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 .
Claims 21-38 are cancelled.
Applicant’s election without traverse of Group I, claims 1-19, and the species of enzyme from claim 4 in the reply filed on 07/29/2025 is acknowledged.
Claim 20 is withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected Group/invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 07/29/2025.
Claims 1-19 are pending and under examination on the merits.
Priority
This application is a 371 of PCT/US2021/015965, filed 01/31/2021, which claims benefit of 62/968,929, filed 01/31/2020.
IDS
The IDS submissions dated 07/29/2022 and 02/27/2024 have been considered, except where otherwise indicated (where items noted with strike through were not considered because there was no copy provided by Applicant, as required).
Claim Interpretation
The recitation of a protein or sensing molecule (as recited in part b of claim 1, for example) is being interpreted as synonymous with a recitation of a sensing probe (as recited in part c of claim1,for example) in light of the specification failing to disclose similar attachment of a protein which is not the sensing probe or to even mention a sensing molecule in the instant specification. This interpretation is made in the good faith interest of advancing prosecution.
Claim Objections
Claim 8 is objected to because of the following informalities: “TPR1” should be fully spelled out for clarity as this is the first instance of this acronym in the related chain of dependency.
Claim 8 is objected to because of the following informalities: “DTPR2” should be fully spelled out for clarity as this is the first instance of this acronym in the related chain of dependency.
Claim 12 is objected to because of the following: claim 1, from which claim 12 depends, recites that ‘the molecular wire is selected from the group consisting of a single nucleic acid duplex, a nucleic acid duplex duo…[etc]’. Claim 12 recites ‘[t]he system of claim 11, wherein the single nucleic acid duplex…; and the nucleic acid duplex duo…’. This connection of the terms by ‘and’ appears inconsistent with Applicant’s intent in light of the specification which does not appear to employ such arrangement of multiple, simultaneous embodiments of the molecular wire. The Examiner suggests replacement of the term ‘and’ with the term ‘or’.
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 1-19 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 recites that the length of the nucleic molecular wire is a length comparable to the length of the nanogap. There is no definition of the term ‘comparable’ or example to demonstrate the metes and bounds of said recitation. Colloquially, any length would be comparable as it is capable of being compared. At best, as drafted, term “comparable” in claim 1 is a relative term which renders the claim indefinite. The term “comparable” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. Artisans are left to dispute the degree and relation of said comparable lengths (noting that the only guidance provided is the disclosure that said molecular wire has a length of ranging from 2 to 1000 nm (see for example paragraph 00048 of the instant specification)). Moreover, one artisan may decide that a comparable length is only one where the nanogap is shorter, or longer, or equivalent to the length of the molecular wire. Therefore, the metes and bounds of the claim, as presently drafted, are indefinite.
Claims 2-19 depend from, and thereby incorporate the above noted deficiency and fail to remedy said deficiency.
Claim 12 recites that the molecular wire comprises a functionalized nucleic acid base at a pre-defined position on each strand. Neither the claims nor the specification define the term or process for ‘predefining’ the position of said functionalized nucleic acid base. No clear examples are provided so as to give sufficient guidance for how or what the position that is predetermined is. Artisans are left to craft their own, potentially conflicting, understanding of this term/process/position such that the metes and bounds of the claim are indefinite as presently drafted. Therefore, the metes and bounds of the claim are indefinite.
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 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.
Claim(s) 1-7, 11-12, 14-16, and 18-19 is/are rejected under 35 U.S.C. 103 as being unpatentable over Roswell (US 2019/0094175 A1; as cited on the 07/29/2022 IDS).
Regarding claim 1, Roswell teach electronic sensor devices that comprise one or more biomolecule components in a measurement circuit, and can be used for nucleic acid sequencing (see for example paragraphs 0002 and 0269).
Regarding part a: One exemplary device of Roswell, as illustrated in FIG. 1 of Roswell, comprises a sensor device [100] comprising a sensor [101] may be used to perform a nucleic acid sequencing reaction (the space between said electrodes is deemed to provide the nanogap as recited in instant claim 1, part 1).
