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 .
Application Status
This action is written in response to applicant’s correspondence received 02/18/2026. Claims 1-2, 4-6, 8-10, 12-17, 19-22, and 24-25 are currently pending. Claims 19-21 and 24-25 are withdrawn from prosecution as being drawn to non-elected subject matter. Accordingly, claims 1-2, 4-6, 8-10, 12-17 and 22 are examined herein. Applicant elected the invention of group I, an isolated heat-repressible Split-T7 polymerase fusion protein, without traverse in the reply filed 02/18/2026
Election/Restrictions
Applicant's election without traverse of the invention of group I, an isolated heat-repressible Split-T7 polymerase fusion protein (claims 1-2, 4-6, 8-10, 12-17 and 22) in the reply filed on 02/18/2026 is acknowledged. The requirement is still deemed proper and is therefore made FINAL. Claims 19-21 and 24-25 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Priority
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code. Please see pages 12, 35, and 37-39. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
Drawings
The drawings are objected to because the figures are not properly labeled.
37 CFR 1.84 (u)(1) states “The different views must be numbered in consecutive Arabic numerals, starting with 1, independent of the numbering of the sheets and, if possible, in the order in which they appear on the drawing sheet(s). Partial views intended to form one complete view, on one or several sheets, must be identified by the same number followed by a capital letter. View numbers must be preceded by the abbreviation "FIG." Where only a single view is used in an application to illustrate the claimed invention, it must not be numbered and the abbreviation "FIG." must not appear.”
In the instant case, partial views (i.e., a, b, c, etc.) are identified by “FIGURE [X] (continued)” instead of the same number followed by a capital letter, i.e., FIG. 1A, FIG. 1B, etc. Please see Figures 2, 4, 5, 7, 9, 10 and 12.
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
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.
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 2, 4, 8-10 and 12 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.
Regarding claims 2 and 4:
Claim 2 recites “the coiled-coil domain”. Claim 1, from which it depends, recites two coiled-coil domains: one fused to the N-terminal fragment of T7, and one fused to the C-terminal fragment of T7. There are two possible antecedent bases for the term. As a result, it is unclear to which of the coiled-coils claim 2 refers: the N-terminal, C-terminal, or both. This renders the metes and bounds of the claimed structure unclear. Claim 4 is rejected because it depends from claim 2 but does not remedy the indefiniteness therein.
Amending the claim to “the coiled-coil domains are selected from” would obviate this rejection, because it makes clear that the term refers to either or both of the coiled-coils of claim 1.
Regarding claims 8-10 and 12:
The issue of indefiniteness arises here because claims 6-13 all depend from claim 5 and define various alternative embodiments of the C-terminal coiled-coil domain, but some of the limitations introduced in this line of claims are mutually exclusive with certain embodiments, rendering the metes and bounds of the required C-terminal coiled-coil domain unclear.
Claim 5, from which claim 8 depends via claim 6, recites that the C-terminal coiled-coil domain comprises one or more of domains X1-X5, which are encoded by nucleotide sequences with at least 70% identity to SEQ ID NOs: 14-18, respectively. The language is open-ended, such that the C-terminal coiled-coil domain may include other elements in addition to any one or combination of the X1-X5 domains.
Claim 6, which depends from claim 5, recites several alternative embodiments. In the first (lines 1-4), it limits the domain to one which must comprise, at a minimum, an X1 domain at the N-terminal end and an X4 domain at the C-terminal end. The language remains open-ended, so this embodiment would amount to the domain of claim 5 which requires the N-terminal X1 and C-terminal X4 but may also have any combination of the other “X” domains.
In an interpretation where the "or" on line 4 of claim 6 is given weight, the claim encompasses either only the first embodiment, or encompasses only any of the embodiments as set forth in lines 5-7. In an interpretation where the "and" is given weight, three more possible embodiments are added. In one embodiment, the N-terminal X1 domain requires the recited specific substitution but the X4 domain may be generic. In another, the X4 may have the specific substitution and the X1 may be generic. In another, both may be specific.
