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 .
Election/Restrictions
Applicant’s election of “Group I (claims 61-71), drawn to a cyclopentyl-modified PNA having a monomethine dye surrogate base in proximity to a cyclopentyl group” in the reply filed on January 15, 2026 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
MPEP § 818.01(a) states:
“As indicated in the first sentence of 37 CFR 1.143, the traverse to a requirement for restriction must be complete as required by 37 CFR 1.111(b). Under this rule, the applicant is required to specifically point out the reason(s) on which he or she bases his or her conclusion(s) that a requirement to restrict is in error. A mere broad allegation that the requirement is in error does not comply with the requirement of 37 CFR 1.111. Thus the required provisional election (see MPEP § 818.01(b)) becomes an election without traverse if accompanied by an incomplete traversal of the requirement for restriction.”
The response states: “Applicant hereby elects Group I (claims 61-71), drawn to a cyclopentyl-modified PNA having a monomethine dye surrogate base in proximity to a cyclopentyl group, classified in C07K/003, with traverse. The present traversal is based on the fact that both groups share the same special technical feature that defines over the prior art. If the examiner rejects the elected claims, applicant will show during prosecution that the elected claims are allowable. At that point, the unity of invention restriction requirement will have to be reconsidered and withdrawn.”
In the requirement for restriction, a detailed case of prima facie obviousness in view of prior art references available under U.S.C. 102(a)(1) was made regarding the contribution of the shared technical feature (i.e. the PNA according to claim 61) over the prior art (see requirement for restriction, mailed November 17, 2025). The requirement for restriction details that the obviousness of the shared technical feature over the prior art (i.e. lack of an inventive step) does not make a contribution over the prior art and therefore is not a special technical feature shared among the claims.
The mere assertions that: a) “both groups share the same special technical feature that defines over the prior art.” and b) “applicant will show during prosecution that the elected claims are allowable”, do not distinctly and specifically point out the supposed errors in the restriction requirement but rather constitute “broad allegation[s] that the requirement is in error” and therefore does not comply with the requirement of 37 CFR 1.111 as detailed above.
Claims 72-80 were 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. Election was made without traverse in the reply filed on January 15, 2026.
Claim Status and Action Summary
This action is in response to the papers filed on January 15, 2026.
Claims 61-80 are pending in the present application. Claims 72-80 were withdrawn as directed to a nonelected invention. Claims 61-71 are under examination.
Priority/Effective Filing Date
The present application, filed on May 8, 2023, is a 371 of PCT/IL2021/051318, filed on November 8, 2021, which claims benefit of U.S. Provisional Patent Application No: 63/111,370.
It is noted that the dyes designated “Dye 1” or “Dye 2” are not disclosed in the certified copy of the priority document 63/111,370. These dyes are disclosed in PCT/IL2021/051318 (page 6). Therefore, claims requiring these dyes (present claim 63) enjoy the benefit of the filing date of the PCT application (November 8, 2021). Claims 61, 62, and 64-71 enjoy the benefit of the U.S. Provisional application (November 9, 2020).
Nucleotide and/or Amino Acid Sequence Disclosures
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Items 1) and 2) provide general guidance related to requirements for sequence disclosures.
37 CFR 1.821(c) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.821(a) must contain a "Sequence Listing," as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.821 - 1.825. This "Sequence Listing" part of the disclosure may be submitted:
In accordance with 37 CFR 1.821(c)(1) via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter "Legal Framework") as an ASCII text file, together with an incorporation-by-reference of the material in the ASCII text file in a separate paragraph of the specification as required by 37 CFR 1.823(b)(1) identifying:
the name of the ASCII text file;
ii) the date of creation; and
iii) the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(1) on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation-by-reference of the material in the ASCII text file according to 37 CFR 1.52(e)(8) and 37 CFR 1.823(b)(1) in a separate paragraph of the specification identifying:
the name of the ASCII text file;
the date of creation; and
the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(2) via the USPTO patent electronic filing system as a PDF file (not recommended); or
In accordance with 37 CFR 1.821(c)(3) on physical sheets of paper (not recommended).
When a “Sequence Listing” has been submitted as a PDF file as in 1(c) above (37 CFR 1.821(c)(2)) or on physical sheets of paper as in 1(d) above (37 CFR 1.821(c)(3)), 37 CFR 1.821(e)(1) requires a computer readable form (CRF) of the “Sequence Listing” in accordance with the requirements of 37 CFR 1.824.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed via the USPTO patent electronic filing system as a PDF, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the PDF copy and the CRF copy (the ASCII text file copy) are identical.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed on paper or read-only optical disc, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the paper or read-only optical disc copy and the CRF are identical.
Specific deficiencies and the required response to this Office Action are as follows:
Specific deficiency - This application fails to comply with the requirements of 37 CFR 1.821 - 1.825 because it does not contain a "Sequence Listing" as a separate part of the disclosure or a CRF of the “Sequence Listing.”.
Required response - Applicant must provide:
A "Sequence Listing" part of the disclosure; together with
An amendment specifically directing its entry into the application in accordance with 37 CFR 1.825(a)(2);
A statement that the "Sequence Listing" includes no new matter as required by 37 CFR 1.821(a)(4); and
A statement that indicates support for the amendment in the application, as filed, as required by 37 CFR 1.825(a)(3).