Regarding part b: During operation of the device, a voltage may be applied between the first electrode and the second electrode of sensor [101], with interactions of the sensor with a target producing modulation of current flow through a biopolymer bridge molecule [333 of FIG. 3] (see for example paragraph 0126; note that bridge molecule of Roswell is deemed to be encompassed by the nucleic acid molecular wire as recited in part b of instant claim 1 as supported by paragraph 00025 of the instant specification). In various embodiments, a single molecule biosensor (noting that Roswell teaches that a single molecule biosensor device can comprise a first electrode and a second electrode, separated by a sensor gap, where the first and second electrodes can be coupled by a bridge molecule( biopolymer, such as nucleic acid or amino acid polymers) spanning the sensor gap; see for example paragraph 0119) can take the form of a transistor, such as a field effect transistor (FET), with the attached bridge molecule and/or probe (sensing molecule) in close proximity to these components, serving as a channel or conductive path in an electrical circuit (see for example paragraphs 0127 and 0130 referencing FIG. 3). Roswell explains and teaches that the term “biopolymer” can include any molecule comprising at least one monomeric unit that can be produced by a living organism, teaching that exemplary embodiments include polynucleotides, polypeptides, and polysaccharides, including well known forms of these such as DNA, RNA and proteins. Roswell further teaches that bridge molecules that comprise a biopolymer can include complexes that comprise common nucleic acid duplex helices, such as a DNA double helix (see for example paragraphs 0133-0135, 0138). Roswell teaches that this bridge/molecular wire may comprise modified oligos capable of interreacting with (functionalized) the probe (see for example paragraph 0141). Roswell further teaches that a probe in accordance with various embodiments can comprise any suitable molecule or multicomponent molecular complex and may be selected based on the molecule to be detected by the sensor or the biochemical reaction to be monitored. Roswell teaches various examples of probes, including peptides, proteins, enzymes, nucleic acids, ribozymes, catalytic DNAs, and the like.
Regarding part c: Roswell goes on to teach that an enzyme probe (sensing probe per instant claim 1, part c can comprise a lysozyme, a kinase, or a polymerase. In various embodiments, a probe can comprise an enzyme such as polymerase or a reverse transcriptase suitable for interacting with individual DNA or RNA target molecules. Roswell explains that enzymes that catalyze the template-dependent incorporation of nucleotide bases into a growing oligonucleotide strand undergo conformational changes in response to sequentially encountering template strand nucleic acid bases and/or incorporating template-specified natural or analog bases (i.e., an incorporation event). Such conformational changes can modulate electrical current through a bridge molecule to which the probe is coupled, thereby provide a sequence-specific signal pattern in a manner that is dependent on the template molecule. As described above, the signal pattern may be detected by a signal processing system and translated to a sequence data output. Such a label-free, direct sequencing method may permit discrimination of a nucleotide-specific incorporation event in a sequencing reaction using nucleotide base mix comprising a mixture of natural and/or analog bases corresponding to all four bases of DNA. The use of a reverse transcriptase as the probe molecule can similarly enable the direct sequencing of RNA molecules without the need for an intermediate cDNA conversion step. In various embodiments and as described briefly above, a probe can be attached to the bridge molecule via a self-assembling linker (see for example paragraphs 0145-0147 of Roswell). Roswell further teaches that an electrode gap can be carved into a previously established continuous metal nanowire using FIB, thereby creating a first electrode and a second electrode simultaneously with forming the electrode gap (see for example paragraph 0171). Roswell teaches embodiments comprising a sensing probe with two attachment sites attached to the two corresponding functionalized sites on the molecular wire that can interact or perform a chemical or a biochemical reaction with the biopolymer (via a self-assembly chemical reaction) (see for example paragraphs 0145, 0149, 0174, and 0184), wherein the two attachment sites interact with the two functionalized sites on the molecular wire and control the orientation of the sensing probe (see for example paragraphs 0119, 0149, 0174, and 0207).
It would have been prima facie obvious to the person of ordinary skill in the art to arrive at the claimed invention from the disclosure of Roswell. The artisan would have been motivated to make and use the invention as claimed because Roswell teaches this system is effective for sequencing (see for example paragraph 0002). While Roswell does not explicitly compare the length of the nanogap and molecular wire (gap and bridge as discussed by Roswell), it is noted that the instant recitation is indefinite with the only guidance being the disclosure of paragraph 0025 of the instant specification. Roswell teaches the bridge (molecular wire) and gap (nanogap) are comparable as discernibly gleaned from the instant disclosure because the sensor gap has a sensor gap dimension of between about 5 nm and about 30 nm (see for example paragraph 0013) and a high efficiency 20nm dsDNA bridge binding to contact points (see for example paragraphs 0047, 0094, 0132, and 0136-0137). The artisan would have had a reasonable expectation of success based on the cumulative disclosure of Roswell.
Regarding claim 2, Roswell teaches the system of instant claim 1, further comprising:
Regarding part a: a voltage that is applied between the first electrode and the second electrode (see for example paragraphs 0103, 0125]-0126, 0150, and 0208-0211), where in one preferred embodiment, the current is monitored under constant applied voltages (which is understood to be a direct (non-alternating) current which is a bias voltage (see for example paragraph 0280).