This leads to issues in claim 8, which depends from claim 6. Claim 8 limits any of these embodiments to one which comprises a plurality of X5 domains, and/or one which consists of, from N-terminal to C-terminal, domains X1, X2, X3 and X4 or X5. This is interpreted as meaning that the C-terminus coiled-coil may either consist of only domains X1-2-3-4, or it may consist of only domains X1-2-3-5. This embodiment leads to indefiniteness for embodiments of claim 6 in which the specifically substituted X1 and/or X4 domains must be present, because a domain which consists solely of X1-2-3-5 cannot also include domain X4, whether generic or substituted. It also leads to issues of indefiniteness for an embodiment of claim 8 which both comprises a plurality of X5 domains and consists of only X1, X2, X3, and X5 (which is interpreted as only a single X5 rather than a plurality). These are mutually exclusive limitations which render the metes and bounds of the claimed structure unclear, and the claim indefinite.
Likewise, claim 9 recites the protein of claim 8 wherein the C-terminal coiled-coil domain comprises (but does not consist of) domains X1, X5, X5, and X4 or X5. The switch to open-ended language between claims 8 and 9 renders certain embodiments indefinite. How can the coiled-coil consist solely of domains X1-4, for example, but also comprise domains X1, X5, X5, and X5? These would again appear to be mutually exclusive limitations which render the metes and bounds of the claimed structure indefinite.
Claim 10 then raises further issues of indefiniteness for embodiments where the coiled-coil domain consists only of specific domains, such as X1-2-3-5. Claim 10 limits the domain to one encoded by a polynucleotide sequence comprising SEQ ID NO: 12. The specification discloses that SEQ ID NO: 12 encodes a mutated coiled-coil domain of a CT7 (C-terminal T7) fragment, Mut1 (p. 17, Table 3). Figure 8 shows a diagram of Mut1 and shows that it has, from N to C, a sequence with 98% identity to X1, X5, X5, and a sequence with 98% identity to X4 (which comprises X5). Notably, SEQ ID NO: 12 does not comprise X2 or X3, which are required for the structure consisting of X1-2-3-5, and additionally comprises X4, which is excluded from the X1-2-3-5 structure by the closed language. Once again, these limitations appear to be mutually exclusive and render the claim indefinite. A similar issue exists for claim 12, which recites that the C-terminal coiled-coil domain is encoded by SEQ ID NO: 13. In Table 3, the specification discloses that SEQ ID NO: 13 represents Mut2. Figure 8 shows that Mut2 has a sequence with 98% identity to X1 as well as X2, X3, X5, and a sequence with 98% identity to X4 (comprising X5). Consider the embodiment of claim 8 which consists of domains X1-2-3-5: the closed language excludes X4, which however is present in Mut2/SEQ ID NO: 13.
In summary, the narrow, closed embodiments recited in claim 8 are mutually exclusive with other recited limitations in that claim and its dependents, which renders the metes and bounds of those structures indefinite.
Claim Rejections - 35 USC § 112(a) – Written Description
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 1, 5-6, 8-9, 13-17 and 22 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.
MPEP 2163.II.A.3.(a).i) states, “Whether the specification shows that applicant was in possession of the claimed invention is not a single, simple determination, but rather is a factual determination reached by considering a number of factors. Factors to be considered in determining whether there is sufficient evidence of possession include the level of skill and knowledge in the art, partial structure, physical and/or chemical properties, functional characteristics alone or coupled with a known or disclosed correlation between structure and function, and the method of making the claimed invention”.
For claims drawn to a genus, MPEP § 2163 states the written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice, reduction to drawings, or by disclosure of relevant, identifying characteristics, i.e., structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the applicant was in possession of the claimed genus. See Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406.
Claim 1 recites an isolated Split-T7 polymerase fusion protein comprising a split T7 RNAP and a polypeptide coiled-coil domain. The preamble states that the fusion protein is “heat-repressible”. MPEP 2111.02 states that, “If the claim preamble, when read in the context of the entire claim, recites limitations of the claim, or, if the claim preamble is ‘necessary to give life, meaning, and vitality’ to the claim, then the claim preamble should be construed as if in the balance of the claim.”. In this case, the preamble is necessary to give life to the claim, as the specification makes clear that this feature is key to the invention (see e.g., p. 1) and the specification indicates that specific TlpA structures are associated with the functional characteristic of the protein being heat repressible (see e.g., p. 12, which describes the design of the split polymerase/TlpA fusion system and its derivation from the prior art). However, the recited “coiled-coil” domain, in contrast to the specific TlpA domains discussed and exemplified in the specification, is generic.
