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 .
Effective Filing Date
The present application, filed on March 18, 2022 is a 371 of PCT/JP2019/036512, filed on September 18, 2019.
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on March 13, 2026 has been entered.
Claim Status and Action Summary
This action is in response to the papers filed March 13, 2026.
Claims 40-63 are pending in the present application.
Claims 40-63 are under examination.
Any objections and rejections not reiterated below are hereby withdrawn.
The 103 rejections of claim 40 and its dependents have been withdrawn in view of the amendment requiring a step of repeating the measurement “a number of times corresponding to the number of sets of the molecular motor binding portion and the primer binding portion”. Claim 40 and its dependents, while not presently rejected over prior art, are/remain rejected under U.S.C. 112 as indefinite.
Claim Rejections - 35 USC § 112(b)
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.
Claims 40-63 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor, regards as the invention.
Claim 40 recites the limitation "the plurality of molecular motor binding portions" in line 10 and “the plurality of primer binding portions” in line 12. There is insufficient antecedent basis for this limitation in the claim.
Furthermore, it is unclear what structures are intended to be encompassed by “molecular motor binding portion to which a molecular motor binds” because the claim requires both “molecular motor binding portions” and “primer binding portions”, and it appears that “molecular motors” such as DNA polymerases bind to double stranded DNA such as “primer binding portions”. Therefore, it is unclear whether “molecular motor binding portion” requires something other than double stranded DNA, and how the “molecular motor binding portions” and “primer binding portions” are intended to be structurally distinguished by the claim language.
Additionally, claim 40 recites the limitations “each molecular motor of the plurality of molecular motors is bound to a respective one of the plurality of molecular motor binding portions ” and “a plurality of primers, wherein each primer of the plurality of primers is hybridized with a respective one of the plurality of primer binding portions”. It is unclear whether these claim limitations are intended to require that: a) all of the molecular motors and primers present in the first liquid tank are bound to biomolecule-adapter molecule complex(es) (i.e. there are no free molecular motors or primers in solution), or b) all of the “molecular motor binding” and “primer binding” portions of the biomolecule-adapter molecule complex(es) are bound by molecular motors and primers (i.e. the “binding portions” are saturated), c) a combination of both a) and b) wherein all of the “binding portions” are occupied and there are no free molecular motors or primers, or d) something else.
A broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the meters and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, claim 40 recites the broad recitations “a biomolecule to be analyzed” and “a molecular motor”, and the claim also recites “the adapter molecule comprises a single-stranded nucleic acid region…” “the measurement being performed by synthesizing a complementary strand from the primer hybridizing with the primer binding portion… to convey the biomolecule… [through the nanopore]” which is the narrower statement of the range/limitation. The claim(s) are considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims. Specifically, as there is no limiting definition in the specification as to the scope of the claim term “biomolecule”, it appears that this term encompasses embodiments wherein the “biomolecule” is: a) any nucleic acid, b) any polypeptide, c) any polysaccharide, or d) any molecule generated by the action of a living thing (“biomolecule”). However, the claim appears to require the use of a “molecular motor” having the functionality “synthesizing a complementary strand from the primer…” (i.e. a nucleic acid polymerase acting on a nucleic acid hybridized to a primer). Therefore, it is unclear if applicant intends to cover methods for analyzing all possible “biomolecules”, or if the claims are meant to be limited to embodiments wherein the biomolecule is a polynucleotide (i.e. DNA or RNA).
Further, claim 40 requires the “biomolecule-adapter complex” is present in a first liquid tank and a second liquid tank is set to a positive potential (relative to the first liquid tank), however, the claim further requires a step of “measuring… comprising… synthesizing a complementary strand… to convey the biomolecule-adapter complex from the second liquid tank to the first liquid tank… and then, conveying the biomolecule-adapter complex… from the first liquid tank to the second liquid tank…”. Because the sequentially recited “synthesizing… and conveying” steps appear to be contradictory to the requirement that the biomolecule-adapter complex is initially present in the first liquid tank, it is unclear how the sequentially recited steps accomplish the claimed method. If the claim is somehow intended to require something more, such as, for example, some unspecified electrochemical step that conveys biomolecule-adapter complex partially or entirely into the second liquid tank prior to the recited “measuring” steps, it is unclear exactly what limitations that unspecified step entails.
Claims 41 and 51-63 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor, regards as the invention.
Claim 41 recites a step of “applying a voltage… where the first liquid tank is filled with an electrolyte solution comprising a biomolecule-adapter complex, a molecular motor bondable to a molecular motor binding portion… and a primer capable of hybridizing with a primer binding portion…” However, the claim does not require any positively recited, active steps that clearly accomplish or require: a) binding a molecular motor to a molecular motor binding portion or b) hybridizing a primer with a primer binding portion. Instead, the claim merely recites that these structures can accomplish “bond[ing]” (i.e. binding) and “hybridizing”.