Regarding part b: Roswell further teaches the system of instant claim 1, for use in any method for measuring changes in electrical conductance of a sensor [101] comprising a bridge molecule can be used to monitor a sensor device described herein. In various embodiments, a voltage of less than about 10 V can be applied to a sensor comprising a biomolecular bridge molecule, and in various embodiments described in greater detail below, a voltage of about 0.5 V is applied. The current flowing through the sensor can be measured as a function of time using integrated circuit [120]. Target binding and/or processing events by a probe (i.e., enzyme activity in the case of an enzymatic probe) in sensor complex [105] can produce changes to the conductivity of the sensor [101], modulating the measured current to produce a signal pattern [122] over time t comprising signal features [123]. Such events, and the associated conformational changes, including structural, chemical, and electronic changes (i.e., charge distributions in an enzyme, substrates, and surrounding solution) can comprise kinetic features of target binding and processing, with the various events producing current fluctuations comprising signal features [123] that can be measured, recorded, discriminated, analyzed or stored using signal processing techniques which are known in the art (see for example paragraphs 0090, 0095, 0150.
Regarding part c: Roswell teach a software for data analysis that identifies or characterizes the biopolymer or a subunit of the biopolymer (see for example paragraphs 0125-0126; see also the guidance as to the meaning of this recitation at paragraph 00026 of the instant specification).
It would have been prima facie obvious to the person of ordinary skill in the art to arrive at the claimed invention from the disclosure of Roswell. The artisan would have been motivated to make and use the invention as claimed because Roswell teaches this system is effective for sequencing (see for example paragraph 0002). While Roswell does not explicitly compare the length of the nanogap and molecular wire (gap and bridge as discussed by Roswell), it is noted that the instant recitation is indefinite with the only guidance being the disclosure of paragraph 0025 of the instant specification. The artisan would have had a reasonable expectation of success based on the cumulative disclosure of Roswell.
Regarding claim 3, as discussed above, Roswell teaches the biopolymer (bridge) may be DNA (see for example paragraphs 0133-0135, 0138).
Regarding claims 4-5, as discussed above, Roswell teaches that the sensing probe may be an enzyme such as polymerase or a reverse transcriptase for DNA or RNA (see for example paragraph 0146).
Regarding claims 6-7, Roswell teaches that in various embodiments, the primary bridge molecule (molecular wire) may comprise a protein, preferably an enzyme (sensing probe), preferably a polymerase enzyme, that has been engineered to directly include binding groups conjugated to contact points into its linear protein sequence, or the sequence of its composite chains if it is a multimeric protein (see for example, paragraph 0221). Roswell further teaches that an enzyme can be engineered directly to form a primary bridge molecule in this system. This can be achieved by engineering the enzyme protein to carry material binding peptide domains or cysteine residues in the manner described above (see for example paragraph 0207). Roswell additionally teaches embodiments where a peptide biopolymer bridge molecule (such as the enzyme sensing probe of the molecular wire) can comprise a modified amino acid consisting of a lysine residue with a covalently attached biotin to provide a conjugation point at a precisely atomically defined location for avidin-based conjugation to probe molecule complexes. A modified lysine can replace a standard lysine (see for example, paragraphs 0142, , 0229, and 0378).
Regarding claim 11, Roswell teaches the system of claim 1 wherein the bridge molecule (molecular wire) may be a nucleic acid duplex, such as, for example, a DNA duplex, a DNA-RNA hybrid duplex, a DNA-PNA hybrid duplex, a PNA-PNA duplex, or a DNA-LNA hybrid duplex (see for example, paragraphs 0012 and 0133). It is noted that the recitation ‘…wherein the nucleic acid strand is either in an A-form, a B-form or a Z-form…’ fails to recite a meaningful limitation as there are only 3 forms of DNA (the A-form, B-form, or the Z-form). Likewise, the recitation ‘…the nucleic acid bases are either natural or unnatural…’ also fails to recite a meaningful limitation as all possible options are encompassed. Therefore, because the system of Roswell uses a DNA bridge/molecular wire (which can be a DNA duplex) (which is presumed to be an A-form, B-form, or Z-form) and an enzymatic probe (comprised of amino acids), the system of Roswell meets the requirements of instant claim 11 as drafted.