The specification discloses the wild-type TlpA coiled-coil domain (Table 2, amino acid SEQ ID NO: 3) and two mutants which are heat repressible, Mut1 and Mut2, whose coding sequences are represented by SEQ ID NOs: 12 and 13, respectively (Table 3, p. 17-18). The specification also discloses five consensus nucleotide regions, X1-X5, which were present in the mutant TlpA coiled-coil domains (Table 5, p 28-29), and notes that the X1 and X4 domains at either end of the coiled-coil were present in both proteins. Presumably, then, at least the X1 and X4 domains are required for the coiled-coil to function as a heat-repressible coiled coil, but it is not clear from the specification what else is required, in what configuration. Mut1 comprises a domain with 98% identity to X1, three X5 domains, and one X4 domain, but also comprises additional sequences whose functional contribution is unclear. Mut2 comprises all five domains (with X1 and X4 at 98% identity), so again, it is not clear if X1 and X4 are all that are required, or if other elements are necessary, and if so, what they are and how their structure correlates with their functional role in the protein.
In contrast to what is disclosed in the instant specification, the prior art shows that the genus of coiled-coil proteins is vast, and within that genus, there is significant variability unpredictability when it comes to sequence and function. Per Szczepaniak, “Coiled coils are widespread and diverse domains that can be found in approximately %5 of proteins.” and, “there are ~50,000 coiled-coil sequences and structures in the Protein Data Bank” (Szczepaniak et al. A library of coiled-coil domains: from regular bundles to peculiar twists. Bioinformatics, 36(22-23), 2020, 5368–5376.). Truebestein teaches that, “coiled-coil domains of some proteins are almost invariant in sequence in length,” but, “Other coiled-coils are divergent in sequence, but conserved in length, thereby functioning as molecular spacers.” (Coiled‐coils: The long and short of it. Bioessays. 2016 Aug 5;38(9):903–916. Abstract.). Truebestein further describes coiled-coils as having diverse functions, acting as molecular spacers, molecular rulers, scaffolding proteins, and acting to propagate conformational changes (Id.). Truebestein also notes that, “coiled-coils represent a significant proportion of the eukaryotic proteome, there are relatively few high-resolution structures of full-length coiled-coil-containing proteins. Furthermore, crystal structures of coiled-coils illustrate the plasticity of the motif, with many coils exhibiting non canonical packing interactions.” (p. 913).
The instant specification itself points to significant unpredictability regarding the correlation between structure and function in coiled-coil domains, on. p 21:
The inventors also investigated the suitability of various lengths of the Tlpa's coiled- coil acting as fusion partners for the NT7 and CT7 protein fragments. The coiled-coil consists mainly of heptad repeats and separated into two distinctive coiled-coil regions: the longer N- terminal based 164-residues coil and the shorter C-terminal based 114-residues coil (Fig. 2a) [Mason and Ardnt, Chembiochem 5:170-176 (2004)]. By selective removal of the shorter coiled-coil (258 - 371th residues) from the T7 fusion partners, the truncated (Tlpa*) system which retained the longer coil (94 - 257th residues) suffered from minimal expression and no observable fold change to temperature changes (Fig. 2d). A possible explanation for its poor performance could be due to the perturbation of interfacial ionic interactions when essential amino acids residues located at the shorter coiled-coil which governed the C2 symmetry of the parallel coiled structure are absent [Piraner, D.I., Wu, Y. and Shapiro, M.G. ACS Synthetic Biology, 8: 2256-2262 (2019)]. This suggests that engineering a high-performance temperature-sensitive split-polymerase system cannot be easily accomplished through mere excisions of conserved regions of the coiled-coil but rather allude to the need for developing a comprehensive mutagenesis workflow, in which the inventors performed, to identify consensus sequences in Tlpa that governs its thermal performance within the fusion polymerase.
