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 .
DETAILED ACTION
Claims 5, 8, 16, and 18-20 are canceled. Claims 1-4, 6-7, 9-15, 17 are pending.
In light of the amendment filed 12/01/2025, the following objections/rejections are withdrawn: the objections to the specification and drawings; the objections to claims 1, 7 and 20; the § 112(a) rejection of claim 16, § 112(b) rejection of claims 1-4, 6-17 and 20; the § 112(d) rejection of claims 2 and 8; and the §103 rejection of claim 20 over Moysey in view of Biswas.
The instant claims are entitled to the effective filing date of 12/24/2019.
Drawings
The replacement sheet received on 12/01/2025 only includes amended figures 1, 2 and 6, which are unacceptable, because in order to comply with 37 CFR 1.121 “[a]ny amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended”.
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.
(Maintained) Claims 1-3, 6, 9-15, and 17 are rejected under 35 U.S.C. 103 as being unpatentable over Biswas (ACS nano, 2015 9(10), 9652-9664; as provided with the IDS filed 03/31/2023) in view of Gundlach (US 2017/0199149; as cited in the IDS filed 06/17/2022) with evidence from Innovagen (Pepcalc.com, pepcalc.com/).
Regarding claim 1, Biswas teaches attaching a PolyT-20 oligonucleotide to the N-termini of peptides and translocating the DNA-peptide conjugates through solid-state nanopores. See the first passage of the right column on page 9652. The peptide P-1 (YLGEEYVK) has a -1 net charge at a pH of 7. See table 1. Evidentiary reference Innovagen indicates that the EEYVK C-terminal subsequence has a -1 net charge at a pH of 7 as well. Therefore, Biswas teaches a PolyT-20 oligonucleotide attached to the N-terminal of peptide P-1, wherein P-1 includes a negatively charged subsequence EEYVK at its C-terminal (i.e. [oligonucleotide-YLG polypeptide- EEYVK negatively charged element]). The peptide-PolyT20 conjugates, including P-1-T20, gave frequent blockade signals indicating that they translocated. See the left column on page 9658 and figure 4, C-iii. As shown in figure 1, the P-1-T20 complex translocated through a pore with a diameter of 3.0 nm.
Biswas does not teach the presence of an oligonucleotide translocase, wherein the oligonucleotide translocase is associated to the oligonucleotide during at least part of the translocation, and wherein the oligonucleotide translocase comprises Hel308 or a mutant thereof.
Biswas does not teach a nanopore provided by a nanopore protein, wherein the nanopore protein comprises MspA or a mutant thereof.
Gundlach teaches a method of characterizing a protein (e.g. helicase protein [0051]) in a nanopore system comprising a nanopore disposed in a membrane that separates a first conductive liquid medium from a second conductive liquid medium, wherein the nanopore comprises a tunnel that provides liquid communication between the first conductive liquid medium and the second conductive liquid medium, and wherein the protein (e.g. helicase) is physically associated with a polymer in the first conductive liquid medium, the method comprising: (a) applying an electrical potential between the first conductive liquid medium and the second conductive liquid medium to cause the polymer to interact with the nanopore tunnel, wherein at least one dimension of the protein (e.g. helicase) exceeds a diameter of the nanopore tunnel; (b) measuring an ion current through the nanopore during the interaction of the polymer with the nanopore tunnel to provide a current pattern; (c) determining a position and/or movement of at least one polymer subunit in the nanopore tunnel from the current pattern; and (d) associating the position and/or movement of the at least one polymer subunit with a characteristic of the protein. See claim 1. The polymer is a nucleic acid, peptide nucleic acid (PNA), or a combination thereof. See claim 2. Gundlach defines “polymer” as referring to any macromolecule that comprises two or more linear units, wherein each subunit may be the same or different; non-limiting examples include nucleic acids, peptides and proteins. See [0059]. The protein is a molecular motor, wherein the molecular motor is selected from a group that includes helicase. See claims 6-8. Exemplary helicases include Hel308. See [0051]. The protein nanopore is selected from a group that includes MspA. See claim 21. Gundlach teaches a MspA nanopore mutated such that the mutation results in a MspA nanopore comprising a diameter from about 2 to about 6 nm. See [0083]. Thus, Gundlach teaches a characterization method in which a Hel308 helicase is associated with a peptide nucleic acid polymer as the peptide nucleic acid polymer moves through a MspA nanopore.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to apply the characterization method of Gundlach to the P-1-T-20 oligonucleotide-peptide conjugate of Biswas. Doing so is merely applying a known technique to a known peptide nucleic acid conjugate. One would be motivated to apply the characterization method of Gundlach because Gundlach suggests that the ability to observe the mechanistic functioning of complex bio-molecules can accelerate health care; and Gundlach discloses that the characterization method can be applied to peptide nucleic acids. See [0004] and claim 2 of Gundlach. Moreover, one would be motivated to use the P-1-T-20 complex of Biswas because Biswas indicates that the complex is specifically designed to facilitate their translocation through nanopores. See the first paragraph in the conclusion section of Biswas. There would be a reasonable expectation of success because Biswas demonstrates translocating the P-1-T-20 complex through a nanopore with a diameter of 3nm and Gundlach teaches MspA nanopores with diameters between 2-6 nm.
Regarding claim 2, Biswas teaches attaching an PolyT-20 oligonucleotide to the N-termini of peptides and translocating the DNA-peptide conjugates through solid-state nanopores. See the first passage of the right column on page 9652 and the caption of figure 6. As such, Biswas describes the P-1-T20 conjugate as a peptide-DNA conjugate.
As discussed above, Gundlach teaches helicase. See claims 6-8.
Regarding claim 3, Biswas teaches loading an analyte solution in the cis-side of the nanopore. See the paragraph spanning pages 9657 to 9658. As shown in figure 4A the peptide oligonucleotide conjugates are provided to the cis side of the nanopore.
Gundlach teaches providing the protein analyte and the associated polymer to the cis region. See [0089].
Regarding claim 6, Gundlach discloses that MspA can be mutated such that the constriction zone has a diameter from about 0.3 to about 3 nm. See [0083]. Gundlach discloses that the protein (e.g. helicase) provides an anchor to the polymer (e.g. anchor point). See [0042]. Gundlach indicates that the protein (e.g. helicase) does not translocate through the nanopore because the protein exceeds a diameter of the nanopore tunnel. See claim 1. Gundlach discloses that the vestibule and a constriction zone together form the tunnel. Optionally, the length is at least about or at most about 3.0 nm or 6.0 nm. See [0084].