The limitation recited by amended claim 41, “a molecular motor detachment induction portion configured to induce detachment of the molecular motor from the adapter molecule, wherein the molecular motor detachment induction portion has a binding affinity to the molecular motor that is lower than the binding affinity between the molecular motor and the biomolecule to be analyzed” has been evaluated under the three-prong test set forth in MPEP § 2181, subsection I, but the result is inconclusive. Thus, it is unclear whether this limitation should be interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph because it is unclear what particular structures are intended to be encompassed by the claim, as the structures are only identified by their intended function “detachment induction” or “induce detachment” and the relative term/property “has a binding affinity to the molecular motor that is lower than the binding affinity between the molecular motor and the biomolecule to be analyzed”. It appears that this limitation encompasses a “detachment induction” portion that is merely a 5’ end of a nucleic acid template (i.e. the detachment is induced by the absence of further template; so-called polymerase run-off; i.e. binding affinity = 0).
It is unclear whether these claim limitations which recite an unspecified “detachment induction portion” structure (i.e. a generic placeholder), identified only by its intended function “induce detachment” linked by the transitional phrase “configured to” should be interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph.
In response to this rejection, applicant must clarify whether this limitation should be interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph. Mere assertion regarding applicant’s intent to invoke or not invoke 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph is insufficient. Applicant may:
(a) Amend the claim to clearly invoke 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, by reciting “means” or a generic placeholder for means, or by reciting “step.” The “means,” generic placeholder, or “step” must be modified by functional language, and must not be modified by sufficient structure, material, or acts for performing the claimed function;
(b) Present a sufficient showing that 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, should apply because the claim limitation recites a function to be performed and does not recite sufficient structure, material, or acts to perform that function;
(c) Amend the claim to clearly avoid invoking 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, by deleting the function or by reciting sufficient structure, material or acts to perform the recited function; or
(d) Present a sufficient showing that 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, does not apply because the limitation does not recite a function or does recite a function along with sufficient structure, material or acts to perform that function.
Claim 42 recites “the adapter molecule further comprises a spacer to which the molecular motor is not bound… between the molecular motor binding portion and the primer binding portion”. It is unclear whether this claim limitation requires the presence of such a spacer: a) between each pair of a primer binding portion on a side of a 3’ terminus of the adapter molecule from its corresponding molecular motor binding portion, b) between the molecular motor binding site of a 3’ pair of binding sites and the primer binding site of a 5’ pair of binding sites, or c) a combination of a) and b):
a)
3’--[motor binding]--[spacer]-[primer binding]-[motor binding]-[spacer]-[primer binding]-5’
b)
3’-[motor binding]-[primer binding]-[spacer]-[motor binding]-[primer binding]-[spacer]…-5’
c)
3’-[motor binding]-[spacer]-[primer binding]-[spacer]- [motor binding]-[spacer]-[primer binding]-[spacer]…-5’
Claim 44 recites “the fall-off prevention portion is a molecule bondable to the single stranded nucleic acid region or a hairpin structure formed in a complementary region in the single-stranded nucleic acid region.” It is unclear whether the claim requires that the fall-off prevention portion is: a) a molecule that can bind to the single stranded nucleic acid region or to a hairpin structure…; b) a molecule that can bind to the single stranded nucleic acid region or is a hairpin structure…; or c) something else.
Claim 44 recites “a hairpin structure formed in a complementary region in the single-stranded nucleic acid region”. It is unclear to what nucleic acid structure or sequence the “region” is complementary to. The claim limitation may be interpreted at least as: a) the single-stranded nucleic acid region comprises self-complementary (i.e. stem) regions separated by an intervening (i.e. loop) region, or b) the single-stranded nucleic acid region comprises regions that are complementary to the “fall-off prevention portion”, which is a polynucleotide, and the regions are separated by an intervening (i.e. loop) region.
Claim 47 recites: “a single-stranded nucleic acid region having a 5’ terminus… comprises a three-dimensional structure formation domain”. The specification does not provide a limiting definition of a “three-dimensional structure” or a “three-dimensional structure formation domain”. The metes and bounds of this claim limitation are unclear because all single stranded nucleic acids form some three-dimensional structure, whether that be specific secondary structures (such as a hairpin) or a so-called “unstructured domain” which does not have a stable secondary structure, but moves through a set of different three-dimensional conformations. It is unclear whether the claim is intended to require that the single stranded 5’ end comprises a sequence that can adopt a particular secondary structure (or structures), or some higher-order assembly of structures, or whether the claim merely requires that the 5’ end is not a two-dimensional molecule (e.g. graphene).
Claim 51 recites, “the molecular motor detachment induction portion is a carbon chain that does not contain a phosphodiester bond or an abasic sequence portion”. It is unclear whether this claim limitation is intended to require that the portion is: a) a carbon chain that contains neither a phosphodiester bond nor an abasic sequence portion or b) the portion is either a carbon chain that does not contain a phosphodiester bond or is an abasic sequence portion.
