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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 8/19/2025 has been entered.
Claim Status and Formal Matters
Claims 1-2, 4-5, 7, 10-11, 13-16, 18, 21-22, 24-25 and 28 are pending.
Claims 4-5, 10-11, 13-14, 16 have been amended.
The instant response is not compliant with 37 CFR 1.121 as claims 4 and 10 have been amended, but are indicated as original. Further claims 9 is identified as previously presented, but has been amended. In order to promote compact prosecution and customer service the instant response will be examined. However future amendments which are non-compliant with 37 CFR 1.121 may not be entered or examined.
Applicant’s election without traverse of group 2, in the reply filed on least 60 percent of the nucleotides are guanine nucleotides; SEQ ID NO: 26; iCy3 and a shift in fluorescence wavelength. is acknowledged.
Claims 1-2, 16-18, 21-22, 24-25 and 28 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 6/24/2024.
Claims 4-5, 7, 9-11, 13-15 are being examined.
The sequence compliance issues have been withdrawn in view of the amended specification filed 1/7/2025.
The previous objection to the claims and specification have been withdrawn in view of amendment.
Priority
The instant application was filed 03/22/2021 and is a national stage entry of PCT/IB2019/058058 with an international filing date: 09/23/2019 and claims priority from provisional application 62734694 , filed 09/21/2018 and claims priority from provisional application 62774648 , filed 12/03/2018.
Information Disclosure Statement
The listing of references in the specification 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.
Claim Rejections - 35 USC § 112
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 4-5, 7, 9-11, 13-15 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.
As set forth in In re Alonso 88 USPQ2d 1849 (Fed. Cir. 2008), at 1851:
The written description requirement of 35 U.S.C. § 112, ¶ 1, is straightforward: “The specification shall contain a written description of the invention ….” To satisfy this requirement, the specification must describe the invention in sufficient detail so “that one skilled in the art can clearly conclude that the inventor invented the claimed invention as of the filing date sought.” Lockwood v. Am. Airlines, Inc., 107 F.3d 1565, 1572 [41 USPQ2d 1961] (Fed. Cir. 1997); see also LizardTech, Inc. v. Earth Res. Mapping, Inc., 424 F.3d 1336, 1345 [76 USPQ2d 1724] (Fed. Cir. 2005); Eiselstein v. Frank, 52 F.3d 1035, 1039 [34 USPQ2d 1467] (Fed. Cir. 1995).
Alonso at 1852:
A genus can be described by disclosing: (1) a representative number of species in that genus; or (2) its “relevant identifying characteristics,” such as “complete or partial structure, other physical and/or chemical properties, functional characteristics when coupled with a known or disclosed correlation between function and structure, or some combination of such characteristics.” Enzo, 323 F.3d at 964.
In applying the test as set forth in Alonso, it is noted that applicant is claiming A reporter-containing oligonucleotide, comprising (a) a nucleotide sequence selected from the group consisting of: AGGAGGGACGG/X/GGCAGGAGGAG (SEQ ID NO: 26);GGAGGGACGG/X/GGCAGGAGGAG (SEQ ID NO: 27);AGGAGGGACGG/X/GGCAGGAGGA (SEQ ID NO: 28);GAGGGACGG/X/GGCAGGAGGAG (SEQ ID NO: 29);GGAGGGACGG/X/GGCAGGAGGA (SEQ ID NO: 30);AGGAGGGACGG/X/GGCAGGAGG (SEQ ID NO: 31);AGGGACGG/X/GGCAGGAGGAG (SEQ ID NO: 32); GAGGGACGG/X/GGCAGGAGGAG (SEQ ID NO: 33);GGAGGGACGG/X/GGCAGGAGG (SEQ ID NO: 34);AGGAGGGACGG/X/GGCAGGAG (SEQ ID NO: 35);GGGACGG/X/GGCAGGAGGAG (SEQ ID NO: 36);AGGGACGG/X/GGCAGGAGGA (SEQ ID NO: 37);GAGGGACGG/X/GGCAGGAGG (SEQ ID NO: 38);GGAGGGACGG/X/GGCAGGAG (SEQ ID NO: 39);AGGAGGGACGG/X/GGCAGGA (SEQ ID NO: 40);GGGACGG/X/GGCAGGAGGA (SEQ ID NO: 41);AGGGACGG/X/GGCAGGAGG (SEQ ID NO: 42);GAGGGACGG/X/GGCAGGAG (SEQ ID NO: 43);GGAGGGACGG/X/GGCAGGA (SEQ ID NO: 44);GGGACGG/X/GGCAGGAGG (SEQ ID NO: 45);AGGGACGG/X/GGCAGGAG (SEQ ID NO: 46);GAGGGACGG/X/GGCAGGA (SEQ ID NO: 47);GGGACGG/X/GGCAGGAG (SEQ ID NO: 48);AGGGACGG/X/GGCAGGA (SEQ ID NO: 49);GGGACGG/X/GGCAGGA (SEQ ID NO: 50); wherein X is a fluorophore as a built-in reporter, wherein the fluorophore is covalently incorporated in the sugar- phosphate backbone of the oligonucleotide and is an environmentally sensitive fluorophore comprising a polymethine bond, wherein reporter-containing oligonucleotide has100 nucleotides or less.