Regarding claim 12, Roswell discloses the system of claim 11. Roswell further teaches that a double-stranded DNA molecular wire (bridge) can comprise one or more thiol-modified oligo comprising a thiol-modified nucleotide or base (see for example paragraph 0091) where the thiol. A thiol-modified nucleotide can comprise a self-assembling anchor configured to bind to a gold nanobead or similar surface contact (Roswell further teaches that in various embodiments of the present disclosure, a sensor comprises: a source electrode; a drain electrode spaced apart from the source electrode by a sensor gap; a gate electrode, wherein the source, drain and gate electrode cooperate to form an electrode circuit; and a bridge molecule bridging across said sensor gap, connecting the source and drain electrodes; and a probe coupled to the bridge molecule, wherein interaction of the probe with a nucleic acid is detectible by monitoring at least one parameter of the electrode circuit. In various examples, the nucleic acid comprises DNA or RNA, or variants thereof (see for example, paragraphs 0005, 0010, 0080, 0137-0138, 0141, 0149, 0174, 0194, and Figures 1, 3, and 57). Roswell further teaches that a double-stranded DNA molecule bridge can also further comprise a biotin linker component to facilitate linking a probe molecule to the bridge with a complementary avidin-type linker component. In various embodiments and as illustrated in the reverse oligonucleotide sequence described above, a biotin-modified oligonucleotide can be incorporated into one of the oligos of a double-stranded DNA molecule bridge. In various embodiments, the biotin-modified oligo is an internal modification, such as via a modified thymidine residue (biotin-dT). A variety of biotin modification configurations may be used, including attachment to thymidine via a C6 spacer, attachment via a triethyleneglycol spacer, attachment via a photocleavable spacer arm, dual biotin modifications, desthiobiotin modifications, and biotin azide modifications. Other modifications that are now known to a person of skill in the art or may be hereinafter devised and may be made to an oligonucleotide to facilitate linkage to a probe molecule are within the scope of the present disclosure. From the figures cited herein, it is clear that the probe is connected, in a functionalized manner, to a nucleic acid of the molecular wire (bridge) (noting that there is no requirement that the connection is direct in the claims as presently drafted) and that the dsDNA bridge (molecular wire) has 2 attachment points at either end for attachment with the electrodes (further supported, for example, by the citations cited herein).
Roswell does not teach the inclusion of said functionalized nucleotide on each strand of the ds DNA molecular wire (bridge).
However, Roswell teaches the same components functioning as claimed to achieve the claimed goal of sequencing. Therefore, absent evidence to the contrary, incorporation of such a functionalized nucleotide on both strands of the dsDNA duplex molecular wire (bridge) versus incorporation of only one such nucleotide on a single strand, would appear to be an obvious variant yielding no more than predictable results. As part of determining obviousness, it is to be considered that the combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results. When a work is available in one field of endeavor, design incentives and other market forces can prompt variations of it, either in the same field or a different one. If a person of ordinary skill can implement a predictable variation, § 103 may bar its patentability. When considering obviousness of a combination of known elements, the operative question is thus “whether the improvement is more than the predictable use of prior art elements according to their established functions.” (see MPEP 2141. I.)). Here, the components are performing their known functions, predictable achieving the same result of sequencing. Therefore, this variation would have been obvious to the artisan, who would have had a reasonable expectation of success.
Regarding claims 14-15, Roswell teaches that this bridge/molecular wire may comprise modified oligos capable of interreacting with (functionalized) the probe (see for example, paragraphs 0140-0141). A variety of biotin modification configurations may be used, including attachment to thymidine via a C6 spacer, attachment via a triethyleneglycol spacer, attachment via a photocleavable spacer arm, dual biotin modifications, desthiobiotin modifications, and biotin azide modifications (see for example, paragraph 0141).
Regarding claim 16, Roswell further teaches that bridge molecules that comprise a biopolymer also include common nucleic acid duplex helices, such as a DNA double helix, which is two DNA single strand molecules bound into a helical double strand by hydrogen bonding, PNA-PNA duplexes, as well as DNA-RNA, DNA-PNA, and DNA-LNA hybrid duplexes (see for example, paragraphs 0133 and 400 and claims 12-13).
Regarding claim 18, Roswell further teaches that the gap (nanogap) defined by the first electrode and the second electrode can be configured with a precise electrode gap dimension. In various embodiments, the electrode gap dimension may be between about 3 nm and about 30 nm (see for example paragraph 0163 and claim 11).
Regarding claim 19, Roswell further teaches the system of claim 1, wherein the nanogap comprises a plurality of nanogaps, each comprising a pair of electrodes, a molecular wire, a sensing probe, and any feature associated with a single nanogap (see for example paragraphs 0086 and 0128 and FIG. 63).
