitive, transient binding of short, labeled (e.g., fluorescently labeled) oligonucleotides (e.g., DNA oligonucleotides), or “imager” strands, to complementary “docking” strands, which are attached to targets of interest, in some embodiments, through an intermediate molecule such as an antibody such as a primary or a secondary antibody, to obtain stochastic switching between fluorescent ON- and OFF-states (FIGS. 1A and 1B; Paragraph 4, lines 1-10).
Regarding claim 17, Jungmann teaches that pluralities of BP-NA conjugates (e.g., protein-nucleic acid conjugates) and imager strands are provided herein. A plurality may be a population of the same species or distinct species where a plurality of BP-NA conjugates of the same species may comprise conjugates that all bind to the same target (e.g., biomolecule) (e.g., the same epitope or region/domain) and conversely, a plurality of BP-NA conjugates of distinct species may comprise conjugates, or subsets of conjugates, each conjugate or subset of conjugates binding to a distinct epitope on the same target or to a distinct target (Paragraph 169, lines 1-5). Jungmann also teaches that for example, a plurality may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200 or more distinct species (Paragraph 169, lines 20-25).
Regarding claim 19, Jungmann teaches that by linking a short docking strand to a binding partner (e.g., a protein-binding moiety or a nucleic acid-binding moiety, whether primary or secondary), such as an antibody including a primary and a secondary antibody, different species of targets (e.g., biomolecules, optionally in a cellular environment) can be labeled and subsequently detected by introducing fluorescently-labeled imager strands that are complementary to and bind to the docking strands through transient Watson-Crick interactions (Paragraph 5, lines 1-5).
Regarding claims 20-21, 23-25, Jungmann teaches that far-field fluorescence microscopy has seen major advances since the advent of methods that circumvent the classical diffraction limit, e.g., super-resolution microscopy and most implementations switch molecules between fluorescent ON- and OFF-states to allow consecutive localization of individual molecules (Paragraph 3, lines 1-5). Specifically, Jungmann teaches that in some embodiments, a method may be carried out involving a plurality of docking strand and imager strand pairs where such a method can be used to detect a plurality of targets (e.g., with each docking strand-imager strand pair corresponding to one target) and the docking strand-imager strand pairs in the plurality must share an approximately equal probability of hybridizing under a single environment or condition (as defined for example by temperature, salt concentration, strand molarity, etc.), such that if there is an observed difference between the level of binding (and thus the detection) of a population of imager strands, an end user can conclude that such difference is a function of the amount of docking strand and thus ultimately the amount of target (Paragraph 8, lines 1-5). Further, Jungmann teaches that with this range of thermal stability, it is possible to select at least 200 orthogonal (e.g., different) sequences to be used in these multiplexing methods and in some embodiments, a protein is an antibody such as a primary antibody or a secondary antibody, an antigen-binding antibody fragment, or a peptide aptamer (Paragraph 8, lines 10-15). Jungmann also teaches that by linking a short docking strand to a binding partner (e.g., a protein-binding moiety or a nucleic acid-binding moiety, whether primary or secondary), such as an antibody including a primary and a secondary antibody, different species of targets (e.g., biomolecules, optionally in a cellular environment) can be labeled and subsequently detected by introducing fluorescently-labeled imager strands that are complementary to and bind to the docking strands through transient Watson-Crick interactions (Paragraph 5, lines 1-5). Also, Jungmann teaches that the methods, compositions and kits of the present disclosure take advantage of repetitive, transient binding of short, labeled (e.g., fluorescently labeled) oligonucleotides (e.g., DNA oligonucleotides), or “imager” strands, to complementary “docking” strands, which are attached to targets of interest, in some embodiments, through an intermediate molecule such as an antibody such as a primary or a secondary antibody, to obtain stochastic switching between fluorescent ON- and OFF-states (FIGS. 1A and 1B; Paragraph 4, lines 1-10).
Jungmann also teaches that in addition to using the inter-event lifetime τ.sub.d or binding frequency to determine the number of available binding sites and to barcode molecules, the fluorescence ON-time or τ.sub.b and thus the dissociation constant k.sub.off can also be used to encode information. k.sub.off can be precisely tuned by the base-composition and/or length of the duplex of docking and imager strand (Paragraph 277, lines 1-5). Further, Jungmann teaches that centroid fitting, or Bessel fitting on the diffraction-limited image to obtain a high-resolution image of the sample, calibrating k.sub.on. using a sample with a known number of targets, wherein k.sub.on is a second order association constant, and c.sub.imager is the concentration of fluorescently-labeled imager strands in the sample, including unbound imager strands, determining variable τ.sub.d by fitting the fluorescence OFF-time distribution to a cumulative distribution function, and determining the number of targets in the sample based on the equation, number of targets (Paragraph 199, lines 5-10).