Biswas and Gundlach do not teach a distance between the constriction and the anchor point that is at least 3 nm during at least part of the translocation.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to adjust the length of the nanopore tunnel of Gundlach, and in the process adjust the distance between the anchor protein (e.g. helicase) at the top of the tunnel and the constriction zone at the bottom of the tunnel. Doing so is mere optimization through experimentation. There would be a reasonable expectation of success because Gundlach teaches nanopore tunnels that are 3.0 nm and 6.0 nm in length. As such, one could reasonably expect to arrive at a protein anchor point at the top of the tunnel that is at least 3 nm away from the constriction zone at the bottom. MPEP 2144.05(II) discloses that "[w]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955)
Regarding claim 9, Biswas teaches reacting an azidoacetic anhydride reagent with a peptide bearing a lysine residue at its C-terminus (e.g. P-1 YLGEEYVK), generating an N-azidoacetylated peptide. Then, the azide group reacts with aza-dibenzocyclooctyne (ADIBO) functionalized PolyT20 through a click reaction, resulting in the desired DNA-peptide conjugate. See scheme 1 and the bottom of page 9653. Thus, Biswas teaches a complex formation step comprising linking the peptide and the oligonucleotide.
Regarding claim 10, Gundlach teaches applying a potential difference of +180 mV to the nanopore in figure 1A. See [0024] for the description of figure 1A. Furthermore, Gundlach teaches applying an electrical potential between the first conductive liquid medium and the second conductive liquid medium to cause the polymer to interact with the nanopore channel. See claim 1.
Regarding claim 11, Gundlach teaches an electrical potential that is applied between 10 mV and 1V. See claim 25 of Gundlach. As shown in figure 1A, a potential difference of +180 mV is applied to the nanopore. Gundlach teaches applying an electrical field that includes a direct or constant current between about 10 mV and about 1V. See [0090]. Gundlach teaches that by varying electric potential in the nanopore system and simultaneously monitoring the resulting current, the stretching of the DNA within the nanopore can be characterized. See [0067]. As shown in figures 2E and 2F, the current (pA) varies between consecutive steps along nucleotides. See [0027].
Regarding claim 12, Biswas teaches measuring translocation using ionic current traces. Frequent blockade signals (e.g. translation related signal) indicate that the peptide-PolyT20 conjugates translocated. See figure 4 and the left column on page 9658.
Gundlach teaches measuring an ion current through the nanopore during the interaction of the polymer with the nanopore tunnel to provide a current pattern (e.g. translation related signal), and determining a position and/or movement of at least one polymer subunit in the nanopore tunnel from the current pattern. See claim 1.
Regarding claim 13, Biswas teaches attaching an PolyT-20 oligonucleotide to the N-termini of peptides and translocating the DNA-peptide conjugates through solid-state nanopores. See the first passage of the right column on page 9652. For example, Biswas teaches a P-1-T20 conjugate that includes P-1 (YLGEEYVK) and the PolyT-20 oligonucleotide. See scheme 1 step 2 and table 1. For translocation measurements, Biswas teaches applying an Ag/AgCl electrode in the trans reservoir, while the electrode in the cis reservoir is kept grounded. Then, the ion current is current is recorded. See the ‘translocation measurements’ section. The bias is 500 mV (e.g. potential difference). See the caption of figure 4. The peptide-PolyT20 conjugates, including P-1-T20, gave frequent blockade signals (e.g. a translocation related signal/ electric current signal). indicating that they translocated. See the left column on page 9658 and figure 4, C-iii.
Biswas does not teach translocation in the presence of an oligonucleotide translocase, wherein the oligonucleotide translocase is associated to the oligonucleotide during at least part of the translocation.
Gundlach teaches a method in which a protein (e.g. helicase) is physically associated with a polymer in the first conductive liquid medium, the method comprises: (a) applying an electrical potential between the first conductive liquid medium and the second conductive liquid medium to cause the polymer to interact with the nanopore tunnel, wherein at least one dimension of the protein (e.g. helicase) exceeds a diameter of the nanopore tunnel; (b) measuring an ion current through the nanopore during the interaction of the polymer with the nanopore tunnel to provide a current pattern; (c) determining a position and/or movement of at least one polymer subunit in the nanopore tunnel from the current pattern; and (d) associating the position and/or movement of the at least one polymer subunit with a characteristic of the protein. See claim 1. The polymer is a nucleic acid, peptide nucleic acid (PNA) (e.g. peptide-nucleotide complex), or a combination thereof. See claim 2. Furthermore, Gundlach discloses that a “polymer” includes nucleic acids, peptides and proteins. See [0059]. The protein is a molecular motor, wherein the molecular motor is selected from a group that includes a translocase and helicase. See claims 6-8. Gundlach teaches applying an electrical potential between 10 mV and 1V. See claim 25. As shown in figure 1A, a potential difference of +180 mV is applied to the nanopore. Figure 2C illustrates the current levels corresponding to a DNA sequence observed from the nanopore system at 180 mV and at 140 mV. See [0027] and figure 2C.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to apply the method of Gundlach to the P-1-T-20 oligonucleotide-peptide conjugate of Biswas. Doing so is merely applying a known technique to a known peptide nucleic acid conjugate. In the process, one would arrive at a method in which the conjugate of Biswas is physically associated with a translocase or helicase protein of Gundlach. One would be motivated to apply the method of Gundlach because Gundlach discloses that the method can be applied to peptide nucleic acids. See [0004] and claim 2 of Gundlach. There would be a reasonable expectation of success because Biswas and Gundlach demonstrate sensing ionic current signals throughout translocation.
Regarding claim 14, Biswas teaches a schematic illustration of the nanopore device for translocation measurements, which includes ionic current traces (e.g. an optical read-out). See figure 4.
Gundlach teaches assays that incorporate Forster Resonance Energy Transfer (FRET), which provide detectable signals when moieties attached to predetermined protein domains interact within a spatial range. See [0003]. Gundlach teaches investigating protein characteristics at spatial and temporal resolutions with existing technologies such as with molecular tweezers and FRET analysis. See [0038] and table 1. In figure 1B, Gundlach teaches a current pattern that indicates that DNA is moved through the nanopore. See [0027].