Claims 42-63 are additionally rejected under 35 U.S.C. 112(b) as indefinite because they depend from, and include indefinite limitations of the claims upon which they depend or repeat indefinite claim terms identified in the claims rejected above.
Response to arguments
The arguments in the response that claim 41 does not invoke 35 USC 112(f) because of the presence of structures capable of performing the claimed function in dependent claims or the presence of exemplary structures capable of performing the claimed function in the specification have been thoroughly reviewed and are not persuasive because 35 USC 112(f) is invoked by claim language, not by the contents of the disclosure. As such, a claim reciting a generic placeholder that is modified by functional language and is not modified by sufficient structure, material, or acts for performing the claimed function in the claim language may invoke 112(f). Here, the analysis is inconclusive because the claim language appears to encompass embodiments wherein a “detachment induction portion” is merely a terminus of a polynucleotide and the detachment is induced by polymerase translocation off of the end of the template polynucleotide (so-called run-off) (see 112(b) above).
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 40 and 42-50 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
This is a New Matter rejection.
Independent claim 40 filed on 02/13/2026 recites amended limitation “the measurement is repeated a number of times corresponding to the number of sets of the molecular motor binding portions and the primer binding portion, the measurement being performed ---”. There is no support for this amended limitation in the specification originally filed on 03/18/2022. In this regard, instant specification discloses “--- the signal, the measurement is repeated, where the measurement being performed by synthesizing a complementary strand from the primer hybridizing with the primer binding portion by the molecular motor located closest to the nanopore to convay the biomolecule-adapter molecule complex from ---" (See [0057], US 2023/0220450 A1, publication of 17/761,833). The specification additionally discloses an example (Example 4) wherein a measurement is “repeated three times”. This example includes Table 3 (see below) which illustrates an exemplary “primer oligo” and “Tandem primer template” (i.e. an adapter molecule). Adapter sequences that are complementary to the primer are highlighted below.
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There is no identification of a “motor binding portion” on a side of a 5’ terminus from each of the corresponding “primer binding portions”. Even if the disclosure can somehow be shown to identify “motor binding portions” as poly-T sequences (i.e. the only nucleic acid sequence between the primer binding portions in Table 3), the example above would only comprise 2 “sets” of primer binding portions and “motor binding portions”, as the 5’ most primer binding portion is not linked to a corresponding poly-T sequence or any other sequence described as a “motor binding portion” in the disclosure. Even if this example can somehow be shown to comprise 2 “sets” of primer binding portions and “motor binding portions”, this does not support the claimed “repeated a number of times corresponding to the number of sets of the molecular motor binding portion and the primer binding portion” because the example states the measurement is “repeated three times”.
Additionally, if this newly added limitation to claim 40 can be shown to somehow be supported by the disclosure, adequate written description of the structures required by the language of the claimed method is still lacking in the disclosure for at least the following reasons:
Relevant to the lack of particular structural limitations in the rejected claims drawn to methods requiring adapter molecules having “a plurality of molecular motor binding portions”,
MPEP 2163 states:
The claimed invention as a whole may not be adequately described if the claims require an essential or critical feature which is not adequately described in the specification and which is not conventional in the art or known to one of ordinary skill in the art.
Additionally, at 2163(II)(A)(3)(a), the MPEP states:
“…describing a composition by its function alone typically will not suffice to sufficiently describe the composition. See Eli Lilly, 119 F.3 at 1568, 43 USPQ2d at 1406 (Holding that description of a gene’s function will not enable claims to the gene “because it is only an indication of what the gene does, rather than what it is.”); see also Fiers, 984 F.2d at 1169-71, 25 USPQ2d at 1605-06 (discussing Amgen Inc. v. Chugai Pharm. Co., 927 F.2d 1200, 18 USPQ2d 1016 (Fed. Cir. 1991)). An adequate written description of a chemical invention also requires a precise definition, such as by structure, formula, chemical name, or physical properties, and not merely a wish or plan for obtaining the chemical invention claimed. See, e.g., Univ. of Rochester v. G.D. Searle & Co., 358 F.3d 916, 927, 69 USPQ2d 1886, 1894-95 (Fed. Cir. 2004) (The patent at issue claimed a method of selectively inhibiting PGHS-2 activity by administering a non-steroidal compound that selectively inhibits activity of the PGHS-2 gene product, however the patent did not disclose any compounds that can be used in the claimed methods. While there was a description of assays for screening compounds to identify those that inhibit the expression or activity of the PGHS-2 gene product, there was no disclosure of which peptides, polynucleotides, and small organic molecules selectively inhibit PGHS-2. The court held that “[w]ithout such disclosure, the claimed methods cannot be said to have been described.”).
The claims are broadly drawn to methods requiring adapter molecules having “a plurality of molecular motor binding portions”.