If the "Sequence Listing" part of the disclosure is submitted according to item 1) a) or b) above, Applicant must also provide:
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required incorporation-by-reference paragraph, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter.
If the "Sequence Listing" part of the disclosure is submitted according to item 1) c) or d) above, applicant must also provide:
A CRF in accordance with 37 CFR 1.821(e)(1) or 1.821(e)(2) as required by 1.825(a)(5); and
A statement according to item 2) a) or b) above.
Specific deficiency – Nucleotide and/or amino acid sequences appearing in the specification are not identified by sequence identifiers in accordance with 37 CFR 1.821(d).
Required response – Applicant must provide:
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required sequence identifiers, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on September 28, 2023 has been considered by the examiner.
The listing of references in the specification (on page 2) is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Specification
The disclosure is objected to because of the following informalities: Table 3 (page 11) and Table 4 (spanning page 11-12) are cut off by the right margin of the specification.
Appropriate correction is required.
Applicant is reminded that NO NEW MATTER may be added.
The use of the terms: “Sepharose”, “Phenomenex”, “Merck”, “Shimadzu”, “Thermo Fisher Scientific”, “TSQ Quantum”, “Cytation”, and “Greiner”, which are each a trade name or a mark used in commerce, has been noted in this application. Each term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Objections
Claim 63 is objected to because of the following informalities: The graphical representation of the structures of “Dye 1” and “Dye 2” recited in the claim are not legible. The claim is not rejected as indefinite over the illegibility of the structures in the claim because legible structures defining “Dye 1” and “Dye 2” are present on page 5 of the specification. Appropriate correction is required.
Claim 69 is objected to because of the following informalities: the phrase “The PNA according to claim 67, wherein the charged nucleobases having the structures: …, wherein each of Z, independently, is an alkyl having between 1 and 5 carbon atoms” appears to be a typographical error for: “…wherein the charged nucleobases have the structures: …, wherein each of Z, independently, is an alkyl having…” Appropriate correction is required.
Claim Rejections - 35 USC § 112(a)-Scope of Enablement
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.
Claim 63 is rejected under 35 U.S.C. 112(a) because the specification, while being enabling for …monomethine dye is a cyanine dye being BisQ or a Dye 1 or Dye 2 wherein each X represents a halogen atom selected from the group consisting of Chlorine and Bromine, does not reasonably provide enablement for the entirety of the genus “a halogen atom”. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make the invention commensurate in scope with these claims.
Factors to be considered in determining whether a disclosure meets the enablement requirement of 35 U.S.C. 112(a) have been described by the court in In re Wands, 8 USPQ2d 1400 (CAFC 1988). Wands states at page 1404, “Factors to be considered in determining whether a disclosure would require undue experimentation have been summarized by the board in Ex parte Forman. They include (1) the quantity of experimentation necessary, (2) the amount of direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skill of those in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claims.”
The nature of the invention and breadth of the claims
Claim 63 is drawn to a cyclopentyl-modified PNA comprising a surrogate base in proximity to one or more cyclopentyl groups, wherein the surrogate base is a monomethine dye that is a cyanine dye being BisQ or a dye herein designated Dye 1 or Dye 2, wherein each X represents a halogen atom:
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The specification describes the genus “halogen atom” on page 6 as comprising the following embodiments: “wherein each X represents a halogen atom (e.g., F, Cl, Br, or I)”
The unpredictability of the art and the state of the prior art
The invention is in a class of invention which the CAFC has characterized as “the unpredictable arts such as chemistry and biology.” Mycogen Plant Sci., Inc. v. Monsanto Co., 243 F.3d 1316, 1330 (Fed. Cir. 2001).
The prior art teaches a variety of peptide nucleic acid (PNA) modifications and surrogate base dyes. For example, Peled et al., “Predictive Model for the Sequence-Dependent Fluorogenic Response of Forced-Intercalation Peptide Nucleic Acid”, ACS Omega, 3(4), 3813-3818 (2018) (NPL citation number 9 on IDS filed September 28, 2023) teach forced-intercalation PNA probes comprising BisQ as a surrogate base located adjacent to a variant nucleotide of interest (Peled et al., abstract Scheme 1, and table 2), reproduced below for convenience).
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Similarly, Kolevzon et al., “Single point mutation detection in living cancer cells by far-red emitting PNA-FIT probes” Chem. Commun., 2016, (cited on September 28, 2023 IDS as NPL cite No. 2) teach a variety of forced-intercalation PNA probes comprising the BisQ surrogate base dye at a position adjacent to a variant nucleotide of interest (i.e. the point mutation to be detected) (Kolevzon et al., Table 1, reproduced below for convenience, and abstract)
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Additionally, the prior art, for instance Pokorski et al., “Cyclopentane-modified PNA improves the sensitivity of nanoparticle-based scanometric DNA detection” Chem. Commun., 2005, 2101-2103 (cited on September 28, 2023 IDS as NPL cite No. 6) teaches incorporation of a tcypPNA monomer at the site of a nucleotide variant of interest provides increased mismatch discrimination (greater difference in melting temperature between tcypPNA complementary to the target and tcypPNA with a single nucleotide mismatch) compared to the aminoethylglycine PNA without the cyclopentyl modification (aegPNA) (Pokorski et al., 2005, table 1, reproduced below for convenience).