.Thus the claim encompass any nucleic acid sequence of any length less than 100 nucleotides comprise the recited sequences with any fluorophore comprising a polymethine bond as a built in reporter, which is incorporated by any means into the sugar phosphate background. Further the clams requires, “and is an environmentally sensitive fluorophore comprising a polymethine bond.” The claims encompass environmental sensitivity relative to any concentration of any environmental ion or metal in view of the dependent claims. This is an enormous genus of anything from 14 nucleotides to a 100 nucleotides.
It is unclear if the recitation of “wherein the fluorophore is covalently incorporated in the sugar- phosphate backbone of the oligonucleotide and is an environmentally sensitive fluorophore comprising a polymethine bond” requires the label to be at the X position or encompasses any position along the sugar phosphate backbone including the 3’ or 5’ terminus. The specification on page 3 states:
AGGAGGGACGG/X/GGCAGGAGGAG SEQ ID NO. 26 GAGGGACGG/X/GGCAGGAGG SEQ ID NO. 38
where X represents a fluorescence dye as the built-in reporter. In a preferred embodiment, the built-in reporter is incorporated into 5 the sugar-phosphate backbone of the aptamers. The built-in reporter may be an environmentally sensitive fluorescence dye, internally coupled to the aptamers. Exemplary environmentally sensitive dyes include cyanine dyes, such as Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Alexa 555, Alexa 647, and derivatives thereof.
The specification teaches, “The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. For example, reference to "a 10 compound" includes a plurality of compounds and reference to "the compound" is a reference to one or more compounds and equivalents thereof known to those skilled in the art.” (page 9)
The specification states, “0048] “Environmentally sensitive fluorescence dye” refers to fluorescence dyes having photophysical properties that are responsive to physicochemical changes in the local environment including pH, viscosity, biological or non-biological analytes, and solvent polarity. The photophysical properties may include the fluorescence wavelength, the fluorescence intensity, fluorescence life time, and combinations thereof. For example, the fluorescence intensities of certain dyes may be highly sensitive to the polarity of their local environments; their fluorescence signals may be more intense and blue-shifted when they are in a less polar environment.” Thus the specification appears to be a relative term which is dependent on local environment including pH, viscosity, biological or non-biological analytes, and solvent polarity and thus does not appear to be an inherent property of the fluorophore and thus the metes and bounds are unclear.
The specification teaches, “"Reporter" or "sensor" as used herein refers to a self-contained analytical device that can provide information about the chemical composition of its environment. For example, it can be a chemical moiety that detects and/or measures a change in the physical property of the environment, the parent molecule, or the overall system, and records, indicates, or otherwise responds to it.” (Page 11). Thus the claims require the reporter can provide information about any chemical in the environment. This is an enormous genus. While the claims comprise the recited sequence, there is no evidence the recited sequences will provide information about any compound, known or unknown.
The specification on pages 12-13 provide potassium ion sensing aptamers, SEQ ID NO 1-25.