Claim(s) 8 is/are rejected under 35 U.S.C. 103 as being unpatentable over Roswell, as applied to claims 1-7, 11-12 ,14-16, and 18-19, above, in further view of Meli et al (DNA Polymerase Conformational Dynamics and the Role of Fidelity-Conferring Residues: Insights from Computational Simulations. Front Mol Biosci. 2016 May 27;3:20. doi: 10.3389/fmolb.2016.00020).
Regarding claim 8, Roswell teaches the system of claim 5. Roswell further teaches an embodiment of a fully assembled sensor comprising an alpha-helix bridge coupled to a neutravidin molecule via the known biotin-neutravidin binding reaction, and also the polymerase attached via an additional biotin-maleimide linker that has been conjugated to a surface cysteine on the polymerase via the known maleimide-cysteine covalent coupling reaction (see for example paragraphs 0111, 0378-0380 and FIG. 83B). Roswell goes on to teach that where the sensing probe is an enzyme configured to engage the target molecule during a reaction in a solution comprising a plurality of different target molecules, wherein the reaction comprises a time period t, and wherein contacting the target molecule produces a plurality of conformation changes in the enzyme in response to the plurality of target molecule features, wherein each of the plurality of configuration changes modulates an electrical current in the sensor to produce a signal feature (see for example paragraph 0412).
Roswell does not explicitly teach that the two engineered sites on the DNA or RNA polymerase are configured with one site in a finger domain, and the other site in either an exonuclease, or a palm, or a thumb, or a TPR1 or a DTPR2 domain.
However, Meli et al teach that the finger domain (blue), palm domain (red), and thumb domain (green) undergo a conformational change upon interaction with a DNA target (activation) (see for example page 2 , Fig. 1 and its caption).
It would have been prima facie obvious to the person of ordinary skill in the art to arrive at the claimed invention from the disclosures of Roswell and Meli et al. The artisan would have been motivated to make and use the invention as claimed because Roswell teaches this system is effective for sequencing (see for example paragraph 0002) where the enzymatic sensing probe undergoes a conformational change which is detected by the system. This guides one of ordinary skill in the art to look to attach the probe to the molecular wire, so as to interact with the electrodes and system, through a portion of the polymerase which undergoes a conformational change, such as the finger and palm or thumb domains. As part of determining obviousness, it is to be considered that the combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results. When a work is available in one field of endeavor, design incentives and other market forces can prompt variations of it, either in the same field or a different one. If a person of ordinary skill can implement a predictable variation, § 103 may bar its patentability. When considering obviousness of a combination of known elements, the operative question is thus “whether the improvement is more than the predictable use of prior art elements according to their established functions.” (see MPEP 2141. I.)). Here, the attachment of the polymerase sensing probe to the molecular wire for detection of conformational change of the probe through domains of the probe known in the art to undergo conformational change upon interaction with target through the art-known cysteine-maleimide coupling reaction yields no more than predictable results (such as the results and uses taught by Roswell). The artisan would have had a reasonable expectation of success based on the cumulative disclosures of Roswell and Meli et al.
Claim(s) 9 is/are rejected under 35 U.S.C. 103 as being unpatentable over Roswell and Meli et al, as applied to claim 8, above, in further view of Young et al (Second-Harmonic Generation (SHG) for Conformational Measurements: Assay Development, Optimization, and Screening. Methods Enzymol. 2018;610:167-190. doi: 10.1016/bs.mie.2018.09.017. Epub 2018 Oct 19).
Regarding claim 9, Roswell teaches the system of claim 5. Roswell and Meli et al teach the system of claim 8 comprising coupling of the polymerase sensing probe to the DNA molecular wire through cysteine-maleimide coupling. Roswell does teach a 66 amino acid sequence for use in the system with cysteine-maleimide coupling (see for example references to SEQ ID NO: 14 and paragraphs 0378-0380). Note that SEQ ID NO: 14 of Roswell only has 2 Cys residues.
Roswell and Meli et al do not explicitly teach that the polymerase only has 2 cysteine residues.
However, Young et al, concerned with general considerations for maleimide chemistry, teach r important consideration for maleimide labeling is the presence of free surface-exposed cysteine residues. On average, cysteine residues are less abundant in the primary amino acid sequence of a protein than lysines, and they may be solvent-exposed, buried, or paired in disulfide bonds. Therefore, the total available reactive cysteine residues in a target protein are often limited, so maleimide reactions generally produce more selective and less heterogeneous conjugates than SE reactions (see for example section 3.2.2 bridging pages 175-176).