Regarding claim 15, Gundlach teaches characterizing a variety of analytes, such as small molecules and polymers, via general methods that involve passing the target analyte through the nanoscopic opening while monitoring a detectable signal, such as an electrical signal. The signal is influenced by the physical properties of the target analyte as it passes through, such that the signals can be associated with a structural feature (e.g. characteristics) of the analyte. See [0037]. In other words, the ion current through the nanopore is measured to provide a current pattern reflective of the structure of the portion of the polymer interacting with the nanopore tunnel. See the abstract. Thus, Gundlach teaches characterizing the structure of polymers based on a translocation-related signal.
Regarding claim 17, Biswas teaches attaching an PolyT-20 oligonucleotide to the N-termini of peptides and translocating the DNA-peptide conjugates through solid-state nanopores. See the first passage of the right column on page 9652. The peptide P-1 YLGEEYVK has a -1 net charge at a pH of 7. See table 1. Evidentiary reference Innovagen indicates that the EEYVK C-terminal subsequence has a -1 net charge at a pH of 7 as well. Thus, Biswas teaches a P-1-T20 conjugate: [oligonucleotide-YLG polypeptide- EEYVK negatively charged element]. As shown in figure 1, the P-1-T20 complex translocated through a solid-state nano-pore.
Biswas does not teach using an oligonucleotide translocase.
Biswas does not teach a nanopore provided by a nanopore protein, wherein the nanopore protein comprises MspA or a mutant thereof.
Gundlach teaches a method of characterizing a protein in a nanopore system comprising a nanopore disposed in a membrane. See claim 1. The protein is a molecular motor, wherein the molecular motor is selected from a group that includes translocase and helicase. See claims 6-8. The protein nanopore is selected from a group that includes MspA. See claim 21. Furthermore, Gundlach teaches mutated MspA nanopores. See [0083].
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to use the method of Gundlach on the conjugate of Biswas. One would be motivated to use the P-1-T-20 complex of Biswas because Biswas indicates that the complex is specifically designed to facilitate their translocation through nanopores. See the first paragraph in the conclusion section of Biswas. There would be a reasonable expectation of success because Biswas demonstrates using the P-1-T-20 complex by translocating it through a nanopore with a diameter of 3nm and Gundlach teaches using helicase and to translocate peptide nucleic acids through MspA nanopores with diameters between 2-6 nm.
(Maintained) Claims 4 and 7 are rejected under 35 U.S.C. 103 as being unpatentable over Biswas (ACS nano, 2015 9(10), 9652-9664) and Gundlach (US 2017/0199149), as applied to claims 1-3, 6, 9-15, and 17 above, and further in view of Stava (US 2015/0152495).
The teachings of Biswas and Gundlach with respect to instant claim 1 are discussed above.
Regarding claim 4, Gundlach teaches a protein that is physically associated with a polymer in a first conductive liquid medium. The protein is selected from a list that includes helicase. See claim 8. The polymer is selected from a list that includes nucleic acid, PNA(peptide nucleic acid) and combinations thereof. See claim 2. Furthermore, Gundlach discloses that polymers include nucleic acids, peptides and proteins. See [0059]. Furthermore, Gundlach discloses that a “polymer” includes nucleic acids, peptides and proteins. See [0059]. Gundlach teaches taking helicase data at the experimental conditions of 300 mM KCl, 5 mM MgCl2 and 180 mV. See [0030].
Biswas and Gundlach do not teach providing the oligonucleotide translocase at a concentration sufficient to provide multi-loading of the peptide-oligonucleotide complex by oligonucleotide translocase.
Stava teaches detecting polynucleotide translocation through pores via transient current responses. Upon polynucleotide translocation detection, the voltage is set to 0V, and 1mM MgCl2 and 115 nM Hel30 helicase are injected into the cis well and the voltage is set to a holding potential of 140 mV or 180 mV. See [0175]. Stava suggests that the relative times at which the full or fractional [translocation] steps occur, and thus the times at which the signal samples the idealized signal, suitably can be adjusted by varying any suitable parameter. See [0223].
Biswas, Gundlach and Stava to not teach multi-loading.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to optimize the helicase concentration of Gundlach in view of Stava. One would be motivated to optimize the concentration of the helicase because Stava suggests that varying any suitable parameter may affect the signal timing. There would be a reasonable expectation of success because Gundlach and Stava demonstrate translocating nucleotides through nanopores in the presence of helicase Hel308 and MgCl2 and at a voltage of 180 mV. MPEP 2144.05(II) states that “[g]enerally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical” and that “where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
Regarding claim 7, Biswas teaches translocating DNA-peptide conjugates. See the first passage of the right column on page 9652. Biswas discloses that DNA is uniformly negatively charged along its phosphate backbone. See the first paragraph in the left column on page 9653.
Biswas does not teach providing a complementary oligonucleotide, comprising a complementary region that is complementary to the oligonucleotide, wherein the complementary oligonucleotide is linked to a tag, wherein the tag is configured to associate with the membrane.
Gundlach teaches a nanopore disposed in a membrane. See claim 1 and [0070]. Gundlach teaches annealing a complementary strand to DNA samples. When the single stranded 5’ end overhang is filed through Spa’s constriction, the complementary strand is removed, letting the DNA pass through the pore until it is held by the helicase. See [0109].
Biswas and Gundlach do not teach a complementary oligonucleotide that is linked to a tag, wherein the tag is configured to associate with the membrane.
Stava teaches a controlled polynucleotide translocation by Hel308 helicase based on a ternary polynucleotide complex with a Hel308 helicase 3’ overhang binding site and cholesterol bilayer (e.g. membrane) anchor. See [0026], [0027], [0110], [0112] and figures 13A-13E and 14A-14D. Stava teaches annealing DNA to a cholesterol containing moiety. See [0307].
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to replace the in the DNA in the DNA-peptide conjugate of Biswas with the DNA and complementary DNA combination of Gundlach and to further combine that complementary strand of Gundlach with the cholesterol anchor moiety of Stava. Doing so is merely substituting and combining prior art elements. One would be motivated to use the DNA-complementary DNA of Gundlach because Gundlach suggests that the complementary DNA is removed by the MspA’s constriction, which lets the DNA pass through the pore until it is held by helicase. There would be a reasonable expectation of success because Biswas suggests that all DNA have the structural similarity of a negatively charged phosphate backbone. One would be further motivated combine the DNA with the cholesterol moiety of Stava because Stava suggests that the cholesterol bilayer anchor contributes to a controlled polynucleotide translocation by Hel308. There would be a reasonable expectation of success because Stava demonstrates annealing a cholesterol moiety to DNA. As such, one would reasonably expect the cholesterol moiety to serve the same function when annealed to the complimentary DNA of Gundlach within the modified DNA-peptide conjugate of Biswas and Gundlach.