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Regarding the structures of Dye 1 and Dye 2 comprising a halogen-modified, deprotonated monomethine bond, the prior art appears to offer few examples of halogenated monomethine dyes wherein the halogen atom is substituted in place of the hydrogen bonded to the monomethine carbon (see annotated BisQ structure vs. Dye 2 structure below; arrow points to the monomethine carbon).
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The art, for example, Kejík et al., “Cyanine dyes in the mitochondria-targeting photodynamic and photothermal therapy” Communications Chemistry (2024)7:180 (post filing date art) provides examples of polymethine cyanine dyes comprising a brominated or chlorinated methine group (see Figure 5A, reproduced below for convenience).
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Mahoney et al., “Tailoring Cyanine Dark States for Improved Optically Modulated Fluorescence Recovery” J Phys Chem B. 2015 Mar 25; 119(13):4637-4643 teach examples of polymethine cyanine dyes comprising a brominated or chlorinated methine group (see figure 1, reproduced below for convenience).
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Finally, Steiger “The Reversible Halogenation of Cyanine Dyes” Photographic Science and Engineering Vol 25, No. 1, Jan/Feb 1981 pages 10-20 teach syntheses of monomethine cyanine dyes that are brominated or chlorinated on the monomethine carbon and are subsequently deprotonated, resulting in a substitution similar to the claimed halogenation on the monomethine carbon (Steiger, Figure 1, reproduced below for convenience).
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Steiger teaches successful synthesis and isolation of stable unprotonated meso-halogen derivatives (i.e. the bottom right compound in figure 1) including brominated and chlorinated derivatives, but that “meso-iodo compounds could not be synthesized” (Steiger, page 11, column 2, paragraph 3). Steiger does not appear to have described an attempt to fluorinate the monomethine carbon.
Guidance in the specification
Other than the structures of the claimed genera “Dye 1” and “Dye 2”, the specification provides no guidance as to how a person skilled in the art would synthesize any of the halogenated monomethine cyanine dyes encompassed by the structures of “Dye 1” or “Dye 2”.
Working examples
The specification provides no working examples utilizing any halogenated monomethine cyanine dye, including any dye encompassed by the structures of “Dye 1” or “Dye 2”. Rather, all of the empirical data (i.e. working examples) provided appear to comprise the monomethine cyanine BisQ that does not comprise a halogen substitution at the indicated monomethine carbon.
The (lack of) guidance provided in the specification amounts for an invitation for the skilled artisan to try to apply synthetic steps known in the art to produce halogenated monomethine cyanine dyes recited by claim 63.
Quantity of experimentation
Given the negative findings in the prior art (the report by Steiger) that Iodo-derivatives of monomethine cyanine dyes wherein the monomethine carbon is substituted with an Iodine atom were not possible using the synthetic pathways taught by Steiger, and the lack of other examples in the art of monomethine cyanine dyes that are halogenated at the monomethine carbon, it appears that there is no synthetic pathway described in the art to produce an iodinated or fluorinated members of the claimed genus of monomethine cyanine dyes that are halogenated at the monomethine carbon.
The quantity of experimentation in this area is extremely large since there are a significant number of parameters which would have to be studied to overcome the chemical limitations described by Steiger and the lack of subsequent examples of monomethine cyanine dyes comprising an Iodine- or Fluorine- substituted monomethine carbon.
The lack of known synthetic steps to make the full scope of species within the claimed genus would require significant inventive effort, with each of the many intervening steps, upon effective reduction to practice, not providing any guarantee of success in the succeeding steps.
Level of skill in the art
The level of skill in the art is deemed to be high.
Conclusion
Given the broad claim to a genus of halogenated monomethine cyanine dyes wherein the halogenated carbon is the monomethine carbon in an art whose nature is identified as unpredictable, the lack of guidance in the specification, the absence of working examples in the present disclosure, the large quantity of research required to define the unpredictable variables underlying the apparently undisclosed synthesis of these members of the claimed genus and the negative teachings in the prior art balanced only against the high level of skill in the art, it is the position of the examiner that it would require undue experimentation for one of skill in the art to make the invention of the claim as broadly written.
Claim Rejections - 35 USC § 112(b)- Indefiniteness
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 61-64 and 66-71 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.
The term “in proximity to” in claims 61 and 62 is a relative term which renders the claims indefinite. The term “proximity” is not defined by the claims, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention.
The specification and dependent claims provide examples and particular embodiments wherein cyclopentyl groups are present at particular positions in the structures of exemplary PNAs. However, the broader claim language “one or more cyclopentyl groups are provided in proximity to said surrogate base” is not limited to any of these specific embodiments. Rather, the claim language encompasses PNAs comprising a surrogate base at any distance (i.e. having any “proximity” to the surrogate base) including embodiments wherein the cyclopentyl group is a constituent of the surrogate base or is directly linked to the surrogate base (i.e. distance = 0), or wherein the cyclopentyl group is a constituent of another PNA monomer in the PNA polymer chain separated from the PNA monomer having the surrogate base by an arbitrary number of PNA monomer units (i.e. the upper bound of the distance in primary PNA sequence space encompassed by “proximity” is not limited). Furthermore, the “proximity” of the cyclopentyl group(s) is not limited to the primary structure of the claimed PNA molecule (i.e. the linear sequence of PNA subunits). The recited relative term “proximity” further includes a distance between a cyclopentyl group and a surrogate base separated in three dimensional space in the context of higher-order structures (e.g. hairpins, loops, as a constituent of a flexible 5’ or 3’ spacer group, etc.). As such, it is unclear what structural limitations the term “in proximity to” is intended to require of the claimed products.