Regarding claim 26, Jungmann teaches in some embodiments, a kit comprises (a) at least one docking strand linked to a binding partner such as a protein (e.g., a protein that binds to a target) and (b) at least one (e.g., at least 2, at least 3, at least 4, at least 5, at least 10, at least 100) labeled imager strand that is capable of transiently binding (e.g., transiently binds) to a docking strand where a docking strand may comprise, for example, at least two domains or at least three domain, wherein each domain binds to a respective complementary labeled imager strand and the number of labeled imager strands may be, for example, less than, greater than or equal to the number of docking strands (Paragraph 206, lines 1-5). Further, Jungmann teaches that the binding partner may be a protein such as, for example, an antibody (e.g., monoclonal antibody), an antigen-binding antibody fragment, or a peptide aptamer. In some embodiments, a kit comprises at least two different binding partners (e.g., proteins), each specific for a different target and a binding partner (e.g., protein), in some embodiments, is linked to a docking strand through an intermediate linker such as, for example, a linker that includes biotin and streptavidin (e.g., a biotin-streptavidin-biotin linker) where in some embodiments, a docking strand is modified to contain an affinity molecule that can be used to link the docking strand to a binding partner. In some embodiments, the affinity molecule is a secondary antibody and an imager strand, in some embodiments, is labeled with at least one fluorescent label (e.g., at least one fluorophore) (Paragraph 206, lines 5-10). Further, Jungmann teaches that in some embodiments, the length of an imager strand is 4 to 30 nucleotides, or longer and for example, the length of an imager strand may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides and in some embodiments, the length of an imager strand is 8 to 10 nucleotides. In some embodiments, a kit comprises at least two imager strands, each different from one another. In some embodiments, the thermal stability of a docking strand transiently bound to its complementary labeled imager strand is within 0.5 kcal/mol of the thermal stability of other docking strands transiently bound to their respective labeled imager strands (Paragraph 206, lines 15-20).
Jungmann does not teach or suggest the application of specifically-labeled FRET oligonucleotides in the generation of a kinetic profile via fluorescence via a specified length (at least 12 nucleotides in length). Further, Jungmann does not teach or suggest the specific sequences of SEQ ID NO: 1-7.
Dai teaches methods for generating super-resolution patterns of molecules on substrates (Abstract). Specifically, Dai teaches that as a next step, the purified protein with DNA oligo are reconstituted in lipids droplets and DNA-PAINT super-resolution imaging is used to understand the exact location and copy number of ion channels per proteo-liposome and then using Action-PAINT, DNA oligo coupled to lumitoxin is placed on individual ion channel of interest via patch-clamp measurements before and after selective silencing or measuring activity of individual ion channel (using techniques such as single-molecule patch-clamp FRET microscopy are used to analyze the activity of individual ion channels (Column 125, lines 5-10). Further, Dai teaches that in important embodiments, the probes are nucleic acids that are transiently bound to their targets at room temperature. In some embodiments, the probe is 7-12 nucleotides in length (Column 22, lines 1-5). Dai also teaches that the combinatorial diversity of specifically-labelled residues allows for unique identification of proteins (library size >10.sup.7) and in step 4, this barcode information is compared and matched to the library of all genetically identified protein coding sequences from whole genome sequencing, and the identity of the current protein is then determined (Column 111, lines 10-15).
Saetrom teaches an artificial sequence (SEQ ID NO: 1187884), specifically a synthetic oligonucleotide comprising SEQ ID NO: 1, as shown in Figure 1 below.
PNG
media_image1.png
148
548
media_image1.png
Greyscale
[AltContent: textbox (Figure 1: SEQ ID NO: 1 is a 100% similarity match to Saetrom’s SEQ ID NO: 1187884. )]
Williams teaches an artificial DNA sequence (SEQ ID NO: 250470), comprising SEQ ID NO: 2, as shown in Figure 2 below.
PNG
media_image3.png
121
543
media_image3.png
Greyscale
[AltContent: textbox (Figure 2: SEQ ID NO: 2 is a 100% similarity match to Williams’ SEQ ID NO: 250470. )]
Saetrom further teaches an artificial sequence (SEQ ID NO: 1187885), specifically a synthetic oligonucleotide, comprising SEQ ID NO: 7, as shown in Figure 3 below.
