DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Effective Filing Date
The present application, filed on November 12, 2021 claims the benefit of priority to U.S. Provisional Application No: 63/114,302, filed on November 16, 2020. Therefore, the effective filing date of the present application is determined to be November 16, 2020.
Election/Restrictions
Applicant’s election of “Group I, claims 1-7, 9-19, and 24-28, drawn to an incorporation mix, a kit, and a labeled nucleotide” in the reply filed on April 30, 2025 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)).
Claim Status/Action Summary
Claims 1-29 are pending in the present application.
Claims 8, 20-23, and 29 are withdrawn as directed to a non-elected invention.
Claims 1-7, 9-19, and 24-28 are under examination.
Any objections and rejections not reiterated below are hereby withdrawn.
The rejections of record under 35 U.S.C. 112(b) have been withdrawn in view of the amendments to the claims.
Drawings
The drawings filed on November 12, 2021 are acceptable.
Claim Interpretation
The claims recite the term “about” linked to several values. The specification defines: “when "about" and/or "substantially" are/is utilized to describe a value, they are meant to encompass minor variations (up to +/- 10%) from the stated value.” (specification, paragraph 0275). Therefore, ranges modified by the term “about” in the claims have been interpreted to encompass minor variations up to +/- 10% from the stated values. Therefore, for example, a range of “about 3 nm to about 12 nm” has been interpreted to be limited to a range of “2.7 nm to 13.2 nm”.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-4 and 6-7 remain/are rejected under 35 U.S.C. 103 as being unpatentable over Ju et al., WO 2017/176679 A1 (published on October 12, 2017) in view of Peris et al., US 2010/0311144 A1 (Published December 9, 2010).
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Regarding claim 1, Ju et al. teaches reagents for DNA sequencing by synthesis (i.e. an incorporation mix) comprising: a liquid carrier, a polymerase, a gold nanoparticle (i.e. a plasmonic nanostructure) linked to the polymerase, and a labeled nucleotide comprising a 3’ OH blocking group attached to the sugar of the nucleotide and a Raman cluster tag label attached to a base of the nucleotide (Ju et al., Figure 5 (see below) and paragraphs 0048-0050). Ju et al. teaches that while Raman tags produce very sharp Raman spectrum peaks compared to fluorescent signals produced by fluorescent tags, the Raman tag signals are substantially weaker than fluorescent signals.
Ju et al. does not teach an incorporation mix comprising a labeled nucleotide with a dye label attached to the base of the nucleotide.
However, Peris et al. teaches similar reagents for DNA sequencing by synthesis comprising a liquid carrier, a polymerase linked to a nanoparticle capable of energy transfer, and a fluorescently labeled terminator nucleotide (i.e. a labeled nucleotide with a 3’ OH blocking group attached to a sugar of the nucleotide and a dye label attached to a base of the nucleotide) (Peris et al., paragraphs 0009-0010 and 0036). Peris further teaches that said polymerase-linked nanoparticle can be made from any suitable metal and/or non-metal atoms for forming semiconductor nanoparticles (Peris et al., paragraphs 0139-0144).
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 substituted the nucleotides comprising 3’ OH blocking groups and Raman tag labels attached to the base of the nucleotide in the nucleotide incorporation mix taught by Ju et al. with the fluorescently labeled terminator nucleotides in the incorporation mix taught by Peris et al.
The ordinary artisan would have been motivated to substitute the Raman tag-labeled terminator nucleotides, taught by Ju et al., for the fluorescent tag-labeled nucleotides, taught by Peris et al., because of the teachings of Ju et al. and Peris et al. Ju et al. teaches that spontaneous Raman signals from the Raman tag-labeled nucleotides are much weaker than fluorescent signals, but are enhanced by local surface plasmons (i.e. the plasmonic nanostructure attached to the polymerase) (Ju et al., paragraphs 0009-0010). Peris et al. teaches that energy transfer between the nanoparticle and the fluorescent label produces fluorescent signals that have improved properties (increased photostability, high quantum yield, modulated blinking, etc. (Peris et al., paragraphs 0156-0161).
The ordinary artisan would have been reasonably confident that substituting the fluorescently-labeled terminator nucleotides taught by Peris et al. into the incorporation mix taught by Ju et al. would have resulted in improved fluorescence detection due to the close proximity of the fluorescent nucleotide label and the nanoparticle-coupled to the polymerase because of the teachings of Peris et al. (and Ju et al.) that such nanoparticles increase detectable signals through plasmonic resonance.
Regarding claim 2, Peris et al. teaches plasmonic nanoparticles (i.e. nanostructures) can be composed of any suitable metal and/or non-metal including: Groups II-V1 or the periodic table including GaAs (Gallium Arsenide) and Silicon (Peris et al., paragraphs 0142-0144). Furthermore, Ju et al. teaches plasmonic nanoparticles (i.e. nanostructures) can be composed of various noble metals, most commonly gold or silver (Ju et al., paragraph 0010).