What the above indicates is that the heat repressibility of a single coiled-coil domain, TlpA, could not readily have been predicted from its structure alone, such that truncating the system unexpectedly led to poorer performance, and effective variants could only be identified through mutagenesis, i.e., trial and error. As discussed above, the two variants discovered only share an X1 and X4 domain, but they also comprise other domains, and it is unclear exactly what combination of domains is needed for the coiled-coil domain to maintain its heat-repressible dimerization functionality when fused to a split protein like T7.
Claim 5 limits the coiled-coil domain to one comprising one or more of the recited domains, X1-X5, represented by SEQ ID NOs: 14-18, respectively. However, the claims only require these domains with at least 70% sequence identity, whereas the specification only discloses the consensus sequences with at least 98% identity to the wild-type TlpA, shown in Table 5. The specification does not provide a correlation between structure and function sufficient for the ordinary artisan to know what nucleotides in those sequences may be changed while retaining the function of being heat-repressible. Additionally, claim 5 only requires “one or more” of those domains, but as the specification notes, “the X1 and X4 domains which are located at either ends of the coiled-coil were present in both the wild-type and the mutated fusion proteins. It is likely that these preserved regions are highly essential to allow proper folding and guidance of the split polymerase units.”. However, the claim encompasses structures wherein only, for example, X3 is present. Would the coiled-coil domain be expected to function as a heat-repressible regulator of the split T7 RNAP if it consisted of the X3 domain alone? This is not clear from the specification or the art, although the preponderance of evidence and the two mutants described in the instant specification, which have at least X1 and X4/5 (see Figure 8), indicate that this structure may not be sufficient.
Claim 6 limits the fusion protein to one in which the protein either must comprise X1 and X4, and/or to one which, if it comprises X1 or X4, the nucleotide sequences must have certain substitutions. It is important to note that the second wherein clause does not necessarily limit the protein to one comprising X1 and/or X4, but is viewed as a conditional alternative embodiment, such that if either is present, they have those substitutions. However, due to the various alternative embodiments recited in claims 5 and 6, claim 6 encompasses embodiments wherein, for example, the fusion protein only comprises X3, or, e.g., comprises only X1 and X3 wherein X1 has the recited substitution, but does not comprise X4. As discussed above, the inventors discovered that X1 and X4 were both necessary for the fusion protein to be heat-repressible. The specification does not provide written support for other structures in which they are absent which have the same heat-repressibility, nor does it provide a correlation between structure and function sufficient for the ordinary artisan to envision the structures that would work.
Claim 8 limits the coiled-coil domain to one which comprises a plurality of X5 domains, but since this encompasses embodiments wherein the “key” identified domains are absent (e.g., where the coiled-coil domain is simply a repeat of multiple X5 domains), this is not considered sufficient to show possession of the full genus of coiled-coil domains with the required heat-repressible functionality, for the same reasons described above. Claim 9 limits the domain to one comprising X1, X5, X5, and X4/X5, but the specification does not provide support for a coiled-coil domain comprising X1, X5, X5, and X5 without X4, and in fact states that X4 appears to be required for the heat-repressing functionality.
Other claims included in the rejection but not discussed are rejected because they do not further limit the scope of the claimed coiled-coil domain, and therefore do not remedy the issue.
Claims 2 and 4 are excluded from this rejection because they recite specific coiled-coil domains/sequences. Claims 10 and 12 are excluded because they recite specific sequences corresponding to the disclosed mutant coiled-coil domains, Mut1 and Mut2, and are interpreted as requiring those sequences with 100% identity.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
Determining the scope and contents of the prior art.
Ascertaining the differences between the prior art and the claims at issue.
Resolving the level of ordinary skill in the pertinent art.
Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-2, 4 and 14-17 are rejected under 35 U.S.C. 103 as being unpatentable over Baumschlager (Baumschlager et al. plus Supplemental Information. ACS Synthetic Biology 2017 6 (11), 2157-2167.) in view of Han (ACS Synth. Biol. 2017, 6, 357−366.; of record, applicant’s submission) and Piraner (Piraner et al. plus Supplemental Information. ACS Synthetic Biology 2019 8 (10), 2256-2262.).