Claim 65 is 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 65 recites “The PNA according to claim 64, wherein the c[yclo]p[entyl] group is positioned on the PNA backbone between the position of the surrogate base and a position of the next nucleotide base, across a nucleotide base or on the PNA unit carrying the surrogate base.”
The specification (and claim 69) describe and recite particular embodiments wherein the cyclopentyl group comprises the β and γ carbons of the PNA backbone (see “Scheme 1” with annotated backbone carbons α β γ for clarity below).
The specification provides the following description of the structures depicted in Scheme 1: “In some embodiments, the cp group is positioned on the PNA backbone between the position of the surrogate base and the position of the next nucleotide base (as in Position A in Scheme 1), across a nucleotide base (as shown in position C in scheme 1) or on the FIT-PNA unit carrying the surrogate base (as in Position B in Scheme 1).” (page 6)
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However, the claim language in claim 65 “between”, “across” and “on the PNA unit” is not clearly limited by the examples as shown in in the specification. It is unclear whether the claim is intended to be limited by the language and exemplary structures in the specification, or if the claim further encompasses alternative structures wherein the cyclopentyl group comprises only one of the backbone carbons (see example 1 below, corresponding to “Position A” in Scheme 1 (1,3=next PNA unit in chain; 2=BASE; 4=BisQ)), or wherein the cyclopentyl group is “on the PNA backbone” as a “pendant” group (i.e. as exemplified by the second example below, corresponding to “Position A” in Scheme 1).
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Claim 71 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 71, which depends from claim 61, recites alternatives “(v)” and “(vi)” wherein: “The PNA according to claim 61…compris[es]… [a] c[yclo]p[entyl]-modified PNA unit, said unit being a BisQ-bearing unit; the PNA further comprising an oxetane-modified PNA base…”. Neither the claims nor the specification provide a structural definition of a “PNA base” nor an “oxetane-modified PNA base”. It is unclear whether an “oxetane-modified PNA base” refers to a structure:
a) wherein the PNA backbone of the PNA unit bearing the BisQ surrogate base comprises an oxetane modification in addition to the surrogate base on a backbone carbon that is not the backbone carbon to which the surrogate base is linked (i.e. “an oxetane-modified PNA unit”)(as required by claim 70 and as exemplified by Scheme 5 in the specification, annotated backbone carbons α β γ for clarity) AND that is not a backbone carbon that is part of the cyclopentyl ring (e.g. as in the second structure below, annotated backbone carbons α β γ for clarity);
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b) wherein any PNA unit in the PNA polymer other than the PNA unit bearing the BisQ comprises an oxetane modification at any position on the PNA backbone;
c) wherein any PNA unit in the PNA polymer other than the PNA unit bearing the BisQ comprises an oxetane modification at any position on a nucleotide base that is attached to the PNA backbone;
d) wherein the surrogate base comprises an oxetane modification; or
e) something else
Claims 63, 64, and 66-71 are rendered indefinite because of their dependence from, and inclusion of the indefinite limitation(s) of claim 61.
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.
The following rejections under 35 U.S.C. 103 are being made over the scope of the claims identified as being enabled by the specification and prior art in the Scope of Enablement rejection under 35 U.S.C. 112(a) above.
Claims 61-65 and 71 are rejected under 35 U.S.C. 103 as being unpatentable over Peled et al., “Predictive Model for the Sequence-Dependent Fluorogenic Response of Forced-Intercalation Peptide Nucleic Acid”. ACS omega, 3(4), 3813-3818 (2018) (NPL citation number 9 on IDS filed September 28, 2023) in view of Pokorski et al., “(S,S)-trans-Cyclopentane-Constrained Peptide Nucleic Acids. A General Backbone Modification that Improves Binding Affinity and Sequence Specificity” Journal of the American Chemical Society, 126(46), 15067-15073 (2004) (NPL citation number 7 on IDS filed September 28, 2023).
Regarding claims 61 and 62, Peled et al. teach forced-intercalation “FIT” aminoethylglycine peptide nucleic acid “aegPNA” probes comprising an aegPNA backbone and a plurality of pendant nucleobases (e.g. “A”,”T”,”C”,”G”), wherein at least one of the pendant nucleobases is replaced with a “surrogate base” that is the monomethine cyanine dye bisquinoline “BisQ” (Peled et al., Scheme 1, see below and page 3815, column 2, paragraphs 2-3).
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Peled et al. further teach “a simple thermodynamic-based model that allows predicting the mismatch discrimination of FIT-PNAs…allow[ing] a simple means to design highly fluorogenic FIT-PNAs with superior mismatch discrimination” (Peled et al., page 3817, column 1, paragraph 2).