In the case of the instant claims, the functionality of “molecular motor binding” is a critical feature of the adapter molecules (i.e. compositions) required by the claimed methods.
The specification does not teach any particular compounds, nucleic acid sequences, or other chemical structures that accomplish the claimed “molecular motor binding”.
While the skilled artisan may be capable of making an adapter comprising a portion with the claimed functionality of “molecular motor binding”, possession may not be shown by merely describing how to obtain possession of members of the claimed genus or how to identify their common structural features. See University of Rochester, 358 F.3d at 927, 69 USPQ2d at 1895. In the present case, there is no guidance as to what chemical structures may be encompassed by the claim beyond their function “molecular motor binding”.
The claims encompass a method encompassing use of a genus of structurally undefined adapters comprising “a plurality of sets of primer binding portion, with which a primer hybridizes, wherein each primer binding portion is located on a side of a 3’-terminus of the adapter molecule from its corresponding molecular motor binding portion, and wherein the molecular motor binding portions and primer binding portions are arranged sequentially and linearly along the adapter molecule…” and “the measurement is repeated a number of times corresponding to the number of sets of the molecular motor binding portion and the primer binding portion.”
For claims drawn to a genus (i.e. methods using adapters having any number of sets of primer binding portion and undefined molecular motor binding portion to repeat a measurement of an attached biomolecule a number of times corresponding to the any number of sets), the written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species. A “representative number of species” means that the species which are adequately described are representative of the entire genus. Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus. See AbbVie Deutschland GmbH & Co., KG v. Janssen Biotech, Inc., 759 F.3d 1285, 1300, 111 USPQ2d 1780, 1790 (Fed. Cir. 2014) (Claims directed to a functionally defined genus of antibodies were not supported by a disclosure that “only describe[d] one type of structurally similar antibodies” that “are not representative of the full variety or scope of the genus.”). The disclosure of only one species encompassed within a genus adequately describes a claim directed to that genus only if the disclosure “indicates that the patentee has invented species sufficient to constitute the gen[us].” See Enzo Biochem, 323 F.3d at 966, 63 USPQ2d at 1615. Further, University of California v. Eli Lilly and Co., 43 USPQ2d 1398, 1404, 1405 held that:
To fulfill the written description requirement, a patent specification must describe an invention and do so in sufficient detail that one skilled in the art can clearly conclude that “the inventor invented the claimed invention.” Lockwood v. American Airlines, Inc., 107 F.3d 1565, 1572, 41 USPQ2d 1961, 1966 (1997); In re Gosteli, 872 F.2d 1008, 1012, 10 USPQ2d 1614, 1618 (Fed. Cir. 1989) (“ [T]he description must clearly allow persons of ordinary skill in the art to recognize that [the inventor] invented what is claimed.”). Thus, an applicant complies with the written description requirement “by describing the invention, with all its claimed limitations, not that which makes it obvious,” and by using “such descriptive means as words, structures, figures, diagrams, formulas, etc., that set forth the claimed invention.” Lockwood, 107 F.3d at 1572, 41 USPQ2d at 1966.
An adequate written description of a DNA, such as the cDNA of the recombinant plasmids and microorganisms of the '525 patent, "requires a precise definition, such as by structure, formula, chemical name, or physical properties," not a mere wish or plan for obtaining the claimed chemical invention. Fiers v. Revel, 984 F.2d 1164, 1171, 25 USPQ2d 1601, 1606 (Fed. Cir. 1993). Accordingly, "an adequate written description of a DNA requires more than a mere statement that it is part of the invention and reference to a potential method for isolating it; what is required is a description of the DNA itself." Id. at 1170, 25 USPQ2d at 1606.
Thus considering the breadth of the compounds required by the claimed methods, their specific required functionalities, and the teachings of the instant specification, it is the conclusion that the specification does not provide an adequate written description of the broadly claimed subject matter.
Claims 42-50 depend from claim 40 and are likewise not adequately described for the same reasons as those described above.
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 41, 51-54, and 57-61 are/remain rejected under 35 U.S.C. 103 as being unpatentable over Davis et al., US 2013/0244340 A1 (published September 19, 2013) in view of Rand., US 2018/0030525 A1 (published February 1, 2018).