The specification on page 26 asserts, “In the presence of 50 mM KCl, the O-328 (22) oligonucleotide containing an internal iCy3 (sequence: AGGAGGGACGG/iCy3/GGCAGGAGGAG) (SEQ ID NO:107) exhibited the longest fluorescence lifetime among the 46 DNA oligonucleotides labeled with iCy3 (Figure 2). Thus, it is evident that the sequence of O-328 is highly specific for K⁺.” Thus th
Dependent claim 5 has been amended to require, “wherein the ligand is a metal ion, optionally selected from the group consisting of potassium, lithium, magnesium, manganese, calcium, cesium, zinc, sodium, potassium, and strontium, or a non-metal cation, optionally an ammonium..” Thus this is a functional limitation. This dependent claims encompass any metal ion, this is an enormous genus further even the Markush group listed provides for 12 distinct species (lithium, magnesium, manganese, calcium, cesium, zinc, sodium, potassium, and strontium, or ammonium) as well as the genus of any non-metal cation.
Claim 7 recites, “comprising between 14 and 22, inclusive, nucleotides and/or wherein the oligonucleotide is a DNA oligonucleotide Thus the claims encompass a DNA oligonucleotide comprising the recited sequences of up to 100 nucleotides in length. Further in view of the comprising (having) language the claims encompass the recited SEQ ID NO to 100 nucleotides.
Claim 10 depends from claim 9 and recites, “wherein the environmentally sensitive fluorophore is a cyanineluorophore.” Thus the claim has same issue as claims from which it depends.
Claim 11 depends from claim 10 and thus has the same issues.
Claim 13 depends from claim 4 and has been amended to require, “wherein the oligonucleotide undergoes a conformational change upon binding to a ligand of the oligonucleotide, wherein the conformational change of the oligonucleotide induces a photophysical change of the fluorophore flurophore, wherein the ligand is a metal ion, optionally selected from the group consisting of potassium, lithium, magnesium, manganese, calcium, cesium, zinc, sodium, potassium, and strontium, or a non-metal cation, optionally an ammonium..” This appears to require the oligonucleotide or aptamer for environmental sensitivity, furthering the written description issues with any flurophore and environmental sensitive as addressed with claims 10-11. However review of the specification is limited to the detection of potassium, Na+ and NH4+ by an iCy3 attached internally via phosphoramidite chemistry. Thus is a single species of ligand encompassed by a single species of oligonucleotides encompassed by the claim as well as a single fluorescent label.
Claim 14 continues providing functional limitations of, “wherein the oligonucleotide undergoes a conformational change upon binding to a ligand of the oligonucleotide, wherein the conformational change of the oligonucleotide induces a photophysical change of the fluorescence dye,wherein the photophysical change of the fluorescence dye is a shift in the fluorescence wavelength, a change in the fluorescence signal intensity, a change in the fluorescence life time, or combinations thereof.”
Further claim 15 recites, “wherein the fluorophore is fluorescent-quenched prior to binding of the ligand and becomes fluorescent or fluorescent-enhanced upon binding of the ligand.” This provides the function of being fluorescent and then provides the functional limitation of “non-fluorescent or fluorescent-quenched prior to binding of the ligand and becomes fluorescent or fluorescent-enhanced upon binding of the ligand.” However review of the specification is limited to the detection of potassium by an iCy3 attached internally via phosphoramidite chemistry. Thus is a thress species of ligand encompassed by a the limited species of SEQ ID NO 26 to SEQ ID NO 50 species of oligonucleotides encompassed by the claim as well as a single fluorescent label.
Thus the claims lack adequate written description.
Response to Arguments
The response traverses the rejection by asserting, “claim 4 has been amended to specify that the fluorophore is an environmentally sensitive fluorophore containing a polymethine bond. This amendment clarifies that the structure function relationship that differentiates an environmental sensitive from an environmental insensitive fluorophore is the polymethine bond. The fluorescence of the environmental sensitive fluorophore is dependent on the cis-trans photoisomerization rate around the polymethine bond, as illustrated in FIG. 1, and this photoisomerization rate in turn hinges on the overall local environment of the fluorophore. See Specification page 18 lines 8-19. Therefore, the local environment has a direct impact on the photoisomerization rate and henceforth the fluorescence properties of the environmentally sensitive dyes. Based on this disclosure, it is clear that the scope of the environmentally sensitive dyes is not limited to a single species, but in fact encompasses any environmentally sensitive fluorescent compound containing polymethine bonds.” It is noted the rejection has been modified in view of the amendments.