Peled et al. teach positioning the surrogate base at a specific number of PNA units away from a SNP-discriminating mismatch in the PNA relative to a fully matched control sequence, “If” results in predictable variation in the fluorescence of the FIT-PNA probes “I/If” that correlates with the well-known “nearest-neighbor interaction” factor in calculating the stability of duplex formation between two polynucleotide sequences (i.e. Tm, “melting temperature”) (Peled et al., Table 3, reproduced below, and page 3816-3817 bridging paragraph). Note in table 3, a value of SNP duplex fluorescence (I)/fully-matched control fluorescence (If) approaching 1.0 means that the PNA probe binds equally well to the control sequence as to the sequence containing the SNP (i.e. the probe does not discriminate well between the two sequences).
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Peled et al. demonstrate that “a mismatch in a PNA:DNA duplex causes the unstacking of the base step situated 5′ to 3′ from the DNA mismatched base. In the current binding model, an “R”-positioned mismatch will cause the unstacking of one of the two base steps which create the BisQ external quinoline binding groove- - restricting its rotational freedom. In contrast, the “L”-positioned mismatch will cause an unstacking of the base step further away from the DNA base triad opposite BisQ, resulting in a higher rotational freedom. This model can then explain this directional bias” (Peled et al., page 3815, column 2, paragraph 4).
Peled et al. do not teach that the PNA backbone comprises one or more cyclopentyl groups in proximity to the surrogate base.
However, Pokorski et al. teach PNA probes wherein one or more of the PNA units comprise a cyclopentane modification to the PNA backbone for improved detection of single nucleotide polymorphisms in target nucleic acids relative to DNA probes and relative to aminoethylglycine (aeg)PNA probes. Pokorski et al. teach replacing a portion of the aegPNA backbone (specifically the β-γ carbon linkage) with a cyclopropyl group significantly increases binding affinity and sequence specificity (i.e. SNP discrimination) to complementary DNA (Pokorski et al., abstract).
Furthermore, Pokorski et al. teach positioning the PNA unit comprising the cyclopentyl group within the PNA polymer such that the residue comprising the cyclopentyl group is in close proximity (distance in primary sequence space from 0-4 PNA units) to a single nucleotide mismatch (Pokorski et al., tables 2 and 4).
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As a proof of concept, Pokorski et al. compare the Tm observed for the hybridization of a known polymorphic site in the human gene encoding p53 to PNA probes specific for the SNP, wherein probe “11” is an all-aegPNA sequence and probe “12” comprises a single cyclopropyl PNA unit at the site of the SNP. Pokorski et al. report a striking increase in Tm (i.e. stability of the base-pairing interaction) of 8.0°C (Pokorski et al., table 2).
Pokorski et al. conclude that incorporation of one or more cyclopentyl group into PNA represents a standard backbone modification relative to all-aegPNA that allow one to tune the oligomers’ DNA binding properties by calculating the number of cyclopentanes required to fine-tune the binding properties of the PNA to a complementary nucleic acid sequence (Pokorski et al., page 15073, column 2, paragraph 2).
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 have modified the FIT-PNAs comprising a monomethine dye surrogate base (i.e. BisQ) at a site complementary to a particular polymorphic nucleotide within a given sequence of interest, taught by Peled et al., to comprise a cyclopentyl backbone modification at the site of the polymorphic nucleotide, as taught by Pokorski et al. (i.e. in proximity to the surrogate base) because both Pokorski et al. and Peled et al. teach inclusion of these PNA modifications are beneficial for detection of nucleic acid sequences of interest. The ordinary artisan would have been motivated to modify the FIT-PNAs taught by Peled et al. with the cyclopentyl groups taught by Pokorski et al. because of the teaching of Pokorski et al. that cyclopentyl groups provide the predictable advantage of increasing the binding affinity of PNA for complementary nucleic acids and increases specificity in mismatch discrimination compared to unmodified PNA probes (Pokorski et al., table 4 and page 15072, column 2, paragraph 2). The ordinary artisan would therefore have been reasonably confident that inclusion of the cyclopentyl group modification at a residue complementary to a SNP of interest in the SNP-specific FIT-PNA probes comprising a monomethine surrogate base, wherein the surrogate base is complementary to a SNP of interest would have predictably yielded the advantages (increased specificity, stability) taught by Pokorski et al. and Peled et al.
Regarding claim 63, Peled et al. teach the monomethine dye is a cyanine dye being BisQ.
Regarding claims 64 and 65, Pokorski et al. teach exemplary PNA probes comprising only one cyclopropyl-modified PNA unit that is complementary to a SNP and Peled et al. teach several forced-intercalation PNA probes wherein the PNA monomer comprising the BisQ surrogate base is positioned directly adjacent to a PNA monomer that is complementary to a SNP (Peled et al., table 2 and page 3815, column 2, paragraph 4) (i.e. the cyclopropyl-modified PNA unit is complementary to a SNP, as taught by Pokorski et al., and the PNA unit comprising the BisQ surrogate base is directly adjacent to the SNP-discriminating cpPNA unit).