Regarding claim 41, Davis et al. teaches a biomolecule analysis method comprising steps of: applying a voltage between a first and second liquid tank, each filled with electrolyte solutions and connected by a nanopore (Davis et al., Figures 3 and 23), wherein the first tank solution: comprises a biomolecule-adapter complex, a molecular motor that can bind to the biomolecule-adapter complex (Davis et al., Figure 23 and paragraph 0282), and a primer capable of hybridizing to the biomolecule-adapter complex (Davis et al., paragraph 0451). Davis et al. teaches the “cis chamber” (i.e. the first tank) is set to a negative potential, while the “trans chamber” (i.e. the second tank) is set to a positive potential (Davis et al., figure 3). Davis teaches the biomolecule-adapter complex comprises an adapter molecule bound to the termini of the biomolecule (Davis et al., figure 28 and paragraphs 0451-0452). Davis further teaches measuring signals generated when the biomolecule-adapter complex is conveyed between the two liquid tanks through the nanopore. Davis teaches that this measurement is repeated (Davis et al., figure 23), facilitated by DNA polymerase synthesizing a complementary strand from a primer bound to the complex (Davis et al., paragraphs 0282 and 0391) (i.e. a molecular motor binding and primer binding portion).
Davis et al. does not teach that the adapter molecule comprises a “molecular motor detachment induction portion”. However, Rand teaches a method of rereading individual nucleic acid molecules with a nanopore device using adapters ligated at either end of a biomolecule to be analyzed. (Rand, abstract) Rand further teaches that the adapter contains a sequence that inhibits an enzyme from translocating on the strand (i.e. a molecular motor detachment induction portion) (Rand, Abstract). Rand teaches that upon encountering the detachment portion, the enzyme dissociates into solution, but the nucleic acid molecule is retained in the pore and can then be reread by the same pore, affording similar advantages of averaging reads of a population of molecules in terms of accuracy but characterizes one molecule of DNA and increases the accuracy of calling methyl and hydroxymethyl cytosine modification at CpG dinucleotides (Rand, Figure 1, and paragraphs 0058 and 0065).
Therefore, it would have been prima facie obvious prior to the effective filing data of the claimed invention for one of ordinary skill in the art to combine the method of analyzing biomolecules by controlled reciprocation of the biomolecule through a nanopore, facilitated by molecular motors and adapter molecules comprising bulky structures, taught by Davis et al., with the molecular motor detachment induction sequences taught by Rand. The ordinary artisan would have been motivated to combine the method and adapters taught by Davis et al. with the motor-detachment sequences taught by Rand because of the teaching of Rand that the motor detachment sequence successive single-molecule, single-pore read cycles and increases the accuracy of detecting modified CpG dinucleotides (Rand, paragraph 0058 and 0065). The ordinary artisan would have been reasonably confident that inclusion of the molecular motor detachment induction sequences, taught by Rand, in the adapters taught by Davis et al. would have successfully improved the method taught by Davis et al. for multipass detection of a single nucleic acid molecule in a single nanopore because both the methods of Rand and Davis et al. allow for multipass detection of nucleic acid complexes “trapped” in single nanopores for more accurate detection of individual nucleotides.
Regarding claim 51, Rand teaches that “any linker that does not allow for the enzyme to translocate” can be used as the molecular motor detachment induction portion, such as alkane or PEG linkers (i.e. carbon chains that do not contain a phosphodiester bond or an abasic sequence portion) (Rand, paragraph 0053).
Regarding claim 52, Rand teaches the adapter comprises a G-quadruplex (i.e. a three-dimensional structure formation domain consisting of a single-stranded nucleotide) proximal to the nucleic acid terminus prevents enzyme translocation (Rand, paragraph 0053). Davis et al. teach pre-bulky structures for trapping nucleic acids in single nanopores comprise hairpin structures (i.e. a three-dimensional structure formation domain consisting of a single-stranded nucleotide at either end of a nucleic acid to be analyzed) It would likewise have been obvious to one of ordinary skill in the art to position the “stopper” structure closer to the nucleic acid terminus (i.e. on a side of a 5’ terminus) relative to the molecular motor detachment induction portion. The ordinary artisan would have been motivated to position the elements in this way because Rand teaches that dissociation of the enzyme facilitates the end of one read and resetting the molecule-pore complex for the next cycle (Rand, figure 1).
Regarding claim 53, Rand teaches the adapter further comprises a double stranded region that binds to the biomolecule to be analyzed at one end and comprises a single stranded nucleic acid region having a 3’ terminus and including the molecular motor detachment induction portion (Rand, figure 3). Rand further teaches that the adapters may be used in either polarity configuration, depending on which type of molecular motor (3’ to 5’ helicase, 5’ to 3’ helicase) (Rand, paragraph 0050). Therefore, Rand teaches the adapter further comprises a double stranded region that binds to the biomolecule to be analyzed at one end and comprises a single stranded nucleic acid region having a 5’ terminus and including the molecular motor detachment induction portion.
Regarding claim 54, Davis et al. teaches an adapter comprising: a) a double stranded region comprising an end that binds to the biomolecule and b) a pair of single stranded nucleic acid regions, comprising first and second “pre-bulky structures” linked to the other end of the double stranded region consisting of sequences that are not complementary to each other (Davis et al., figure 28). Rand teaches the molecular motor detachment induction portion is in a single stranded nucleic acid region at either terminus of a nucleic acid depending on the molecular motor in question (Rand, paragraph 0050). It would have been obvious to a person of ordinary skill in the art that the detachment induction portion could be located at the 5’ end of the adapter because methods taught by Davis use nucleic acid polymerases as the molecular motor. Because nucleic acid polymerases translocate along the template strand in a 3’ to 5’ direction while synthesizing a complementary strand, it would have been obvious that the detachment induction portion taught by Rand would have been located at the 5’ terminus of the pair of single-stranded nucleic acid regions.