It would have been prima facie obvious to the person of ordinary skill in the art to arrive at the claimed invention from the disclosures of Roswell, Meli et al, and Young et al. The artisan would have been motivated to make and use the invention as claimed because Roswell teaches this system is effective for sequencing (see for example paragraph 0002) where the probe and wire are connected through cysteine-maleimide coupling for which Young et al teach that reduction of Cys residues which are not intended to participate in the cysteine-maleimide reaction are desirably reduced, as discussed above. The artisan would have had a reasonable expectation of success based on the cumulative disclosures of Roswell, Meli et al, and Young et al.
Claim(s) 10 is/are rejected under 35 U.S.C. 103 as being unpatentable over Roswell, as applied to claims 1-7, 11-12 ,14-16, and 18-19, above, in further view of Liu et al (Utilizing Selenocysteine for Expressed Protein Ligation and Bioconjugations. J Am Chem Soc. 2017 Mar 8;139(9):3430-3437. doi: 10.1021/jacs.6b10991. Epub 2017 Feb 27).
Regarding claim 10, Roswell teaches the system of claim 5. Roswell teaches coupling of the molecular wire (bridge) to the sensing probe (polymerase) through cysteine-maleimide coupling and does teach a 66 amino acid sequence for use in the system with cysteine-maleimide coupling (see for example references to SEQ ID NO: 14 and paragraphs 0378-0380).
Roswell does not explicitly teach that the polymerase is engineered to comprise at least a selenocysteine or that at least one cysteine is replaced with a selenocysteine.
However, Liu et al teach that selenocysteine (Sec) uses a diselenide bod in selenocysteine-maleimide coupling/reactions and that the diselenide bond has a lower redox potential than that of disulfide (typical of cysteine-maleimide reactions) bond and can be retained under mild reducing conditions for downstream applications such as activity assays or for keeping the diselenide linkage while reducing undesired disulfide bonds. Lui et al further teach that it is possible to selectively target Sec over Cys in a mixture, further demonstrating the well-documented ability to selectively modify Sec, even when cysteines are present, while further corroborating that no side reactions, such as Sec elimination, takes place (as detected by mass spectrometry) (see for example, paragraph 1 of the Site-specific conjugation of Post-translational Modifications section at page 5).
It would have been prima facie obvious to the person of ordinary skill in the art to arrive at the claimed invention from the disclosures of Roswell and Liu et al. The artisan would have been motivated to make and use the invention as claimed because Roswell teaches this system is effective for sequencing (see for example paragraph 0002) where the probe and wire are connected through cysteine-maleimide coupling for which Liu et al teach selenocysteine is preferably compatible under milder conditions with less risk of unintended interaction with buried or remaining cysteines, as discussed above. The artisan would have had a reasonable expectation of success based on the cumulative disclosures of Roswell and Liu et al.
Claim(s) 13 and 17 is/are rejected under 35 U.S.C. 103 as being unpatentable over Roswell, as applied to claims 1-7, 11-12 ,14-16, and 18-19, above, in further view of Vecchioni et al (Construction and characterization, obtained from: https://doi.org/10.1038/s41598-019-43316-1 (published May 06, 2019)) and Troha et al (Surface-Adsorbed Long G-Quadruplex Nanowires Formed by G:C Linkages. Langmuir. 2016 Jul 19;32(28):7056-63. doi: 10.1021/acs.langmuir.6b01222. Epub 2016 Jul 8).
Regarding claims 13 and 17, Roswell teaches the system of claim 11, as discussed above.
Roswell does not explicitly teach that the nucleic acid duplex (dsDNA) is palindromic.
However, there is nothing in Roswell to suggest that a palindromic sequence (GC-rich or otherwise) would not function in the system of Roswell. Thus, a palindromic sequence, absent evidence to the contrary, is presumed to be an obvious variant of the more generically suggested nucleic acid bridge (molecular wire) taught by Roswell, which one of ordinary skill in the art would have had a reasonable success of predictably using in the system of Roswell.
As part of determining obviousness, it is to be considered that the combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results. When a work is available in one field of endeavor, design incentives and other market forces can prompt variations of it, either in the same field or a different one. If a person of ordinary skill can implement a predictable variation, § 103 may bar its patentability. When considering obviousness of a combination of known elements, the operative question is thus “whether the improvement is more than the predictable use of prior art elements according to their established functions.” (see MPEP 2141. I.)).
Roswell does not teach desirability of a GC-rich palindrome.