The response continues by asserting, “he Examiner further alleges that the specification is limited to the detection of potassium. Applicant respectfully disagrees. The specification demonstrates the detection of other ions beyond potassium. For example, the fluorescence lifetime of 0-328 (18) increased in the presence of Na+, NH4+, and Sr2+ suggesting that these cations induced and stabilized a DNA secondary structure. The fluorescence lifetimes of the 0-328 (18) sensor in increasing concentrations of Na+ and NH4+ cations were measured. The response curves for these two cations exhibited similar sigmoidal behavior to that of K+ (FIG. 7B).” This argument has been thoroughly reviewed but is not considered persuasive as the independent claims are not limited to the asserted cations. Further dependent claims encompass any metal ion, this is an enormous genus further even the Markush group listed provides for 12 distinct species (lithium, magnesium, manganese, calcium, cesium, zinc, sodium, potassium, and strontium, or ammonium) as well as the genus of any non-metal cation. Thus while the claims encompass any metal ion or any non-metal cation the response alleges support for 5 of the genus of thousands of metal ions and non-metal cations.
The response continues argument any ligand by providing arguments with respect to claims 13 and 14. This argument has been thoroughly reviewed but is not considered persuasive as the independent claims are not limited to the Markus groups recited in these claims. However the independent claim is not limited to these species. Further the species recited encompass any metal ion including those recited and any non-metal cation. Thus this is an enormous genus which is much broader than K+, Na+, NH4+, and Sr2+ argued by the response.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 4-5, 7, 9-11, 13-15 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 4 recites, “A reporter-containing oligonucleotide, comprising (a) a nucleotide sequence selected from the group consisting of: AGGAGGGACGG/X/GGCAGGAGGAG (SEQ ID NO: 26);GGAGGGACGG/X/GGCAGGAGGAG (SEQ ID NO: 27);AGGAGGGACGG/X/GGCAGGAGGA (SEQ ID NO: 28);GAGGGACGG/X/GGCAGGAGGAG (SEQ ID NO: 29);GGAGGGACGG/X/GGCAGGAGGA (SEQ ID NO: 30);AGGAGGGACGG/X/GGCAGGAGG (SEQ ID NO: 31);AGGGACGG/X/GGCAGGAGGAG (SEQ ID NO: 32); GAGGGACGG/X/GGCAGGAGGAG (SEQ ID NO: 33);GGAGGGACGG/X/GGCAGGAGG (SEQ ID NO: 34);AGGAGGGACGG/X/GGCAGGAG (SEQ ID NO: 35);GGGACGG/X/GGCAGGAGGAG (SEQ ID NO: 36);AGGGACGG/X/GGCAGGAGGA (SEQ ID NO: 37);GAGGGACGG/X/GGCAGGAGG (SEQ ID NO: 38);GGAGGGACGG/X/GGCAGGAG (SEQ ID NO: 39);AGGAGGGACGG/X/GGCAGGA (SEQ ID NO: 40);GGGACGG/X/GGCAGGAGGA (SEQ ID NO: 41);AGGGACGG/X/GGCAGGAGG (SEQ ID NO: 42);GAGGGACGG/X/GGCAGGAG (SEQ ID NO: 43);GGAGGGACGG/X/GGCAGGA (SEQ ID NO: 44);GGGACGG/X/GGCAGGAGG (SEQ ID NO: 45);AGGGACGG/X/GGCAGGAG (SEQ ID NO: 46);GAGGGACGG/X/GGCAGGA (SEQ ID NO: 47);GGGACGG/X/GGCAGGAG (SEQ ID NO: 48);AGGGACGG/X/GGCAGGA (SEQ ID NO: 49);GGGACGG/X/GGCAGGA (SEQ ID NO: 50); herein X is a fluorophore as a built-in reporter, wherein the fluorophore is covalently incorporated in the sugar- phosphate backbone of the oligonucleotide and is an environmentally sensitive fluorophore comprising a polymethine bond, wherein reporter-containing oligonucleotide has100 nucleotides or less..” The specification teaches, “The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. For example, reference to "a 10 compound" includes a plurality of compounds and reference to "the compound" is a reference to one or more compounds and equivalents thereof known to those skilled in the art.” (page 9) Thus the metes and bounds are unclear if the fluorophore is at the position of X, “/X/” or any where in the phosphate backbone.