Regarding claim 57, Davis et al. teaches adapter molecules comprise a single stranded region having a 3’ terminus including a bulky structure (i.e. a fall-off prevention portion having a larger diameter than that of a nanopore) (Davis et al., figure 28 and paragraphs 0054-0056).
Regarding claim 58, Davis et al. teaches the fall off prevention portion is a hairpin structure or a molecule bondable to the single-stranded nucleic acid region (Davis et al., paragraph 0055).
Regarding claim 59, Rand teaches adapter molecules comprise a single stranded nucleic acid region having a 3’ terminus comprising a molecular motor binding portion (Rand, figure 3 and paragraphs 0050-0053).
Regarding claim 60, Davis et al. teaches a molecular motor that can bind to the adapter molecule (Davis et al., Figure 23 and paragraph 0282), and a primer capable of hybridizing to the adapter molecule (Davis et al., paragraph 0451) (i.e. the adapter comprises adapter and primer binding regions). Davis et al. further teaches the molecular motor can be a nucleic acid polymerase (i.e. the primer hybridizes on a side of the 3’ terminus from the molecular motor binding portion) (Davis et al., paragraphs 0282 and 0391).
Regarding claim 61, Rand teaches an adapter can comprise a spacer to which the molecular motor is not being bound between the molecular motor binding portion and the sequence to be analyzed (i.e. the primer binding portion) (Rand, figure 3 and paragraph 0052-0053)
Claims 55-56 are/remain rejected under 35 U.S.C. 103 as being unpatentable over Davis et al., US 2013/0244340 A1 (published September 19, 2013) in view of Rand., US 2018/0030525 A1 (published February 1, 2018) as applied to claims 41, 51-54, and 57-61 above, and further in view of Turner et al., US 9,017,937 B1 (patent issued April 28, 2015).
Regarding claim 55, Davis et al. in view of Rand teaches a biomolecule analysis method comprising steps of: applying a voltage between a first and second liquid tank, each filled with electrolyte solutions and connected by a nanopore (Davis et al., Figures 3 and 23), wherein the first tank solution: comprises a biomolecule-adapter complex, a molecular motor that can bind to the biomolecule-adapter complex (Davis et al., Figure 23 and paragraph 0282), and a primer capable of hybridizing to the biomolecule-adapter complex (Davis et al., paragraph 0451). Davis et al. in view of Rand et al. teaches the “cis chamber” (i.e. the first tank) is set to a negative potential, while the “trans chamber” (i.e. the second tank) is set to a positive potential (Davis et al., figure 3). Davis et al. in view of Rand et al. teaches the biomolecule-adapter complex comprises an adapter molecule bound to the termini of the biomolecule (Davis et al., figure 28 and paragraphs 0451-0452). Davis et al. in view of Rand et al. further teaches measuring signals generated when the biomolecule-adapter complex is conveyed between the two liquid tanks through the nanopore wherein the measurement is repeated (Davis et al., figure 23), facilitated by DNA polymerase synthesizing a complementary strand from a primer bound to the complex (Davis et al., paragraphs 0282 and 0391) (i.e. a molecular motor binding and primer binding portion). Davis et al. in view of Rand et al. further teaches that the adapter contains a sequence that inhibits an enzyme from translocating on the strand (i.e. a molecular motor detachment induction portion) proximal to the 5’ nucleic acid terminus relative to the molecular motor detachment induction portion (Rand, Abstract and paragraph 53). Rand teaches that upon encountering the detachment portion, the enzyme dissociates into solution, but the nucleic acid molecule is retained in the pore and can then be reread by the same pore, affording similar advantages of averaging reads of a population of molecules in terms of accuracy but characterizes one molecule of DNA and increases the accuracy of calling methyl and hydroxymethyl cytosine modification at CpG dinucleotides (Rand, Figure 1, and paragraphs 0058 and 0065).
The method taught by Davis et al. in view of Rand et al. does not teach that the adapter further comprises a three-dimensional structure formation inhibiting oligomer comprising a base sequence complementary to at least a portion of the three-dimensional structure formation domain.