PNG
media_image5.png
121
553
media_image5.png
Greyscale
[AltContent: textbox (Figure 3: SEQ ID NO: 7 is a 100% similarity match to Saetrom’s SEQ ID NO: 1187885. )]
It would have been obvious to one of ordinary skill in the art at the time of the invention to modify the multiplexed DNA-PAINT imaging methods of Jungmann with the specifically labeled nucleic acid probes and FRET-based detection techniques taught by Dai. Specifically, Jungmann teaches transient hybridization of complementary oligonucleotide pairs, sequence-dependent tuning of binding and dissociation kinetics, scothastic ON/OFF fluorsecnce behavior, and multiplexed detection using numerous orthogonal oligonucleotide sequences. Further, Dai teaches the use of specifically labeled nucleic acid probes, including FRET microscopy and transiently bound oligonucleotide probes for super-resolution imaging and molecular identification. One of ordinary skill in the art would have been motivated to incorporate Dai’s FRET-labeled oligonucleotide probes into Jungmann’s multiplexed DNA-PAINT framework in order to provide an alternative fluorescence detection mechanism while retaining the known advantages of transient hybridization-based multiplexed imaging, including increased sensitivity, molecular discrimination, and generation of distinguishable fluorescence signatures for target identification.
A person of ordinary skill in the art would have had a reasonable expectation of success because both Jungmann and Dai operate within the same field of nucleic-acid-based super-resolution fluorescence microscopy and rely upon transient hybridization of complementary oligonucleotides to generate detectable fluorescence signals. Jungmann expressly teaches that kinetic behavior can be encoded and tuned through oligonucleotide sequence composition and duplex length, while Dai teaches FRET-based fluorescent detection using transiently bound nucleic acid probes. Because the references employ compatible nucleic acid hybridization principles and fluorescence-based detection systems, incorporation of Dai’s FRET-labeled probes into Jungmann’s transient binding platform would have represented the predictable use of known elements according to their established functions.
It would have further been obvious to substitute the specific oligonucleotide sequences disclosed by Saetrom and Williams for Jungmann’s docking/imager strand sequences because Jungmann teaches the use of numerous orthogonal oligonucleotide sequences for multiplexed imaging and recognizes that sequence composition and strand length may be selected to achieve desired hybridization kinetics. Selection of known artificial oligonucleotide sequences from the prior art for use as docking or probe sequences would have amounted to the routine substitution of one known sequence for another suitable sequence in a system already designed to accommodate multiple orthogonal nucleic acid pairs.
Double Patenting
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 filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual 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/apply/applying-online/eterminal-disclaimer.
Claims 1-3, 5, 7-8, 10-14, 16-17 and 19-21, 23-26 are provisionally rejected on the ground of nonstatutory obviousness-type double patenting as being unpatentable over claims 1, 35, 38-40 and 43-44 of copending Application No: 19/053,631.
The claims of the instant application are not patentably distinct from the claims of the copending application because both are directed to multiplexed fluorescence microscopy utilizing binding agent-T-oligonucleotide conjugates and FRET-oligonucleotides that hybridize with multiple T-oligonucleotides to generate distinguishable fluorescence kinetic profiles through dissociation and reassociation events.
Specifically, the claims of copending application ‘631 recite substantially the same multiplexed fluorescence microscopy methods, kits, compositions, and design methods as presently claimed, including embodiments utilizing the same SEQ ID NOs: 1-8 and corresponding FRET oligonucleotides. To the extent differences exist, such differences are directed to obvious variations of the same inventive concept, including recitation of particular sequence combinations, particular assay formats, kit embodiments, design methods, or selection of subsets of the disclosed T-oligonucleotide/FRET-oligonucleotide pairings. One of ordinary skill in the art would have reasonably expected such modifications to function in the same manner because they employ the same disclosed oligonucleotide sequences, the same hybridization interactions, and the same fluorescence kinetic profiling principles. Accordingly, the differences between the claims would have been obvious and do not render the claims patentably distinct.
Representative corresponding claims include instant claim 1 and ‘631 claim 1, instant claim 19 and ‘631 claims 35, 40, and 44, instant claim 20 and ‘631 claim 39, instant claim 23 and ‘631 claim 43, instant claim 24 and ‘631 claim 44, and instant claim 26 and ‘631 claim 48.
This is a provisional nonstatuatory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Conclusion
No claim is allowed, however SEQ ID NOs: 3, 4, 5, 6, and 8 are free of the prior art.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ELIZABETH ROSE LAFAVE whose telephone number is (703)756-4747. The examiner can normally be reached Compressed Bi-Week: M-F 7:30-4:30.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Heather Calamita can be reached on 571-272-2876. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/ELIZABETH ROSE LAFAVE/ Examiner, Art Unit 1684
/HEATHER CALAMITA/ Supervisory Patent Examiner, Art Unit 1684
Prosecution Insights