Claim 4 has been amended to recite, “environmentally sensitive fluorophore” The specification states, “0048] “Environmentally sensitive fluorescence dye” refers to fluorescence dyes having photophysical properties that are responsive to physicochemical changes in the local environment including pH, viscosity, biological or non-biological analytes, and solvent polarity. The photophysical properties may include the fluorescence wavelength, the fluorescence intensity, fluorescence life time, and combinations thereof. For example, the fluorescence intensities of certain dyes may be highly sensitive to the polarity of their local environments; their fluorescence signals may be more intense and blue-shifted when they are in a less polar environment.” Thus the specification appears to be a relative term which is dependent on local environment including pH, viscosity, biological or non-biological analytes, and solvent polarity and thus does not appear to be an inherent property of the fluorophore and thus the metes and bounds are unclear.
Claim 10 recites, “wherein the environmentally sensitive fluorophore is a cyaninefluorophore.” The specification teaches , “The cyanine dyes exhibit interesting photophysical properties due to their non-rigid structure with a polymethine bond connecting two nitrogen-containing chemical moieties. The fluorescence of these dyes as quantified by fluorescence intensity, fluorescence lifetime or quantum yield is dependent on the cis-trans photoisomerization rate around the polymethine bond, as illustrated in FIG. 1.” Thus the specification appear to suggest polymethine bond are a limitation or define cyanine dyes. Thus it is unclear what is required of this claim or if this claim further limits the independent claims.
Claim 14 recites, “the fluorescence dye” and depends from claim 4. Claim 4 does not provide antecedent basis for “fluorescence dye.” Thus the metes and bounds are unclear. This rejection can be overcome by providing indefinite articles prior to, “fluorescence dye.”
Claim 15 recites, “the ligand” and depends from claim 4. Claim 4 does provide antecedent basis for “the ligand,” however the amendment to claim 4 has removed ligand from claim 4. Thus the metes and bounds are unclear what ligand, “the ligand” is referencing.
Response to Arguments
The response traverses the previous rejections in view of the amendment. The previous rejections have been withdrawn, however the amendment has introduced numerous issues.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claim(s) 4- 7, 9-15 is/are rejected under 35 U.S.C. 103 as being unpatentable over Kwok ( Trends in Biotechnol., 35(10):997-1013 (2017)), Tucker(Current Pharmaceutical Design, 2012, 18, 2014-2026), Tippana (Nucleic Acids Research, 2014, Vol. 42, No. 12, 8106–8114), Li (Anal. Chem. 2010, 82, 7576–7580), Varizhuk (Mini-Reviews in Medicinal Chemistry, 2016, 16, 1321-1329), Wojczewski (Synlett 1999, No. 10, 1667–1678), Domenyuk (WO2017161357), Bentwich (20070003575), Schierle-Arndt(US 20040118700).
Attention is directed to MPEP 2129 [R-6], Admissions as Prior Art, which states in part:
I. ADMISSIONS BY APPLICANT CONSTITUTE PRIOR ART
A statement by an applicant >in the specification or made< during prosecution identifying the work of another as “prior art” is an admission **>which can be relied upon for both anticipation and obviousness determinations, regardless of whether the admitted prior art would otherwise qualify as prior art under the statutory categories of 35 U.S.C. 102. Riverwood Int’l Corp. v. R.A. Jones & Co., 324 F.3d 1346, 1354, 66 USPQ2d 1331, 1337 (Fed. Cir. 2003); Constant v. Advanced Micro-Devices Inc., 848 F.2d 1560, 1570, 7 USPQ2d 1057, 1063 (Fed. Cir.1988).
Further, the courts have held information that an applicant admits is in the prior art "may be considered 'prior art' for any purpose, including use as evidence of obviousness under § 103." In re Nomiya, 509 F.2d 566, 570-71 (CCPA 1975).
The specification on page 15 states:
Additional sequences which can be used to make ligand sensing aptamers with a built-in reporter are the sequences of G-quadruplex forming aptamers, which are known in the art. DNA G-quadruplex forming aptamers are reviewed in Tucker, et al, Current Pharmaceutical Design, 2012, 18, 2014-2026. Exemplary sequences and their targets are listed in Table 1 below. See also Kwok, et al., Trends in Biotechnol., 35(10):997-1013 (2017).