However, Turner et al. teaches a method for nanopore sequencing wherein a “pro-hairpin adapter” (i.e. an adapter comprising a three-dimensional structure formation domain) is bound to a biomolecule to be analyzed and is hybridized to a “splint oligo” comprising a sequence complementary to the hairpin region. (Turner et al., figure 24) Turner further teaches that the splint strand prevents formation of the hairpin (i.e. the three-dimensional structure) (Turner et al., figure 24). Turner teaches that the inclusion of the “splint oligo” inhibits the formation of the hairpin until the pro-hairpin adapter has passed through the nanopore, allowing for the biomolecule to become trapped in the nanopore, thus preventing the loss of the molecule and allowing for multiple sequencing passes across the nanopore (Turner et al., column 50, line 40- column 52, line 15).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to combine the biomolecule analysis method utilizing adapters comprising a molecular motor detachment induction portion, taught by Davis et al. in view of Rand et al., with the three-dimensional structure formation inhibiting “splint oligo” taught by Turner et al.
The ordinary artisan would have been motivated to add the structure formation inhibiting splint oligo taught by Turner et al. to the adapter taught by Davis et al. in view of Rand et al. because of the teaching of Turner et al. that the splint oligo facilitates loading of the molecules into the nanopore for repeat reading of the molecules by providing a convenient method of blocking both ends of the molecule to be analyzed such that the molecule is does not fall out of the pore when it reaches the end of the molecule while being translocated through the pore (Turner et al., column 50, lines 44-55 and column 51, lines 22-53). The ordinary artisan would have been reasonably confident that the three-dimensional structure formation inhibiting splint oligo taught by Turner et al., would have facilitated loading of a molecule comprising pore blocking bulky groups as taught by Davis et al. in view of Rand et al. because they both teach methods of threading “pre-bulky structures” through a nanopore, including strategies comprising hybridizing complementary oligos to the pre-bulky structures.
Regarding claim 56, Turner et al. teaches that the adapter molecule hybridized to the three-dimensional structure formation inhibiting oligomer comprises terminal nucleotides that are not hybridized to the structure formation inhibiting oligomer to allow threading into the nanopore (Turner et al., column 51, lines 24-27).
Claims 62-63 are/remain rejected under 35 U.S.C. 103 as being unpatentable over Davis et al., US 2013/0244340 A1 (published September 19, 2013) in view of Rand., US 2018/0030525 A1 (published February 1, 2018) as applied to claims 41, 51-54, and 57-61 above, and further in view of Heron et al., US 2017/0107569 A1 (published April 20, 2017).
Regarding claim 62, Davis et al. in view of Rand et al. teaches a biomolecule analysis method comprising steps of: applying a voltage between a first and second liquid tank, each filled with electrolyte solutions and connected by a nanopore (Davis et al., Figures 3 and 23), wherein the first tank solution: comprises a biomolecule-adapter complex, a molecular motor that can bind to the biomolecule-adapter complex (Davis et al., Figure 23 and paragraph 0282), and a primer capable of hybridizing to the biomolecule-adapter complex (Davis et al., paragraph 0451). Davis et al. in view of Rand et al. teaches the “cis chamber” (i.e. the first tank) is set to a negative potential, while the “trans chamber” (i.e. the second tank) is set to a positive potential (Davis et al., figure 3). Davis et al. in view of Rand et al. teaches the biomolecule-adapter complex comprises an adapter molecule bound to the termini of the biomolecule (Davis et al., figure 28 and paragraphs 0451-0452). Davis et al. in view of Rand et al. further teaches measuring signals generated when the biomolecule-adapter complex is conveyed between the two liquid tanks through the nanopore wherein the measurement is repeated (Davis et al., figure 23), facilitated by DNA polymerase synthesizing a complementary strand from a primer bound to the complex (Davis et al., paragraphs 0282 and 0391) (i.e. a molecular motor binding and primer binding portion). Davis et al. in view of Rand et al. further teaches that the adapter contains a sequence that inhibits an enzyme from translocating on the strand (i.e. a molecular motor detachment induction portion) proximal to the 5’ nucleic acid terminus relative to the molecular motor detachment induction portion (Rand, Abstract and paragraph 53). Rand teaches that upon encountering the detachment portion, the enzyme dissociates into solution, but the nucleic acid molecule is retained in the pore and can then be reread by the same pore, affording similar advantages of averaging reads of a population of molecules in terms of accuracy but characterizes one molecule of DNA and increases the accuracy of calling methyl and hydroxymethyl cytosine modification at CpG dinucleotides (Rand, Figure 1, and paragraphs 0058 and 0065).
The method taught by Davis et al. in view of Rand et al. does not teach that the Y- or forked adapter further comprises a single stranded nucleic acid region having the 3’-terminus and a plurality of sets of a molecular motor binding portion and a primer binding portion on a side of the 3’ terminus from the molecular motor binding portion.