Please note that while Baumschlager and Piraner minus supplemental information are of record, having been submitted by applicant, the supplemental information was not submitted and was cited in this rejection, which necessitated an additional citation and entry of the supplemental information into the record.
Regarding claim 1:
Figure 1A of Baumschlager teaches a split-T7 polymerase fusion protein, comprising a split T7 RNA polymerase polypeptide (T7RNAP) fused at the C- and N-termini to a regulatory polypeptide (nMag/pMag) via a linker:
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The N-terminal domain of T7RNAP was fused to nMagHigh1, and the C-terminal domain of T7RNAP was fused to pMag, both using a short GGSGG linker, schematically depicted in Figure 1A. (p. 2159)
Baumschlager does not teach that the fusion protein is heat-repressible, or that the regulatory polypeptide is a coiled-coil domain. However, Baumschlager notes that, “due to the modularity of the system, the blue light sensitive Magnets could be exchanged with regulators that dimerize in response to other wavelengths”, thus indicating that the system is modular and the regulators are substitutable.
Baumschlager further cites Han, which teaches a similar split T7 RNAP system using light-sensitive VVD domains or, alternatively, rapamycin-sensitive domains. Han teaches that, “the T7 RNAP has high plasticity for this type of engineering”, and that, “ modularity exists for the combination of the split T7 RNAP fragments with the extra regulatory domains. In the future, this modularity can be exploited to engineer polymerase switches controlled by signals other than the blue light or rapamycin signals tested here”.
Therefore, Baumschlager indicates that the system is modular, and Han further supports its modularity. Combined, the two disclosures would have given the ordinary artisan a reasonable expectation that the regulatory domains could be substituted to create split-T7 switches responsive to various signals.
Piraner teaches heat-repressible regulatory domains: “thermomers” based on a sharply thermolabile coiled-coil protein that, when fused to other proteins, can reversibly control their association (Abstract).
Piraner further teaches that these thermomers are heat-repressible, i.e., the coiled-coil domain undimerizes above a temperature of ~42C (p. 2257). Piraner also provides a teaching, suggestion or motivation to substitute light-inducible dimerization domains, such as those taught by Baumschlager and Han, with their heat-repressible thermomers, stating that, “optically inducible dimerization domains…are limited by the scattering of photons in deep tissue and other complex media”, while, “Temperature offers an alternative mechanism for controlling biological signaling with several advantages over chemicals and light.” (p. 2256). Namely, “Temperature can be applied to biological samples globally using simple heat sources or electromagnetic radiation, and can be targeted locally deep within scattering media using technologies such as focused ultrasound, providing spatial and temporal resolution on the order of millimeters and seconds, respectively” (Id.).
Piraner further teaches that the thermomers are modular and can be used, “to control a variety of protein functions” including “reconstitution of various split proteins or dimerization-dependent protein complexes” (p. 2261). Therefore, Piraner provides a teaching, suggestion or motivation to use the thermomers in a split protein dimerization system such as a split T7 RNAP.
It would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to have substituted the light-inducible VVD domains in the modular split T7 RNAP switch, as taught by Baumschlager and Han, with Piraner’s heat-repressible coiled-coil thermomer domains, to achieve the predictable result of a heat-repressible switch. The ordinary artisan would have had a reasonable expectation of success based on Baumschlager and Han’s teachings that the split T7 RNAP system was modular and the regulatory domains could be substituted. The prior art split T7 polymerase differed from the claimed polymerase only by the substitution of the VVD and rapamycin-responsive regulatory domains with a heat-repressible domain. A search for alternative regulatory domains would have led the ordinary artisan to Piraner, and Piraner’s teachings of modular thermomers which addressed the limitations of light-based systems. All components, from the split T7 RNAP to the various regulatory domains, were known to be modular. One of ordinary skill could have substituted one known element for another, and the results of the substitution would have been predictable.