Kwok provides a review of G-Quadruplexes: Prediction, Characterization,
And Biological Application (title);. Kwok teaches, “Besides proteins and enzymes, specific G-quadruplex-containing aptamers have also been found to recognize small molecules (Table 1). SELEX was used to identify an aptamer called ‘spinach’ that recognizes 3,5-difluoro-4-hydroxybenzylidene imidazolinone, and produces fluorescence upon ligand binding [14]. Recent crystal structural studies showed that the ligand stacked with the unique G-quadruplex [15] (Figure 2B). Notably, the G-quadruplex was found to be in a special structural scaffold in the spinach aptamer that is essential for the ligand binding and fluorescence [15]. Since then, the spinach aptamer has been modified and applied for live cell imaging to track different biomolecules [16,17]. In addition, the sequence requirement for the fluorescence of spinach RNA aptamer was extensively studied [18], and several other fluorescent RNA aptamers, such as ‘mango’ aptamer and other variants of ‘spinach’, have been shown to contain G-quadruplex structures [19–22], providing structural insights for future design and development of brighter fluorescent RNA for imaging purposes.”(998, 3rd paragraph) Kwok teaches on page 1000, “Thanks to the unusual structure and folding of the G-quadruplex, one can experimentally identify G-quadruplex formation and investigate structural properties using biophysical tech niques (Table 2). For example, the topology of the G-quadruplex structure can be determined by monitoring the positive or negative circular dichroism (CD) signals at specific wavelengths [57]. In general, G-quadruplexes with parallel topology (Figure 1B) have negative and positive CD signals at 240 and 262 nm, respectively, whereas antiparallel topology (Figure 1B) places these signals at 262 and 295 nm, respectively. To verify G-quadruplex formation, one should also perform the CD experiments under non-G-quadruplex stabilizing (Li+) and G-quadruplex stabilizing conditions (such as K+or with G-quadruplex ligands), and scan toward the far-UV region ( 180–230 nm)”
Tucker teaches, “G-quadruplex DNA Aptamers and their Ligands: Structure, Function and Application “ (title) Tucker in table 1 multiple ligands of G-Quadraplexes including potassium. Tucker teaches, “G-quadruplex topology and stability was first understood from the perspective of telomeric sequences, and from these examples some generalities were drawn such as: 1) cation ability to induce quadruplex formation follows the trend K+>Rb+>Na+>Cs+>Li+ with mechanistic differences being the most understood between K+ and Na+, 2) many cations move in and out of the quadruplex core if not obstructed, 3) Ka values for divalent cations are significantly greater than those for monovalent cations and 4) stabilizing abilities of divalent cations relate in the order Sr2+>Ba2+>Ca2+>Mg2+ [40]. A number of studies have investigated the influence of cations on DNA aptamer structure, and TBA is again the most deeply studied model for this aspect of aptamers that can act as a case study. Not only do cations have an important role in stabilizing quadruplex aptamers, but this property has even been exploited for the purpose of cation detection. The TBA aptamer itself has also been used to detect Hg2+ and Pb2+ [75]. As a further example, aptamers have been generated for the detection of K+ using fluorometric and nanosilver resonance scattering methods [76, 77]. To some degree, these detection methods owe their approach to previous work in understanding the relationship between cation and DNA G quadruplex aptamer.” (2019, 2nd column, last full paragraph)
Tippana teaches, “The basic formula of [G3N1–7G3N1–7 G3N1–7 G3] allows four sets of G triplets to form into three layers of G tetrads, mediated by the Hoog steen base pairing (10). The GQ structures are stabilized by monovalent cations such as potassium or sodium. These ions occupy the central cavity created by the stacks of G tetrads (11–13). GQ DNA can fold into parallel, antiparal lel and hybrid conformations depending on its loop length and sequence composition (14). Conventional techniques such ascircular dichroism (CD)andthermalmeltingcurves acquired throughUV–visiblespectroscopyareoftenusedto distinguishGQfoldingintoparallelandantiparallelconfor mations (15). CD readings will provide either a characteris tic peak at 260 nmforparallel or 295nmfortheantiparallel state. This allows for qualitative comparison among vari ous GQ forming sequences (16). As demonstrated before, the single molecule FRET (smFRET) technique offers sev eral advantages over ensemble methods. First, the fraction of molecules that fold into different conformations (paral lel and antiparallel) can be quantified with accuracy. Sec ond, unfolded DNA can be distinguished from folded con formations. Third, the real-time imaging ofsingle molecules allows for the monitoring of molecules undergoing transitions from one state to another, thus enabling kinetic analy sis. This approach was applied in studies of telomeric DNA (17–19), modified GQ sequences in various solution condi tions (18), GQbindingligands (20) andprotein interactions with the telomere overhang (21–23).” (8106, 2nd column, full paragraph).