Regarding claim 71, Pokorski et al. further teach that the increased stability of PNA:DNA hybridization due to substitution of one aegPNA unit with a cpPNA unit (in particular at the site of a SNP) is further increased by further substitutions of neighboring aegPNA units with cpPNA units (see, for example, “entry” 2, 5, and 8 in Pokorski et al., Table 2). In these examples, inclusion of a single cpPNA unit at a particular site increased the Tm (i.e. stability) of a complementary PNA:DNA duplex by 6.0°C relative to an all-aegPNA control sequence. Further modification of either of the aegPNA units adjacent to the first cpPNA unit with a second (or third) cpPNA unit increased the Tm relative to the all aegPNA control by 11.3 or 15.5 °C, respectively.
Therefore, given the teachings of Peled et al. and Pokorski et al., it would likewise have been obvious to modify the aegPNA unit(s) comprising the BisQ surrogate base adjacent to the SNP with cpPNA unit(s) comprising the BisQ surrogate base.
Claim 61 is alternatively rejected over Peled et al. in view of Pokorski et al., as applied to claims 61-65 and 71 above, and further in view of Yavin et al., WO 2017/025968 A1 and Steiger “The Reversible Halogenation of Cyanine Dyes” Photographic Science and Engineering Vol 25, No. 1, Jan/Feb 1981 pages 10-20.
This rejection addresses the alternative structures “Dye 1” or “Dye 2” recited by claim 61.
Regarding claim 61, as described above, Peled et al. in view of Pokorski et al. teach forced intercalation PNA probes comprising at least one surrogate base that is a monomethine dye, and wherein one or more of the PNA units comprises a modification to the standard aminoethylglycine PNA backbone wherein a cyclopropyl group comprises the β-γ carbon linkage (i.e. cpPNA).
Peled et al. in view of Pokorski et al. further teach the monomethine dye is “BisQ” (Peled et al., Scheme 1, see below and page 3815, column 2, paragraphs 2-3).
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However, Peled et al. in view of Pokorski et al. do not teach the meso-halogenated derivative of the monomethine cyanine dye “BisQ” recited as an alternative in claim 63 as “Dye 2”.
Furthermore, Peled et al., in view of Pokorski et al. do not teach the meso-halogenated monomethine cyanine dye “Dye 1”.
However, Yavin et al. teach various “long wavelength emitting probes” (LWEP) including the monomethine cyanine dyes from which the meso-halogenated “Dye 1” and “Dye 2” are derived. Yavin et al. further teaches that these LWEPs may expressly be conjugated to a PNA backbone (Yavin et al., Scheme 1, reproduced below, “Dye 1” corresponds to a derivative of Structure B; “Dye 2” corresponds to a derivative of Structure A).
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Yavin et al. teach long-wavelength emission (far red to near infrared) of the LWEP dyes relative to their parent dyes are particularly beneficial for applications involving determining deep- or live- tissue imaging such as during surgical removal of a tumor comprising staining the outer perimeter of the excised tissue with a cancer-specific PNA probe comprising the LWEP dye to determine whether the tumor has been completely removed during the surgery (Yavin et al., page 18, paragraph 4).
Yavin et al. do not further teach that the hydrogen on the monomethine carbon is substituted with a halogen.
However, Steiger et al. teach syntheses of monomethine cyanine dyes that are brominated or chlorinated on the monomethine carbon and are subsequently deprotonated, resulting in a substitution similar to the claimed halogenation on the monomethine carbon (Steiger, Figure 1, reproduced below for convenience).
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Steiger teaches successful synthesis and isolation of stable unprotonated meso-halogen derivatives (i.e. the bottom right compound in figure 1) including brominated and chlorinated derivatives, but that “meso-iodo compounds could not be synthesized” (Steiger, page 11, column 2, paragraph 3). Steiger does not appear to have described an attempt to fluorinate the monomethine carbon.
Finally, Steiger teaches that halogenation of an exemplary monomethine cyanine dye at the monomethine carbon greatly increased the wavelength (by approximately 100 nm) at which the halogenated dye absorbs light relative to the non-halogenated monomethine cyanine dye (Steiger et al., Figure 4).
Therefore, it would have been prima facie obvious prior to the effective filing date for one of ordinary skill in the art to have modified the cyclopentyl-modified PNAs comprising monomethine surrogate base(s), taught by Peled et al. in view of Pokorski et al. with the teachings of Yavin et al. comprising the particular long-wavelength monomethine cyanine dyes “LWEP” that are analogous to the claimed structures “Dye 1” and “Dye 2” (Yavin et al., see above) and Steiger et al. comprising synthetic mechanisms for preparing halogenated derivatives of monomethine cyanine dyes wherein the hydrogen of the monomethine carbon has been substituted with a halogen selected from Chlorine and Bromine. The ordinary artisan would have been motivated to substitute the monomethine cyanine surrogate base LWEP dyes taught by Yavin et al. into the PNAs taught by Peled et al. in view of Pokorski et al. in place of the monomethine cyanine surrogate base “BisQ” because of the teaching of Yavin et al. that longer wavelength dyes are beneficial for detecting nucleic acid sequences indicative of disease (such as particular tumors) in live tissues (such as during surgical resection of said tumor). Furthermore, the ordinary artisan would have been motivated to halogenate the monomethine cyanine surrogate base LWEP dyes at the monomethine carbon as taught by Steiger because of the teaching of Steiger that halogenation at the monomethine carbon greatly increases the wavelength at which a monomethine cyanine dye absorbs (and thus emits) light.