However, Vecchioni et al teach that in a palindromic sequence, the cytosines are exposed to identical chemical shift environments, producing a single peak (see for example page 6), where a duplex heavy in GC base pairs tends to form a quadraplex (see for example, page 12).
Vecchioni et al do not teach the use of palindromes comprising at least 50% GC base pairs for a molecular wire.
However, Troha et al teach that guanine-rich (therefore GC base-paired rich dsDNA) sequences form quadraplexed DNA structures called G-wired, which show properties superior to dsDNA when applied in nanotechnology (see for example, page 7056).
It would have been prima facie obvious to the person of ordinary skill in the art to arrive at the claimed invention from the disclosures of Roswell, Vecchioni et al, and Troha et al. The artisan would have been motivated to make and use the invention as claimed because Roswell teaches this system is effective for sequencing (see for example paragraph 0002) where the molecular wire/bridge may be DNA, where a GC-rich (where at least 50% of the base pairs would be GC in order to promote G-wire formation (also suggested by the sequences taught by Troha et al (see for example, table 1)) where uniform cytosine exposure for a single cytosine peak would understandably be desirable for simplified data analysis downstream, as discussed above. The cited references guide the artisan to use a palindromic, quadraplex DNA sequence where at least 50% of the base pairs are GC in order to form a superior G-wire. The artisan would have had a reasonable expectation of success based on the cumulative disclosures of Roswell, Vecchioni et al, and Troha et al.
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 1-7, 11-12 ,14-16, and 18-19 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-23 of copending Application No. 17/777,877 (reference A) in view of Roswell (US 2019/0094175 A1; as cited on the 07/29/2022 IDS).
This is a provisional nonstatutory double patenting rejection.
Regarding claim 1, reference A claims a system of a conductive or semiconductive molecular wire and a system for identification, characterization, or sequencing of a biopolymer comprising, a. a nanogap formed by a first electrode and a second electrode placed next to each other on a non-conductive substrate or placed overlapping each other separated by a non- conductive layer; b. a nanostructure comprising one or more nucleic acid base pairs that bridges the said nanogap by attaching one end to the first electrode and another end to the second electrode through a chemical bond, wherein at least one nucleic acid base within the nanostructure is modified, and the presence of the modified nucleic acid base improves the conductance of the nanostructure in comparison to a canonical nucleic acid base in the same position; and c. a sensing probe attached to the nanostructure that can interact or perform a chemical or biochemical reaction with the biopolymer, further comprising, a. a bias voltage that is applied between the first electrode and the second electrode; b. a device that records a current fluctuation through the nanostructure caused by the interaction between the sensing probe and the biopolymer; and c. a software for data analysis that identifies or characterizes the biopolymer or a subunit of the biopolymer (see for example claims 1-3 of reference A).
Reference A does not specify certain particular elements of instant claim 1, such as the presence of two functionalized sites on the molecular wire interacting with 2 attachment sites.
However, Roswell teach electronic sensor devices that comprise one or more biomolecule components in a measurement circuit, and can be used for nucleic acid sequencing (see for example paragraphs 0002 and 0269).
Regarding part a: One exemplary device of Roswell, as illustrated in FIG. 1 of Roswell, comprises a sensor device [100] comprising a sensor [101] may be used to perform a nucleic acid sequencing reaction (the space between said electrodes is deemed to provide the nanogap as recited in instant claim 1, part 1).
Regarding part b: During operation of the device, a voltage may be applied between the first electrode and the second electrode of sensor [101], with interactions of the sensor with a target producing modulation of current flow through a biopolymer bridge molecule [333 of FIG. 3] (see for example paragraph 0126; note that bridge molecule of Roswell is deemed to be encompassed by the nucleic acid molecular wire as recited in part b of instant claim 1 as supported by paragraph 00025 of the instant specification). In various embodiments, a single molecule biosensor (noting that Roswell teaches that a single molecule biosensor device can comprise a first electrode and a second electrode, separated by a sensor gap, where the first and second electrodes can be coupled by a bridge molecule( biopolymer, such as nucleic acid or amino acid polymers) spanning the sensor gap; see for example paragraph 0119) can take the form of a transistor, such as a field effect transistor (FET), with the attached bridge molecule and/or probe (sensing molecule) in close proximity to these components, serving as a channel or conductive path in an electrical circuit (see for example paragraphs 0127 and 0130 referencing FIG. 3). Roswell explains and teaches that the term “biopolymer” can include any molecule comprising at least one monomeric unit that can be produced by a living organism, teaching that exemplary embodiments include polynucleotides, polypeptides, and polysaccharides, including well known forms of these such as DNA, RNA and proteins. Roswell further teaches that bridge molecules that comprise a biopolymer can include complexes that comprise common nucleic acid duplex helices, such as a DNA double helix (see for example paragraphs 0133-0135, 0138). Roswell teaches that this bridge/molecular wire may comprise modified oligos capable of interreacting with (functionalized) the probe (see for example paragraph 0141). Roswell further teaches that a probe in accordance with various embodiments can comprise any suitable molecule or multicomponent molecular complex and may be selected based on the molecule to be detected by the sensor or the biochemical reaction to be monitored. Roswell teaches various examples of probes, including peptides, proteins, enzymes, nucleic acids, ribozymes, catalytic DNAs, and the like.