Li teaches, “In this work, we utilize circular dichroism (CD), fluorescence, and UV-vis spectroscopy to characterize the DNA structures and monitor the fluorescence and absorbance changes of PPIX in K+ or Na+solution. Interestingly, we find the fluorescence intensity of PPIX can be remarkably enhanced by DNA binding. More importantly, PPIX exhibits a high selectivity for parallel G quadruplexes over the antiparallel and duplex. The selectivity and fluorescence property enable PPIX to indicate DNA structural changes via modulating fluorescence signal readout by its binding and release. This is illustrated by utilizing PPIX as a specific fluorescent probe for a K+ sensor, which is constructed based on an antiparallel-to-parallel conformation transition of the G-quadruplex.” (7577, 1st column, 2nd paragraph).
Varizhuk teaches methods of designing G4 aptamers.
Wojczewski teaches numerous methods of labeling nucleic acids including 5’ end, 3’ end and internal nucleotides.
Domenyuk teaches SEQ ID NO 33524 which provides 35 nucleotides including the re4vers complement of SEQ ID NO 42.
Bentwich teaches SEQ ID NO 185011 which is 64 nucleotides and comprise SEQ ID NO 42 (nucleotides 38-54) and SEQ ID NO 396894 which is 64 nucleotides and comprise SEQ ID NO 42 (5-21).
Schierle-Arndt teaches SEQ ID NO 160118 which comprise the reverse complement of SEQ ID NO 41( nucleotides 18 to 2)
Designing and labeling G4, which are equivalents to those of the claims is routine experimentation. The prior art teaches the parameters and objectives involved in the selection of oligonucleotides that function as G4 quadraplexes, see Varizhuk, Tucker, Tippana, Li, etc. The prior art is replete with guidance and information necessary to permit the ordinary artisan in the field of nucleic acid detection to design labeled G4 aptamers or G4 quadraplexes. As discussed above, the ordinary artisan would be motivated to have designed and tested new probes to obtain additional oligonucleotides that function to detect specific metal ions and identify oligonucleotides with improved properties. The ordinary artisan would have a reasonable expectation of success of obtaining additional labeled G4 aptamers or G4 quadraplexes. Thus, for the reasons provided above, the ordinary artisan would have designed additional labeled G4 aptamers using the teachings in the art at the time the invention was made. The claimed SEQ ID NOs are obvious over the cited prior art, absent secondary considerations.
With regards to claim 7, Long teaches, “50-Cy5.5-(CH2)6-GGGTGGGTGGGTGGGT-3). Thus long teaches a DNA sequence which is between 14 and 22 nucleotides inclusive.
With regards to claim 10-11, Long teaches Cy5.5 which is a derivative of the structure of claim 11. Thus it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claim that any cyanine 3 or cyanine 5 dye as the fluorescent dye. The artisan would be motivated as Long suggest use of a Cyanine 5.5 dye and artisan would know Cy3 and cy5 has similar structure and properties. The artisan would have a reasonable expectation of success as the artisan is merely using known dyes.
With regards to claim 13, Li teaches K+ and Na+.
With regards to claim 14, Long teaches a change in fluorescence intensity. (Figure 2).
With regards to claim 15, Long teaches fluorescence is increased upon binding of the ligand to the Pb+, K+ and Na+. Thus the fluorescence was quenched prior to binding of Pb+ relative to after Pb+.
Response to Arguments
This is a new grounds of rejection
Summary
No claims are allowed.
Conclusion
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/Steven Pohnert/Primary Examiner, Art Unit 1683
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