Claims 61, 64, and 66 are rejected under 35 U.S.C. 103 as being unpatentable over Sato et al., “Design of Fluorescent Peptide Nucleic Acid Probes Carrying Cyanine Dyes for Targeting Double-Stranded RNAs for Analytical Applications” Bull. Chem. Soc. Jpn. 2020, 93, 406-413 (published January 24, 2020) in view of Pokorski et al., “(S,S)-trans-Cyclopentane-Constrained Peptide Nucleic Acids. A General Backbone Modification that Improves Binding Affinity and Sequence Specificity” Journal of the American Chemical Society, 126(46), 15067-15073 (2004) (NPL citation number 7 on IDS filed September 28, 2023).
Regarding claims 61, 64, and 66, Sato et al. teach off-on fluorescent PNA probes for detecting the 3’ overhang structure in siRNAs wherein an aegPNA comprising an N-terminal (i.e. analogous to the 5’ end of a DNA molecule) surrogate base modification comprising a monomethine cyanine dye (Sato et al., Figure 2). Sato et al. further teach the “light up” property of the PNA probe is due to restriction of the rotation between the two heterocycles in the monomethine dye about the monomethine carbon due to intercalation of the probe into the target siRNA (Sato et al., page 408, column 1, paragraphs 1-2). Sato et al. further teach that the base surrogate cyanine dye may be “QB” (i.e. “BisQ”, see Sato et al., figure 8).
Sato et al. do not teach that the PNA probe comprises a cyclopentyl group in proximity to the surrogate base.
However, Pokorski et al. teach PNA probes wherein one or more of the PNA units comprise a cyclopentane modification to the PNA backbone for improved detection of single nucleotide polymorphisms in target nucleic acids relative to DNA probes and relative to aminoethylglycine (aeg)PNA probes. Pokorski et al. teach replacing a portion of the aegPNA backbone (specifically the β-γ carbon linkage) with a cyclopropyl group significantly increases binding affinity and sequence specificity (i.e. SNP discrimination) to complementary DNA (Pokorski et al., abstract).
Furthermore, Pokorski et al. teach positioning the PNA unit comprising the cyclopentyl group within the PNA polymer such that the residue comprising the cyclopentyl group is in close proximity (distance in primary sequence space from 0-4 PNA units) to a single nucleotide mismatch (i.e. a target nucleotide) (Pokorski et al., tables 2 and 4).
Furthermore, Pokorski et al. teach that “replacing the ethylenediamine portion of a PNA with a carbocyclic ring (i.e. the cyclopentane ring) significantly reduces the conformational flexibility of an unbound PNA, lowering the entropic cost associated with formation of a PNA-DNA complex (Pokorski et al., page 15073, column 1).
Pokorski et al. conclude that incorporation of one or more cyclopentyl group into PNA represents a standard backbone modification relative to all-aegPNA that allow one to tune the oligomers’ DNA binding properties by calculating the number of cyclopentanes required to fine-tune the binding properties of the PNA to a complementary nucleic acid sequence (Pokorski et al., page 15073, column 2, paragraph 2).
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 have modified the N-terminal-modified, cyanine dye-labeled (i.e. “5’-modified”) aegPNA probe for the 3’ overhang of specific siRNA target sequences, taught by Sato et al., with the teachings of Pokorski et al. that introducing at least one cpPNA unit at and/or near the site of a discriminating nucleotide in a PNA probe increases the Tm (i.e. stability) of a PNA for its complementary nucleic acid target sequence by reducing the conformational flexibility of the PNA. The ordinary artisan would have been motivated to modify the N-terminal cyanine labeled aegPNA probe taught by Sato et al. with a cpPNA unit at the site of the PNA unit carrying the terminal cyanine dye because of the teachings of Sato et al. and Pokorski et al. that increasing the conformational constraint in the PNA probe at the site of the intercalating cyanine dye results in more stable binding of the PNA to the target sequence and results in increase in cyanine dye fluorescence due to the restriction of nonradiative energy loss by rotation of the PNA/dye unit in space.
Claims 67-69 are rejected under 35 U.S.C. 103 as being unpatentable over Peled et al. in view of Pokorski et al., as applied to claims 61-65 and 71 above, and further in view of Hibino et al., “Cationic Guanine: positively charged nucleobase with improved DNA affinity inhibits self-duplex formation” Chem. Commun., 2020, 56, 2546 (published February 3, 2020).
Regarding claim 61, as described above, Peled et al. in view of Pokorski et al. teach forced intercalation PNA probes comprising at least one surrogate base that is a monomethine dye, and wherein one or more of the PNA units comprises a modification to the standard aminoethylglycine PNA backbone wherein a cyclopropyl group comprises the β-γ carbon linkage (i.e. cpPNA).
Regarding claim 67, Peled et al. in view of Pokorski et al. do not teach that the PNA further comprises a charged guanine or charged adenine nucleobase.