Response to Arguments
Applicant's arguments filed 12/01/2025 have been fully considered but they are not persuasive.
§ 103 rejection of claims 1-3, 6, 9-15, and 17 over Biswas in view of Gundlach with evidence from Innovagen
Applicant argues that Biswas does not provide any teaching or suggestion that the P-1-T20 can be translocated via any protein nanopore, much less through one comprising MspA or a mutant thereof. Accordingly, one of ordinary skill in the art would understand that Biswas does not disclose nor suggest that an oligonucleotide translocase (e.g. Hel308 or a mutant thereof) can be used to translocate a peptide through a protein nanopore (e.g. MspA or a mutant thereof). See the second paragraph on page 14 of the remarks.
This argument is not persuasive because one cannot show non-obviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). Biswas is not relied upon for teaching the oligonucleotide translocase (Hel308 or a mutant thereof) element or protein nanopore (MspA or a mutant thereof) element. Rather, Gundlach is relied upon for teaching those elements. Yet, Biswas does not teach away from protein nanopores. In the introduction section on page 9652, Biswas references the sequencing resolution of MspA protein nanopores.
Applicant asserts that Gundlach also does not teach that an oligonucleotide translocase can be used to translocate a peptide that is part of a peptide-oligonucleotide complex through a protein nanopore. Applicant notes that peptide nucleic acids are not peptide-oligonucleotide complexes, but are instead synthetic nucleic acids, as acknowledged by Gundlach (paragraph [0061]). In a PNA the typical deoxyribose-phosphate backbone of a nucleic acid is replaced with N-(2-amino-ethyl) glycyl (AEG) units. In contrast, peptides have different chemical, physical and electrical properties compared to PNAs. The claims of Gundlach do not teach translocating a peptide in a peptide-oligonucleotide complex through a protein nanopore. See the paragraph spanning pages 14-15 of the remarks.
This argument is not persuasive because the polypeptide of Gundlach is not exclusive to PNAs. Gundlach defines “polymer” as referring to any macromolecule that comprises two or more linear units, wherein each subunit may be the same or different; non-limiting examples include nucleic acids, peptides and proteins. See [0059]. As such the polymer, as recited in claim 1 of Gundlach, is not limited to synthetic peptide nucleic acids. MPEP 2123 states that “[a] reference may be relied upon for all that it would have reasonably suggested to one having ordinary skill the art, including nonpreferred embodiments”. Merck & Co. v. Biocraft Laboratories, 874 F.2d 804, 10 USPQ2d 1843 (Fed. Cir.), cert. denied, 493 U.S. 975 (1989). Moreover, Gundlach is not relied upon for teaching the instantly claimed peptide-oligonucleotide complex. Rather, Biswas is relied upon for teaching that element.
Applicant argues that the analysis method of Gundlach analyzes the protein performing the translocation of the polymer through the nanopore, not the polymer itself. For example, Gundlach’s method requires the protein analyte “must have at least one dimension that exceeds the internal diameter of the nanopore to prevent passage off the protein through the nanopore” [0043]. Applicant asserts that one would understand that Gundlach’s method characterizes a protein, which necessarily cannot enter the nanopore, based on its activity on the polymer that can enter a nanopore. As such, one would understand that Gundlach does not disclose that an oligonucleotide translocase can be used to translocate a peptide through a nanopore. See the last full paragraph on page 15 of the remarks.
This argument is not persuasive because “[o]ne of ordinary skill in the art need not see the identical problem addressed in a prior art reference to be motivated to apply its teachings.”; In re Linter, 458 F.2d 1013, 173 USPQ 560 (CCPA 1972). The purpose of instant claims 12-15 is to analyze a peptide in the peptide-oligonucleotide complex. In contrast, Gundlach is drawn to a method of characterizing a protein, wherein the protein is an enzyme such as a helicase (claims 1, 6-8 of Gundlach). As shown in figure 1A of Gundlach, the enzyme protein does not pass through the nanopore. However, Gundlach does not teach away from translocating peptide polymers through nanopores, because the term “polymer” includes peptides (see [0059] of Gundlach). Furthermore, the method of Gundlach entails an analysis of the polymer, because the method includes measuring (sensing) an ion current through the nanopore during the interaction of the polymer with the nanopore tunnel (claim 1 of Gundlach). Thus, Gundlach, with respect to instant claims 12-13 (and dependent claims thereof), teaches sensing a translocation-related single during the translocation, wherein the signal comprises an electrical current signal.
Applicant argues that the results of combining Gundlach and Biswas would not have been predictable. Applicant asserts that the proposed application of Gundlach’s method to Biswas’s conjugate would require the solid-state nanopore of Biswas be replaced with the protein nanopore comprising MspA or a mutant thereof and that the electrical translocation of the conjugate of Biswas be replaced with enzymatic translocation by Hel308 or a mutant thereof. Applicant asserts that there would be no reasonable expectation of success. See the first paragraph on page 16 of the remarks. Applicant argues that Biswas does not provide any teaching that its conjugates are compatible with protein nanopores. Instead, Biswas teaches that peptides and proteins have complex electrical properties. One would instead expect that replacing the solid-state nanopore of Biswas with a protein nanopore would at best introduce additional electrical complexities and require substantial re-engineering. See the second paragraph on page 16. Applicant argues that Gundlach does not teach or suggest that a charged peptide such as the P-1-T20 of Biswas is even capable of being translocated through a protein nanopore by any means. See the paragraph spanning pages 16-17 of the remarks.
This argument is not persuasive because the rationale discussed above is not based on replacing the solid-state nanopore of Biswas with the MspA nanopore of Gundlach. Rather, the rational above is based on applying the method of Gundlach to the P-1-T20 conjugate of Biswas. There would be a reasonable expectation of success because Gundlach teaches translocating polymers; and the term “polymer” refers to any macromolecule that comprises two or more linear units (see [0059]). Applicant argues that replacing the solid-state nanopore of Biswas with a protein nanopore would at best introduce additional electrical complexities and require substantial re-engineering. To the extent that Applicant is arguing that the combination of Biswas and Gundlach present electrical complexities that would render the combination inoperable, it is unpersuasive. Instant claims 1-4, 6-7, 9-10, 12-15 and 17 do not require any particular electrical environment, and arguments of counsel cannot take the place of factually supported objective evidence (MPEP 2145 or 716.01(c)). See, e.g., In re Huang, 100 F.3d 135, 139-40, 40 USPQ2d 1685, 1689 (Fed. Cir. 1996); In re De Blauwe, 736 F.2d 699, 705, 222 USPQ 191, 196 (Fed. Cir. 1984).