Regarding claim 2, Baumschlager teaches that the two fragments of the T7 RNA polymerase are derived by splitting the T7 at amino acid position 563 (p. 2159):
Previous studies have shown that T7RNAP can be split at multiple positions….We selected five split positions…563/564 (Figure 1A). The chosen positions (69, 302, 563) all lie in surface-exposed flexible loops of T7RNAP, minimizing structural and steric interference of the additional Magnet domains. Further, the amino acids at the split positions…563(S)/564(E) are preferred residues in natural linkers. We named these variants…Opto-T7RNAP(563)…OptoT7RNAP*(563) showed the highest fold change (>300-fold), with a dark-state expression of ∼5-fold and a lit-state expression ∼1900-fold above the reporter control.
Also regarding claim 2, Piraner teaches wherein the coiled-coil domain is TlpA (“we used TlpA, a temperature-sensitive transcriptional repressor from Salmonella typhimurium with a relatively simple predicted architecture and well-characterized thermal behavior”; p. 2256
Regarding claim 4, Baumschlager teaches a T7RNAP coding sequence on pages 24-26 of the supplemental material. When translated, the resulting amino acid sequence has 99.9% identity to SEQ ID NO: 1:
RESULT 1
AASEQ2_03062026_110835
Query Match 99.9%; Score 4615; DB 1; Length 883;
Best Local Similarity 99.9%;
Matches 882; Conservative 0; Mismatches 1; Indels 0; Gaps 0;
Qy 1 MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLK 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLK 60
Qy 61 AGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIK 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 AGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIK 120
Qy 121 TTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKK 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 TTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKK 180
Qy 181 AFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQD 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 AFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQD 240
Qy 241 SETIELAPEYAEAIA TRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTH 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 SETIELAPEYAEAIA TRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTH 300
Qy 301 SKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREEL 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 SKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREEL 360
Qy 361 PMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNM 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 PMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNM 420
Qy 421 DWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIK 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 DWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIK 480
Qy 481 FIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSC 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 FIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSC 540
Qy 541 SGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDE 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 SGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDE 600
Qy 601 NTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAID 660
||||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||
Db 601 NTGEISEKVKLGTKALAGQWLAYGVTRSVTKSSVMTLAYGSKEFGFRQQVLEDTIQPAID 660
Qy 661 SGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILR 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 661 SGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILR 720
Qy 721 KRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAP 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 721 KRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAP 780
Qy 781 NFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCD 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 781 NFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCD 840
Qy 841 VLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA 883
|||||||||||||||||||||||||||||||||||||||||||
Db 841 VLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA 883
Also regarding claim 4, on page 5 of the supplementary content, Piraner teaches the TlpA amino acid sequence used. The sequence has 98.2% overall identity to SEQ ID NO: 3:
RESULT 1
AASEQ2_03062026_112418
Query Match 98.2%; Score 1309; DB 1; Length 371;
Best Local Similarity 98.6%;
Matches 274; Conservative 1; Mismatches 3; Indels 0; Gaps 0;
Qy 1 AVRAAERRVAEVTRAAGEQTAQAERELADAAQTVDDLEEKLVELQDRYDSLTLALESERS 60
||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||
Db 94 AVRAAERRVAEVTRAAGEQTAQAERELADAAQTVDDLEEKLDELQDRYDSLTLALESERS 153
Qy 61 LRQQHDVEMAQLKERLAAAEENTRQREERYQEQRTVLQDALNAEQAQHINTREDQQKRLE 120
|||||||||||||||||||||||||||||||||:|||||||||||||| ||||| |||||
Db 154 LRQQHDVEMAQLKERLAAAEENTRQREERYQEQKTVLQDALNAEQAQHKNTREDLQKRLE 213
Qy 121 QISAEANARTEELKSERDKVNTLLTRLESQENALASERQQHLATRETLQQRLEQAIA DTQ 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 214 QISAEANARTEELKSERDKVNTLLTRLESQENALASERQQHLATRETLQQRLEQAIA DTQ 273
Qy 181 ARAGEIALERDRVSSLTARLESQEKASSEQLVRMGSEIASLTERCTQLENQRDDARLETM 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 274 ARAGEIALERDRVSSLTARLESQEKASSEQLVRMGSEIASLTERCTQLENQRDDARLETM 333
Qy 241 GEKETVAALRGEAEALKRQNQSLMAALSGNKQTGGQNA 278
||||||||||||||||||||||||||||||||||||||
Db 334 GEKETVAALRGEAEALKRQNQSLMAALSGNKQTGGQNA 371
Regarding claim 14, Piraner teaches that the thermomers are active at temperatures in the range of about 30°C to about 39°C and are thermally repressed above 39°C. For example, see Figure 3, which shows the validation of TlpA heterodimerization. Specifically, part b shows that dimers were formed at 37°C but dimerization was repressed at 45°C. Figure 4 further demonstrates the range of the thermomers, with part a showing a schematic wherein heterodimerization leads to RFP localization at the membrane, and part f showing that the localization (TlpA membrane fusion) began dropping around 38-39°C and was close to zero over 41°C. On p. 2257, Piraner states, “The coiled-coil domain of TlpA undimer izes and unfolds to a random coil above a temperature of ∼42 °C” and “the thermal set-point of TlpA could be tuned through directed evolution without compromising cooperativity”, thus teaching that the precise temperature range could be fine-tuned.