Regarding part c: Roswell goes on to teach that an enzyme probe (sensing probe per instant claim 1, part c can comprise a lysozyme, a kinase, or a polymerase. In various embodiments, a probe can comprise an enzyme such as polymerase or a reverse transcriptase suitable for interacting with individual DNA or RNA target molecules. Roswell explains that enzymes that catalyze the template-dependent incorporation of nucleotide bases into a growing oligonucleotide strand undergo conformational changes in response to sequentially encountering template strand nucleic acid bases and/or incorporating template-specified natural or analog bases (i.e., an incorporation event). Such conformational changes can modulate electrical current through a bridge molecule to which the probe is coupled, thereby provide a sequence-specific signal pattern in a manner that is dependent on the template molecule. As described above, the signal pattern may be detected by a signal processing system and translated to a sequence data output. Such a label-free, direct sequencing method may permit discrimination of a nucleotide-specific incorporation event in a sequencing reaction using nucleotide base mix comprising a mixture of natural and/or analog bases corresponding to all four bases of DNA. The use of a reverse transcriptase as the probe molecule can similarly enable the direct sequencing of RNA molecules without the need for an intermediate cDNA conversion step. In various embodiments and as described briefly above, a probe can be attached to the bridge molecule via a self-assembling linker (see for example paragraphs 0145-0147 of Roswell). Roswell further teaches that an electrode gap can be carved into a previously established continuous metal nanowire using FIB, thereby creating a first electrode and a second electrode simultaneously with forming the electrode gap (see for example paragraph 0171). Roswell teaches embodiments comprising a sensing probe with two attachment sites attached to the two corresponding functionalized sites on the molecular wire that can interact or perform a chemical or a biochemical reaction with the biopolymer (via a self-assembly chemical reaction) (see for example paragraphs 0145, 0149, 0174, and 0184), wherein the two attachment sites interact with the two functionalized sites on the molecular wire and control the orientation of the sensing probe (see for example paragraphs 0119, 0149, 0174, and 0207).
It would have been prima facie obvious to the person of ordinary skill in the art to arrive at the claimed invention from the disclosures of reference A and Roswell. The artisan would have been motivated to make and use the invention as claimed because Roswell teaches this system is effective for sequencing (see for example paragraph 0002). While Roswell does not explicitly compare the length of the nanogap and molecular wire (gap and bridge as discussed by Roswell), it is noted that the instant recitation is indefinite with the only guidance being the disclosure of paragraph 0025 of the instant specification. Roswell teaches the bridge (molecular wire) and gap (nanogap) are comparable as discernibly gleaned from the instant disclosure because the sensor gap has a sensor gap dimension of between about 5 nm and about 30 nm (see for example paragraph 0013) and a high efficiency 20nm dsDNA bridge binding to contact points (see for example paragraphs 0047, 0094, 0132, and 0136-0137). The artisan would have had a reasonable expectation of success based on the cumulative disclosure of Roswell.
Regarding claim 2, reference A and Roswell teaches the system of instant claim 1, further comprising
Regarding part a: a voltage that is applied between the first electrode and the second electrode (see for example paragraphs 0103, 0125]-0126, 0150, and 0208-0211), where in one preferred embodiment, the current is monitored under constant applied voltages (which is understood to be a direct (non-alternating) current which is a bias voltage (see for example paragraph 0280).
Regarding part b: Roswell further teaches the system of instant claim 1, for use in any method for measuring changes in electrical conductance of a sensor [101] comprising a bridge molecule can be used to monitor a sensor device described herein. In various embodiments, a voltage of less than about 10 V can be applied to a sensor comprising a biomolecular bridge molecule, and in various embodiments described in greater detail below, a voltage of about 0.5 V is applied. The current flowing through the sensor can be measured as a function of time using integrated circuit [120]. Target binding and/or processing events by a probe (i.e., enzyme activity in the case of an enzymatic probe) in sensor complex [105] can produce changes