§ 103 rejection of claims 4 and 7 over Biswas and Gundlach and further in view of Stava
Applicant argues that the combination of Biswas and Gundlach is not sufficient to establish a prima facie case of obviousness with respect to the amended claims because none of the references teach that an oligonucleotide translocase could be used to translocate a peptide in a peptide-oligonucleotide complex through a nanopore. Stava does not remedy this deficiency. See paragraph 3 on page 17 of the remarks.
This argument is not persuasive for reasons discussed above.
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.
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(Maintained) Claims 1-4, 6-7, 9-15, and 17 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-21 of copending Application No. 18/789,966 (hereafter Gundlach ‘966) in view of Biswas (ACS nano, 2015 9(10), 9652-9664), Gundlach (US 2017/0199149), and Stava (US 2015/0152495).
This is a provisional nonstatutory double patenting rejection.
Copending claim 1 of Gundlach ‘966 recites a method of characterizing a conformational state of a protein in a nanopore system, the nanopore system comprising: a nanopore disposed in a membrane that separates a first conductive liquid medium from a second conductive liquid medium, wherein the nanopore comprises a tunnel that provides liquid communication between the first conductive liquid medium and the second conductive liquid medium, and wherein the protein is physically associated with a polymer in the first conductive liquid medium, the method comprising: (a) applying an electrical potential between the first conductive liquid medium and the second conductive liquid medium and causing the polymer to interact with the nanopore tunnel, wherein the protein is unable to pass through the nanopore tunnel; (b) measuring an ion current through the nanopore during the interaction of the polymer with the nanopore tunnel to provide a first current pattern; (c) determining a position and/or movement of at least one polymer subunit in the nanopore tunnel from the current pattern; and (d) associating the position and/or movement of the at least one polymer subunit with the conformational state of the protein.
Copending claim 2 of Gundlach ‘966 recites the method of claim 1, wherein the polymer is a nucleic acid, peptide nucleic acid (“PNA”), a peptide, or a combination thereof.
Copending claim 4 of Gundlach ‘966 recites the method of claim 1, wherein the protein is covalently coupled to the polymer (e.g. associated).
Copending claim 8 of Gundlach ‘966 recites the method of claim 1, wherein the protein is a mutant protein or a fusion protein.
Copending claim 9 of Gundlach ‘966 recites the method of claim 8, wherein the fusion protein comprises two or more domains that mutually interact to cause a confirmational change.
Copending claim 10 of Gundlach ‘966 recites the method of claim 1, wherein the conformational state is characterized by measuring the movement of the polymer in the nanopore (e.g. translocation).
Copending claim 11 of Gundlach ‘966 recites the method of claim 10, wherein measuring the movement of the polymer in the nanopore comprises measuring the frequency, duration, and distance of polymer movement.
Copending claim 12 of Gundlach ‘966 recites the method of claim 1, wherein the protein is an enzyme, and wherein the polymer is a nucleic acid.
Copending claim 13 of Gundlach ‘966 recites the method of claim 12, wherein the method characterizes conformational changes associated with the activity of the enzyme.
Copending claim 15 of Gundlach ‘966 recites the method of claim 12, wherein the enzyme is a molecular motor.
Copending claim 16 of Gundlach ‘966 recites the method of claim 15, wherein the molecular motor is a translocase, a polymerase, a helicase, an exonuclease, a viral packaging motor, or a topoisomerase.
Copending claim 18 of Gundlach ‘966 recites the method of Claim 1, wherein the nanopore is a solid-state nanopore, a protein nanopore, a hybrid solid state-protein nanopore, a biologically adapted solid-state nanopore, or a DNA origami nanopore.
Copending claim 19 of Gundlach ‘966 recites the method of Claim 18, wherein the protein nanopore is alpha-hemolysin, leukocidin, Mycobacterium smegmatis porin A (MspA), outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, Neisseria autotransporter lipoprotein (NalP), WZA, Nocardia farcinica NfpA/NfpB cationic selective channel, lysenin or a homolog or variant thereof.
Copending claim 20 of Gundlach ‘966 recites the method of claim 1, wherein the electrical potential applied is between 10 mV and 1 V or between −10 mV and −1 V (e.g. overlaps with the range of instant claim 11).
Copending claims of Gundlach ‘966 lack a method for translocation of a peptide through a nanopore, the peptide is comprised by a peptide-oligonucleotide complex wherein the peptide is linked to an oligonucleotide, and whether the peptide as a first peptide end linked to the oligonucleotide and a second peptide end linked to a negatively charged element; and an oligonucleotide translocase that comprises Hel308 or a mutant thereof; and the use of such complex (relevant to instant claims 1, and 17). Copending claims of Gundlach ‘966 lack a DNA oligonucleotide (relevant to instant claim 2). Copending claims of Gundlach ‘966 lack providing the peptide and the oligonucleotide translocase to a cis side of the nanopore (relevant to instant claim 3). Copending claims of Gundlach ‘966 lack providing the oligonucleotide translocase at a concentration sufficient to provide multi-loading of the peptide-oligonucleotide complex by the oligonucleotide translocase (relevant to instant claim 4). Copending claims of Gundlach ‘966 lack a constriction, wherein the constriction has a circular equivalent diameter selected from the range of 0.5 - 3 nm, and wherein the oligonucleotide translocase associates with the peptide-oligonucleotide complex at an anchor point, wherein a distance between the constriction and the anchor point is at least 3 nm during at least part of the translocation (relevant to instant claim 6). Copending claims of Gundlach ‘966 lack providing a complementary oligonucleotide at least partially complementary to the oligonucleotide, wherein the complementary oligonucleotide is linked to a tag, wherein the tag is configured to associate with the membrane (relevant to instant claim 7). Copending claims of Gundlach ‘966 lack linking the peptide and the oligonucleotide thereby providing the peptide- oligonucleotide complex (relevant to instant claim 9). Copending claims of Gundlach ‘966 lack varying the potential difference between two consecutive steps of the oligonucleotide translocase along the oligonucleotide (relevant to instant claim 11). Copending claims of Gundlach ‘966 lack sensing a translocation related signal during the translocation (relevant to instant claim 12). Copending claims of Gundlach ‘966 lack analysis method for analyzing a peptide, wherein the analysis method comprises the method for translocation of a peptide through a nanopore, wherein the method for translocation comprises translocating the peptide in the presence of an oligonucleotide translocase, wherein the peptide is comprised by a peptide- oligonucleotide complex, sensing a translocation related signal during the translocation, and measuring an electrical current through the nanopore and providing an electrical current signal, wherein the translocation- related signal comprises the electrical current signal (relevant to instant claim 13). Copending claims of Gundlach ‘966 lack an optical read-out related signal, wherein the translocation-related signal comprises the optical read-out related signal (relevant to instant claim 14). Copending claims of Gundlach ‘966 lack characterizing the peptide based on the translocation- related signal (relevant to instant 15).