Regarding claims 15-17, Baumschlager teaches nucleic acid molecules, vectors and host cells expressing the fusion protein (“Plasmids were transformed using a one-step preparation protocol of competent E. coli. We used the pZ-series modular vectors…as the basis for plasmids containing the Opto-T7RNAPs.” P. 2164).
Claims 5 and 6 are rejected under 35 U.S.C. 103 as being unpatentable over Baumschlager, Han and Piraner, as applied to claims 1-2, 4 and 14-17, further in view of U.S. Patent No. 10,975,420 to Shapiro et al. (hereinafter ‘Shapiro’).
Baumschlager, Han and Piraner render obvious the isolated heat-repressible split-T7 fusion protein of claim 1, from which instantly rejected claims 5 and 6 depend, as described above.
Baumschlager, Han and Piraner do not teach wherein the coiled-coil domain comprises one or more domains X1-X5 (SEQ ID NOs: 14-18) with at least 70% identity, or combinations thereof.
However, Piraner does teach fusion proteins comprising the coiled-coil domain of the C-terminal coiled-coil domain of TlpA after truncating the DNA binding domain, as discussed above.
Shapiro teaches the nucleotide coding sequence of TlpA (SEQ ID NO: 460), which comprises a coiled-coil region comprising one or more of the above domains with at least 70% identity, as follows (see FIG. 14, which notes the position of the DNA binding domain and the coiled-coil domains, and the alignments below):
SEQ ID NO: 14 (X1):
RESULT 1
NASEQ2_03092026_132939
Query Match 100.0%; Score 61; DB 1; Length 1116;
Best Local Similarity 100.0%;
Matches 61; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 GCGGTCCGGGCTGCAGAACGCCGGGTTGCGGAAGTCACGCGTGCTGCCGGTGAACAGACC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 280 GCGGTCCGGGCTGCAGAACGCCGGGTTGCGGAAGTCACGCGTGCTGCCGGTGAACAGACC 339
Qy 61 G 61
|
Db 340 G 340
SEQ ID NO: 15 (X2):
RESULT 1
NASEQ2_03092026_133001
Query Match 98.7%; Score 123.4; DB 1; Length 1116;
Best Local Similarity 99.2%;
Matches 124; Conservative 0; Mismatches 1; Indels 0; Gaps 0;
Qy 1 CACAGGCAGAGCGGGAGCTGGCCGACGCCGCGCAGACAGTCGACGACCTGGAAGAAAAAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 341 CACAGGCAGAGCGGGAGCTGGCCGACGCCGCGCAGACAGTCGACGACCTGGAAGAAAAAC 400
Qy 61 TGGTTGAACTGCAGGACAGATATGACAGTTTGACGCTGGCGCTGGAGTCAGAACGTTCAC 120
||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 401 TGGATGAACTGCAGGACAGATATGACAGTTTGACGCTGGCGCTGGAGTCAGAACGTTCAC 460
Qy 121 TGCGT 125
|||||
Db 461 TGCGT 465
SEQ ID NO: 16 (X3):
RESULT 1
NASEQ2_03092026_133020
Query Match 100.0%; Score 27; DB 1; Length 1116;
Best Local Similarity 100.0%;
Matches 27; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 CACTGCGTGGTGAGGCTGAAGCCCTGA 27
|||||||||||||||||||||||||||
Db 1022 CACTGCGTGGTGAGGCTGAAGCCCTGA 1048
SEQ ID NO: 17 (X4):
RESULT 1
NASEQ2_03092026_133102
Query Match 100.0%; Score 64; DB 1; Length 1116;
Best Local Similarity 100.0%;
Matches 64; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 GCGTCAGAACCAGTCACTGATGGCGGCGCTTTCAGGCAATAAACAGACCGGTGGCCAGAA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1050 GCGTCAGAACCAGTCACTGATGGCGGCGCTTTCAGGCAATAAACAGACCGGTGGCCAGAA 1109
Qy 61 TGCG 64
||||
Db 1110 TGCG 1113
SEQ ID NO: 18 (X5, which is merely the 3’ terminal portion of X4):