However, Hibino et al. teach inclusion of a cationic 7-methylguanine nucleobase in a PNA probe destabilizes PNA:PNA duplex (i.e. self-hybridization of the probe) while simultaneously stabilizing the interaction of a PNA probe for a DNA target sequence (Hibino et al., Figure 2 and page 2547, column 2, paragraph 3).
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 have modified the PNA forced intercalation probes comprising one or more monomethine surrogate bases in proximity to cpPNA backbone modifications, taught by Peled et al. in view of Pokorski et al., with the teachings of Hibino et al. that inclusion of a cationic guanine nucleobase into a PNA probe prevents self-hybridization of the probe and increases the affinity of a PNA probe for a complementary DNA sequence (Hibino et al., Figure 2 and page 2547, column 2, paragraph 3). The ordinary artisan would have been motivated to include a cationic guanine nucleobase in the cpPNA-FIT probes taught by Peled et al. in view of Pokorski et al. with a reasonable expectation of success in attaining the predictable benefits of decreased self-hybridization of PNA probe and increased affinity of the PNA probe for its target nucleic acid sequence because of the express teachings of Hibino: “G+ PNA not only improved DNA affinity but also strongly suppressed self-duplex formation, indicating that G+ PNA meets the requirements set for self-avoiding PNA. A similar self-avoiding tendency was observed irrespective of the position of G+.” (Hibino et al., page 2547, column 2, paragraph 3).
Regarding claim 68, Hibino et al. teach the charged guanine nucleobase is an N7-methylguanine nucleobase (i.e. an alkylation product of a free nucleobase G… (Hibino et al., Figure 1 b).
Regarding claim 69, Hibino et al. teach a PNA comprising the charged nucleobase has the structure:
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Wherein Z is an alkyl having 1 carbon atom (i.e. N7-methyl Guanine).
Claim 70 is rejected under 35 U.S.C. 103 as being unpatentable over Peled et al. in view of Pokorski et al., as applied to claims 61-65 and 71 above, and further in view of Wuitschik et al., “Oxetanes as Promising Modules in Drug Discovery” Angew. Chem. Int. Ed. 2006, 45, 7736-7739 and Roesner et al., “Development of oxetane modified building blocks for peptide synthesis” Org. Biomol. Chem., 2020, 18, 5400 (published June 29, 2020).
Regarding claim 61, as described above, Peled et al. in view of Pokorski et al. teach forced intercalation PNA probes comprising at least one surrogate base that is a monomethine dye, and wherein one or more of the PNA units comprises a modification to the standard aminoethylglycine PNA backbone wherein a cyclopropyl group comprises the β-γ carbon linkage (i.e. cpPNA).
Regarding claim 70, Peled et al. in view of Pokorski et al. do not teach the PNA comprising at least one monomethine dye surrogate base in proximity to a cyclopentyl group further comprises a PNA unit of the structure recited in claim 70, wherein a PNA unit comprising a BisQ surrogate base further comprises an oxetane-ring modification on the β carbon of the PNA backbone:
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However, Wuitschik et al. teach generating a series of molecules from a precursor comprising an “oxetane scan” revealing that inclusion of an oxetane ring one or two carbons away from an amino group allows for tuning the basicity of the amino group and increases solubility of the examined compounds (Wuitschik et al., page 7738, column 2, paragraphs 3-4)
Furthermore, Roesner et al. demonstrate facile incorporation of oxetane rings 2 carbons away from the amino terminal group into the peptide backbone analogous to the aegPNA backbone present in the claimed structure (Roesner et al., Figure 1).
Pokorski et al. teach that incorporation of cyclopentane groups into a PNA polymer is expected to increase the hydrophobicity, and thus reduce the aqueous solubility of the PNA polymer (Pokorski et al., page 15071, column 1-2 bridging paragraph).
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 have modified the cyclopentyl-modified PNAs comprising at least one cpPNA backbone modification and at least one monomethine dye surrogate base in proximity to the cpPNA backbone modification taught by Peled et al. in view of Pokorski et al. with the teachings of Wuitschik et al. and Roesner et al. that incorporation of hydrophobic groups (i.e. the cyclopropyl ring and the planar, highly aromatic, BisQ surrogate base) into a PNA polymer would have been expected to decrease the aqueous solubility of the PNA polymer, and that incorporation of an oxetane ring at a position on a peptide backbone would have predictably increased the solubility of the PNA polymer.
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
McLaughlin et al., “Oxetanyl Peptides: Novel Peptidomimetic Modules for Medicinal Chemistry” Org. Lett. 2014, 16, 4070-4073
Powell et al., “Synthesis and structure of oxetane containing tripeptide motifs” Chem. Commun., 2014, 50, 8797
Nanjunda et al., “Halogenated pentamethine cyanine dyes exhibiting high fidelity for G-quadruplex DNA” Bioorganic and Medicinal Chemistry 20 (2012) 7002-7011
Montazersaheb et al., “Potential of Peptide Nucleic Acids in Future Therapeutic Applications” Adv Pharm Bull, 2018, 8(4), 551-563
Bull et al., “Oxetanes: Recent Advances in Synthesis, Reactivity, and Medicinal Chemistry” Chem. Rev. 2016, 116, 12150-12233
Beadle et al., “Solid-Phase Synthesis of Oxetane Modified Peptides” Org. Lett. 2017, 19, 3303-3306
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
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