However, Biswas teaches translating a P-1-T20 complex through a nanopore pore, wherein PolyT20 DNA is attached to the N-terminal of P1, which is a peptide with a negatively charged C-terminal . See table 1. Biswas discloses that frequent blockade signals (e.g. an electrical current signal related to translocation) indicate translocation. See the left column on page 9658 and figure 4, C-iii. Gundlach teaches a method of characterizing a protein comprising associating the position and/or movement of at least one polymer subunit with a characteristic of the protein, wherein the protein is a molecular motor selected from a group that includes helicase. See claims 1 and 6-8. Exemplary helicases include Hel308. See [0051] (relevant to instant claim 1, 2, 9, 12-13 and 17). Biswas teaches loading an analyte solution in the cis-side of the nanopore. See the paragraph spanning pages 9657 to 9658 and figure 4A. Gundlach teaches providing the polymer to the cis region. See [0089] (relevant to claim 3). Stava teaches a concentration of 115 nM Hel30 helicase. See [0175] (relevant to instant claim 4). Gundlach discloses that MspA can be mutated such that the constriction zone has a diameter from about 0.3 to about 3 nm. See [0083]. Gundlach discloses that the protein (e.g. helicase) provides an anchor to the polymer See [0042] (relevant to instant claim 6). Gundlach teaches annealing a complementary strand to DNA samples. See [0109]. Stava teaches annealing DNA to a cholesterol containing moiety. See [0307] (relevant to instant claim 7). Gundlach teaches that by varying electric potential in the nanopore system. See [0067]. As shown in figures 2E and 2F, the current (pA) varies between consecutive steps along nucleotides. See [0027] (relevant to instant claim 11). Biswas teaches a schematic illustration of the nanopore device for translocation measurements, which includes ionic current traces (e.g. an optical read-out). See figure 4 (relevant to instant claim 14). Gundlach teaches characterizing a polymer by monitoring a detectable signal, such as an electrical signal influenced by the physical properties of the target analyte as it passes through. See [0037] (relevant to instant claim 15).
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to replace the polymer and helicase in the method of Gundlach ‘966 with the P-1-T20 complex of Biswas and the Hel308 of Gundlach respectively; to further optimize the concentration of the Hel308 in view of Stava; to further apply the characterizations techniques of Gundlach to the method of Gundlach ‘966 in order to analyze a nanopore system.
(Maintained) Claims 1-4, 6-7, 9-15, and 17 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-20 of US 10,359,395 (hereafter Gundlach ‘395) in view of Biswas (ACS nano, 2015 9(10), 9652-9664), Gundlach (US 2017/0199149), and Stava (US 2015/0152495). The underlined text below is relevant to the amended claims.
Claim 1 of Gundlach ‘395 recites a method of characterizing a protein in a nanopore system comprising a nanopore disposed in a membrane that separates a first conductive liquid medium from a second conductive liquid medium, wherein the nanopore comprises a tunnel that provides liquid communication between the first conductive liquid medium and the second conductive liquid medium, and wherein the protein is physically associated with a polymer in the first conductive liquid medium, the method comprising:
(a) applying an electrical potential between the first conductive liquid medium and the second conductive liquid medium to cause the polymer to interact with the nanopore tunnel, wherein the protein is unable to pass through the nanopore tunnel;
(b) measuring an ion current through the nanopore during the interaction of the polymer with the nanopore tunnel to provide a current pattern;
(c) determining a position and/or movement of at least one polymer subunit in the nanopore tunnel from the current pattern; and
(d) associating the position and/or movement of the at least one polymer subunit with a characteristic of the protein.
Claim 2 of Gundlach ‘395 recites the method of claim 1, wherein the polymer is a nucleic acid, peptide nucleic acid (“PNA”), or a combination thereof.
Claim 4 of Gundlach ‘395 recites the method of claim 1, wherein the protein is an enzyme.
Claim 5 of Gundlach ‘395 recites the method of claim 4, wherein the enzyme is a molecular motor
Claim 6 of Gundlach ‘395 recites the method of claim 5, wherein the molecular motor is a translocase, a polymerase, a helicase, an exonuclease, a viral packaging motor, or a topoisomerase.
Claim 16 of Gundlach ‘395 recites the method of claim 1, wherein the nanopore is a solid-state nanopore, a protein nanopore, a hybrid solid state-protein nanopore, a biologically adapted solid-state nanopore, or a DNA origami nanopore.
Claim 16 of Gundlach ‘395 recites the method of claim 16, wherein the protein nanopore is alpha-hemolysin, leukocidin, Mycobacterium smegmatis porin A (MspA), outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, Neisseria autotransporter lipoprotein (NalP), WZA, Nocardia farcinica NfpA/NfpB cationic selective channel, lysenin or a homolog or variant thereof.
Claim 19 of Gundlach ‘395 recites the method of claim 1, wherein the electrical potential applied is between 10 mV and 1 V or between −10 mV and −1 V.