However, Heron et al. teaches a method for nanopore sequencing of nucleic acids using Y-adapters comprising a plurality of sets of molecular motor binding sequences and primer binding sequences (Heron et al., figure 11 and paragraph 0034). Heron et al. teaches that the method of nanopore sequencing with adapters comprising a plurality of motor and primer binding sequences allows for controlled movement of the polynucleotide through the nanopore and produces increased information obtained from a single double-stranded polynucleotide as well as a mechanism for proof-reading collected sequence information (Heron et al., paragraphs 0109-0110). Furthermore, Heron et al. teaches that the single stranded nucleic acid region may comprise a plurality of sets of molecular motor binding and primer binding portions at either the 5’ or 3’ terminus, depending on the polarity of the particular molecular motor (Heron et al., figure 11 and paragraphs 0140-0142).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to modify the method taught by Davis et al. in view of Rand et al. that uses adapter molecules comprising molecular motor binding and primer binding portions to control the movement of a biomolecule of interest through a nanopore with the Y-adapters comprising multiple binding sites for molecular motors and primers, taught by Heron et al. The ordinary artisan would have been motivated to modify the adapters taught by Davis et al. in view of Rand et al. to further comprise a plurality of molecular motor and primer binding sites because of the teaching of Heron et al. that such adapters provide higher confidence observations (Heron et al., paragraph 110). The ordinary artisan would have been reasonably confident that a simple modification to the adapters taught by Davis et al. to further comprise additional primer and molecular motor binding sites would have facilitated control of translocation of the biomolecule through the pore and improved the accuracy of characterizing the biomolecule, as taught by Heron et al. Similarly, it would have been obvious to a person of ordinary skill in the art that the primer binding portion could be located at the 3’ end of the adapter because methods taught by Davis use nucleic acid polymerases as the molecular motor. Because nucleic acid polymerases translocate along the template strand in a 3’ to 5’ direction while synthesizing a complementary strand, it would have been obvious that the primer binding portion taught by Rand would have been located at the 3’ terminus of the pair of single-stranded nucleic acid regions.
Regarding claim 63, Heron et al. teach that the adapter molecule comprises a spacer to which the molecular motor is not bound provided between the molecular motor binding portion and the primer binding portion (Heron et al., figure 11 and paragraph 0034 and 0298-0316).
Response to arguments
Regarding claim 41 as amended and its dependents, the response argues that Rand does not teach effecting detachment based on differential interaction energy… for controlled cyclic engagement/disengagement and rather functions by physically blocking enzyme translocation.
This argument has been thoroughly reviewed and is not persuasive.
Rand, in fact, explicitly teaches “enzyme displacement sequence that is an abasic series of nucleotide sugars. The enzyme displacement sequences causes the processive enzyme used on the construct to fall off the construct.” (Rand et al., paragraph 0016). Furthermore, Rand figure 1 and paragraph 0058 explicitly demonstrates how this mechanism effects cyclic engagement/disengagement to effect directional, iterative processing (See below).
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Furthermore, the response argues i) that Rand’s displacement sequence functions differently by blocking translocation rather than by differential affinity and ii) the claimed method functions differently from “complete enzyme dissociation into solution” taught by Rand because the claimed method “uses differential binding affinity to enable controlled separation and re-initiation during strand synthesis.
These arguments have been thoroughly reviewed and are not persuasive. First, the assertion that Rand’s displacement sequence “contains a block of abasic residues proximal to the end of the strand” which are “regions of DNA that do not contain bases but are linked through phosphodiester backbone, or other covalent chemical linkage” and that “the enzyme used must not be able to translocate through this region” is somehow different from the claimed “molecular motor detachment portion” is not consistent with the teachings of the present specification and the present claims. As the response notes, Rand teaches the displacement sequence encompasses a block of abasic residues through which the polymerase does not translocate. Present claim 51, which depends from 41, can be interpreted (see 112(b) rejection above) as specifically reciting the “molecular motor detachment induction portion is a carbon chain that does not contain a phosphodiester bond or [is] an abasic sequence portion. In this interpretation, it appears that the structure (the “molecular motor detachment portion” or “displacement sequence”) necessary to accomplish the claimed methods are identical. In the case that the claim is intended to be read as requiring that the detachment induction portion is a carbon chain that does not contain either a phosphodiester bond or an abasic sequence portion, it is noted that Rand et al. further teach “initial experiments use d-spacers (abasic residues), however any linker that does not allow for the enzyme to translocate will work for this purpose, such as alkane or PEG linkers” (Rand et al., paragraph 0053). Alkane and PEG linkers are carbon chains that do not contain phosphodiester bonds and do not contain abasic sequence portions.
Additionally, the assertion that “the claimed invention uses differential binding energy to enable controlled separation and re-initiation…” is i) not commensurate in scope with the claims (i.e. “controlled separation and re-initiation” is not required by the claims) and ii) appears to be synonymous with “dissociate” because there are no limitations that distinguish polymerase “separation and re-initiation” from polymerase “displacement” or “dissociation” and “rebinding” or “binding a new enzyme”.
Therefore, claim 41 and its dependents are/remain properly rejected under U.S.C. 103.
Conclusion
No claim is allowed.
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/Z.M.T./Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682