RESULT 1
NASEQ2_03092026_133136
Query Match 100.0%; Score 28; DB 1; Length 1116;
Best Local Similarity 100.0%;
Matches 28; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 CAATAAACAGACCGGTGGCCAGAATGCG 28
||||||||||||||||||||||||||||
Db 1086 CAATAAACAGACCGGTGGCCAGAATGCG 1113
Regarding claim 6: the broadest reasonable interpretation of the terms “N-terminal end” and “C-terminal end” encompass fragments which are more towards one terminus than the other, but are not limited to only fragments at the extreme termini. For example, if the polypeptide were split in half, it would consist of one C-terminal half which would be considered the C-terminal end, and one N-terminal half considered the N-terminal end. Insofar as Shapiro teaches X1 at the N-terminal end of the coiled-coil region of TlpA and X4/5 at the C-terminal end, it teaches the limitations of the claim.
It would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to have expressed the TlpA amino acid sequence as taught by Piraner, in the context of the heat repressible split T7 fusion protein as taught by Baumschlager, Han and Piraner, from the TlpA coding sequence as taught by Shapiro. The preceding references render obvious a split T7 RNAP with a coiled-coil domain of TlpA fused to the N- and C-terminal ends of the polymerase. Shapiro merely teaches a coding sequence predictably capable of expressing said coiled-coil domain.
Claim 22 is rejected under 35 U.S.C. 103 as being unpatentable over Baumschlager, Han and Piraner, as applied to claims 1-2, 4 and 14-17, further in view of Ahern (Ahern. Biochemical, Reagents Kits Offer Scientists Good Return On Investment. The Scientist, vol. 9, No. 15. pp. 1-9.)
Baumschlager, Han and Piraner render obvious the isolated nucleic acid molecule expressing the heat-repressible split-T7 fusion protein of claim 15, from which instantly rejected claim 22 depends, as described above.
Baumschlager, Han and Piraner do not teach a kit comprising the nucleic acid.
Ahern teaches that, “The major advantages of purchasing products that come already prepared -- such as restriction enzymes, gels, and buffers --are that it enables investigators to save time and money.”.
It would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to have included the nucleic acid encoding the fusion protein in a kit along with key reagents such as a reaction buffer and ribonucleoside triphosphates, to enable the ordinary artisan to express the protein in a reproducible manner that saves time and money.
Subject matter free of the prior art
The prior art does not teach or suggest a polynucleotide encoding a TlpA coiled-coil domain, wherein the polynucleotide comprises SEQ ID NOs: 12 or 13, i.e., the Mut1 or Mut2 coiled-coil domains. While the prior art teaches TlpA coiled-coil domains, it does not teach truncated or mutated TlpA coiled-coil domains with the particular set of conserved domains and sequences claimed, nor does it provide guidance that would have led the ordinary artisan to those sequences from the wild-type TlpA coding sequence.
Conclusion
No claim is allowed at this time.
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/AMANDA M ZAHORIK/Examiner, Art Unit 1636