The patent claims of Gundlach ‘395 lack a method for translocation of a peptide through a nanopore, the peptide is comprised by a peptide-oligonucleotide complex wherein the peptide is linked to an oligonucleotide, and whether the peptide as a first peptide end linked to the oligonucleotide and a second peptide end linked to a negatively charged element; and an oligonucleotide translocase that comprises Hel308 or a mutant thereof; and the use of such complex (relevant to instant claims 1, and 17). The patent claims of Gundlach ‘395 lack a DNA oligonucleotide (relevant to instant claim 2). The patent claims of Gundlach ‘395 lack providing the peptide and the oligonucleotide translocase to a cis side of the nanopore (relevant to instant claim 3). The patent claims of Gundlach ‘395 lack providing the oligonucleotide translocase at a concentration sufficient to provide multi-loading of the peptide-oligonucleotide complex by the oligonucleotide translocase (relevant to instant claim 4). The patent claims of Gundlach ‘395 lack a constriction, wherein the constriction has a circular equivalent diameter selected from the range of 0.5 - 3 nm, and wherein the oligonucleotide translocase associates with the peptide-oligonucleotide complex at an anchor point, wherein a distance between the constriction and the anchor point is at least 3 nm during at least part of the translocation (relevant to instant claim 6). The patent claims of Gundlach ‘395 lack providing a complementary oligonucleotide at least partially complementary to the oligonucleotide, wherein the complementary oligonucleotide is linked to a tag, wherein the tag is configured to associate with the membrane (relevant to instant claim 7). The patent claims of Gundlach ‘395 lack linking the peptide and the oligonucleotide thereby providing the peptide- oligonucleotide complex (relevant to instant claim 9). The patent claims of Gundlach ‘395 lack varying the potential difference between two consecutive steps of the oligonucleotide translocase along the oligonucleotide (relevant to instant claim 11). The patent claims of Gundlach ‘395 lack sensing a translocation related signal during the translocation (relevant to instant claim 12). The patent claims of Gundlach ‘395 lack analysis method for analyzing a peptide, wherein the analysis method comprises the method for translocation of a peptide through a nanopore, wherein the method for translocation comprises translocating the peptide in the presence of an oligonucleotide translocase, wherein the peptide is comprised by a peptide- oligonucleotide complex, sensing a translocation related signal during the translocation, and measuring an electrical current through the nanopore and providing an electrical current signal, wherein the translocation- related signal comprises the electrical current signal (relevant to instant claim 13). The patent claims of Gundlach ‘395 lack an optical read-out related signal, wherein the translocation-related signal comprises the optical read-out related signal (relevant to instant claim 14). The patent claims of Gundlach ‘395 lack characterizing the peptide based on the translocation- related signal (relevant to instant 15).
However, Biswas teaches translating a P-1-T20 complex through a nanopore pore, wherein PolyT20 DNA is attached to the N-terminal of P1, which is a peptide with a negatively charged C-terminal. See table 1. Biswas discloses that frequent blockade signals (e.g. an electrical current signal related to translocation) indicate translocation. See the left column on page 9658 and figure 4, C-iii. Gundlach teaches a method of characterizing a protein comprising associating the position and/or movement of at least one polymer subunit with a characteristic of the protein, wherein the protein is a molecular motor selected from a group that includes helicase. See claims 1 and 6-8. Exemplary helicases include Hel308. See [0051] (relevant to instant claim 1, 2, 9, 12-13 and 17). Biswas teaches loading an analyte solution in the cis-side of the nanopore. See the paragraph spanning pages 9657 to 9658 and figure 4A. Gundlach teaches providing the polymer to the cis region. See [0089] (relevant to claim 3). Stava teaches a concentration of 115 nM Hel30 helicase. See [0175] (relevant to instant claim 4). Gundlach discloses that MspA can be mutated such that the constriction zone has a diameter from about 0.3 to about 3 nm. See [0083]. Gundlach discloses that the protein (e.g. helicase) provides an anchor to the polymer See [0042] (relevant to instant claim 6). Gundlach teaches annealing a complementary strand to DNA samples. See [0109]. Stava teaches annealing DNA to a cholesterol containing moiety. See [0307] (relevant to instant claim 7). Gundlach teaches that by varying electric potential in the nanopore system. See [0067]. As shown in figures 2E and 2F of Gundlach, the current (pA) varies between consecutive steps along nucleotides. See [0027] (relevant to instant claim 11). Biswas teaches a schematic illustration of the nanopore device for translocation measurements, which includes ionic current traces (e.g. an optical read-out). See figure 4 (relevant to instant claim 14). Gundlach teaches characterizing a polymer by monitoring a detectable signal, such as an electrical signal influenced by the physical properties of the target analyte as it passes through. See [0037] (relevant to instant claim 15).
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to replace the PNA and helicase in the method of Gundlach ‘395 with the P-1-T20 complex of Biswas and the Hel308 of Gundlach respectively; to further optimize the concentration of the Hel308 in view of Stava; to further apply the characterizations techniques of Gundlach to the method of Gundlach ‘966 in order to analyze a nanopore system.
(Maintained) Claims 1-4, 6-7, 9-15, and 17 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-20 of US 12,085,533 (hereafter Gundlach ‘533) in view of Biswas (ACS nano, 2015 9(10), 9652-9664), Gundlach (US 2017/0199149), and Stava (US 2015/0152495).
Claim 1 of Gundlach ‘533 recites a method of identifying a molecule/drug effective in modulating the activity and/or conformation state of an enzyme in a nanopore system comprising a nanopore disposed in a membrane that separates a first conductive liquid medium from a second conductive liquid medium, wherein the nanopore comprises a tunnel that provides liquid communication between the first conductive liquid medium and the second conductive liquid medium, wherein the molecule/drug is present in the first conductive medium, and wherein the enzyme is physically associated with a polynucleotide in the first conductive liquid medium, the method comprising:
(a) applying an electrical potential between the first conductive liquid medium and the second conductive liquid medium and causing the polynucleotide to interact with the nanopore tunnel, wherein the enzyme is unable to pass through the nanopore tunnel;
(b) measuring an ion current through the nanopore during the interaction of the polynucleotide with the nanopore tunnel to provide a first current pattern;
(c) comparing the first current pattern to a reference current pattern;
(d) determining a change in position and/or movement of at least one polynucleotide subunit in the nanopore tunnel from the position and/or movement of at least one polynucleotide subunit in the nanopore tunnel determined from the reference current pattern; and
(e) associating the change in position and/or movement of the at least one polynucleotide subunit to identify the molecule/drug effective in modulating the activity or the conformation state of the enzyme,
wherein the reference current pattern is generated in the absence of the molecule/drug or in presence of a different concentration of the molecule/drug from the concentration used to generate the first current pattern in the first conductive medium.
Claim 2 of Gundlach ‘533 recites the method of claim 1, wherein the polynucleotide is a DNA, RNA, a peptide nucleic acid (“PNA”), or a combination thereof.
Claim 4 of Gundlach ‘533 recites the method of claim 1, wherein the enzyme is a molecular motor.
Claim 5 of Gundlach ‘533 recites the method of claim 4, wherein the molecular motor is a translocase, a polymerase, a helicase, an exonuclease, a viral packaging motor, or a topoisomerase.
Claim 14 of Gundlach ‘533 recites the method of claim 13, wherein the protein nanopore is alpha-hemolysin, leukocidin, Mycobacterium smegmatis porin A (MspA), outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, Neisseria autotransporter lipoprotein (NalP), WZA, Nocardia farcinica NfpA/NfpB cationic selective channel, lysenin or a homolog or variant thereof.
The patent claims of Gundlach ‘533 lack a method for translocation of a peptide through a nanopore, the peptide is comprised by a peptide-oligonucleotide complex wherein the peptide is linked to an oligonucleotide, and whether the peptide as a first peptide end linked to the oligonucleotide and a second peptide end linked to a negatively charged element; and an oligonucleotide translocase that comprises Hel308 or a mutant thereof; and the use of such complex (relevant to instant claims 1, and 17). The patent claims of Gundlach ‘533 lack a DNA oligonucleotide (relevant to instant claim 2). The patent claims of Gundlach ‘533 lack providing the peptide and the oligonucleotide translocase to a cis side of the nanopore (relevant to instant claim 3). The patent claims of Gundlach ‘533 lack providing the oligonucleotide translocase at a concentration sufficient to provide multi-loading of the peptide-oligonucleotide complex by the oligonucleotide translocase (relevant to instant claim 4). The patent claims of Gundlach ‘533 lack a constriction, wherein the constriction has a circular equivalent diameter selected from the range of 0.5 - 3 nm, and wherein the oligonucleotide translocase associates with the peptide-oligonucleotide complex at an anchor point, wherein a distance between the constriction and the anchor point is at least 3 nm during at least part of the translocation (relevant to instant claim 6). The patent claims of Gundlach ‘533 lack providing a complementary oligonucleotide at least partially complementary to the oligonucleotide, wherein the complementary oligonucleotide is linked to a tag, wherein the tag is configured to associate with the membrane (relevant to instant claim 7). The patent claims of Gundlach ‘533 lack linking the peptide and the oligonucleotide thereby providing the peptide- oligonucleotide complex (relevant to instant claim 9). The patent claims of Gundlach ‘533 lack a potential difference that is selected from the range of 10 - 400 mV, and varying the potential difference between two consecutive steps of the oligonucleotide translocase along the oligonucleotide (relevant to instant claim 11). The patent claims of Gundlach ‘533 lack sensing a translocation related signal during the translocation (relevant to instant claim 12). The patent claims of Gundlach ‘533 lack analysis method for analyzing a peptide, wherein the analysis method comprises the method for translocation of a peptide through a nanopore, wherein the method for translocation comprises translocating the peptide in the presence of an oligonucleotide translocase, wherein the peptide is comprised by a peptide- oligonucleotide complex, sensing a translocation related signal during the translocation, and measuring an electrical current through the nanopore and providing an electrical current signal, wherein the translocation- related signal comprises the electrical current signal (relevant to instant claim 13). The patent claims of Gundlach ‘533 lack an optical read-out related signal, wherein the translocation-related signal comprises the optical read-out related signal (relevant to instant claim 14). The patent claims of Gundlach ‘533 lack characterizing the peptide based on the translocation- related signal (relevant to instant 15).
However, Biswas teaches translating a P-1-T20 complex through a nanopore pore, wherein PolyT20 DNA is attached to the N-terminal of P1, which is a peptide with a negatively charged C-terminal . See table 1. Biswas discloses that frequent blockade signals (e.g. an electrical current signal related to translocation) indicate translocation. See the left column on page 9658 and figure 4, C-iii. Gundlach teaches a method of characterizing a protein comprising associating the position and/or movement of at least one polymer subunit with a characteristic of the protein, wherein the protein is a molecular motor selected from a group that includes helicase. See claims 1 and 6-8. Exemplary helicases include Hel308. See [0051] (relevant to instant claim 1, 2, 9, 12-13 and 17). Biswas teaches loading an analyte solution in the cis-side of the nanopore. See the paragraph spanning pages 9657 to 9658 and figure 4A. Gundlach teaches providing the polymer to the cis region. See [0089] (relevant to claim 3). Stava teaches a concentration of 115 nM Hel30 helicase. See [0175] (relevant to instant claim 4). Gundlach discloses that MspA can be mutated such that the constriction zone has a diameter from about 0.3 to about 3 nm. See [0083]. Gundlach discloses that the protein (e.g. helicase) provides an anchor to the polymer See [0042] (relevant to instant claim 6). Gundlach teaches annealing a complementary strand to DNA samples. See [0109]. Stava teaches annealing DNA to a cholesterol containing moiety. See [0307] (relevant to instant claim 7). Gundlach teaches a potential difference of +180 mV applied to a nanopore in figure 1A. Gundlach teaches that by varying electric potential in the nanopore system. See [0067]. As shown in figures 2E and 2F, the current (pA) varies between consecutive steps along nucleotides. See [0027] (relevant to instant claim 11). Biswas teaches a schematic illustration of the nanopore device for translocation measurements, which includes ionic current traces (e.g. an optical read-out). See figure 4 (relevant to instant claim 14). Gundlach teaches characterizing a polymer by monitoring a detectable signal, such as an electrical signal influenced by the physical properties of the target analyte as it passes through. See [0037] (relevant to instant claim 15).
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to replace the PNA and helicase in the method of Gundlach ‘533 with the P-1-T20 complex of Biswas and the Hel308 of Gundlach respectively; to further optimize the concentration of the Hel308 in view of Stava; and to further apply the characterizations techniques of Gundlach to the method of Gundlach ‘966 in order to analyze a nanopore system.
Response to Arguments
Applicant's arguments filed 12/01/2025 have been fully considered but they are not persuasive.
Non-statutory Double Patenting rejections of claims 1-4, 6-7, 9-15, and 17
Applicant argues that the following non-statutory double patenting rejections of claims 1-4, 6-7, 9-15, and 17, should be withdrawn in view of the amendments and arguments presented in the remarks filed 12/01/2025: the provisional rejection over the copending claims of Gundlach ‘966 in view of Biswas, Gundlach and Stava; the rejection over the patent claims of Gundlach ‘395 in view of Biswas, Gundlach and Stava; and the rejection over the patent claims of Gundlach ‘533 in view of Biswas, Gundlach and Stava.
This argument is not persuasive for reasons discussed above.
Conclusion
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/K.C.B./Examiner, Art Unit 1657