Regarding claim 3, Ju et al. teaches gold nanoparticles (i.e. plasmonic nanostructures) are conjugated to the polymerase through a cysteine of the polymerase (Ju et al., paragraph 0180). Furthermore, Peris et al. teaches polymerases are linked (i.e. chemical conjugated) to the nanoparticles through thiol bonding between the cysteine residues on the polymerase and sulfur atoms on the nanoparticle surface (Peris et al., paragraph 0076). Peris et al. further teaches polymerases may be linked to nanoparticles through condensation reactions between the amines on the polymerase and carboxy groups on the nanoparticles (i.e. may be chemically conjugated through an amine of the polymerase) (Peris et al., paragraph 0076).
Regarding claim 4, Ju et al. teaches an oligonucleotide may be attached to the polymerase, wherein the oligonucleotide is hybridized to a complementary oligonucleotide that is attached to a gold nanoparticle (i.e. a plasmonic nanostructure) (Ju et al., Figure 7, see below).
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Regarding claim 6, Ju et al. teaches gold nanoparticles (i.e. plasmonic nanostructures) are conjugated to the polymerase through a cysteine of the polymerase (Ju et al., paragraph 0180). Furthermore, Peris et al. teaches polymerases are linked (i.e. chemical conjugated) to the nanoparticles through thiol bonding between the cysteine residues on the polymerase and sulfur atoms on the nanoparticle surface (i.e. the nanoparticles are functionalized with one member of a binding pair corresponding to a second member of a binding par on the polymerase) (Peris et al., paragraph 0076). Peris et al. further teaches polymerases may be linked to nanoparticles through condensation reactions between the amines on the polymerase and carboxy groups on the nanoparticles (i.e. may be chemically conjugated through an amine of the polymerase) (Peris et al., paragraph 0076).
Regarding claim 7, Peris et al. teaches the polymerase can be linked to a linker moiety (i.e. a member of a binding pair) comprising biotin or avidin for conjugation to a nanoparticle (Peris et al., paragraphs 0069-0070 and 0076-0081).
Claim 5 remains/is rejected under 35 U.S.C. 103 as being unpatentable over Ju et al., WO 2017/176679 A1 (published on October 12, 2017) in view of Peris et al., US 2010/0311144 A1 (Published December 9, 2010) as applied to claims 1-4 and 6-7 above, and further in view of Yao et al., “Clicking DNA to gold nanoparticles: poly-adenine-mediated formation of monovalent DNA-gold nanoparticle conjugates with nearly quantitative yield”, NPG Asia Materials (2015) 7, e159, (Published January 30, 2015).
As discussed in the 103 rejections above, Ju et al. in view of Peris et al. teaches an incorporation mix comprising a liquid carrier, a complex including a polymerase linked to a plasmonic nanostructure, and a labeled nucleotide including a 3’ OH blocking group and a dye label attached to the base of the nucleotide. As described above, Ju et al. in view of Peris et al. further teaches that the complex can further comprise an oligonucleotide attached to the polymerase, wherein the oligonucleotide is hybridized to a complementary nucleotide that is attached to a plasmonic nanostructure (the tethered primer attached to a magnetic or gold nanoparticle) (See 103 rejection of claims 1 and 4 in section 12 of this action).
Ju et al. in view of Peris et al. does not teach that the oligonucleotide tether includes an additional portion that is wrapped around the plasmonic nanostructure. However, Yao et al. teach monovalent (i.e. 1:1) conjugates between polyadenylated DNA and gold nanoparticles (i.e. plasmonic nanostructures) wherein the polyadenylated portion of the DNA is wrapped around the nanoparticle. Furthermore, Yao et al. teaches that a non-poly-A portion of the nanoparticle-wrapping oligonucleotide is capable of specifically hybridizing to a complementary DNA sequence (Yao et al., figure 1, see below). Finally, Yao et al. teaches that the monovalent complex formed between a polyadenylated oligonucleotide and a gold
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nanoparticle is useful in DNA-regulated plasmonic applications and biomolecular imaging.
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 gold-nanoparticle-tethering primer, taught by Ju et al. in view of Peris et al. to further comprise a poly-A “additional portion” at the 5’ end of the tether to effectively restrict the nanoparticle:primer complex to a monovalent interaction as taught by Yao et al. The ordinary artisan would have been motivated to modify the tethering primer with a 5’ poly-A portion capable of wrapping around a gold nanoparticle because of the teaching of Yao et al. that monovalent DNA:nanoparticle interaction allows for increased control and uniformity of the resulting complexes (Yao et al., abstract and figure 1). The ordinary artisan would have been reasonably confident that a poly-A modification to the tethering primer would have advantageously reduced the presence of polyvalent DNA:nanoparticle complexes in the sequencing by synthesis reactions taught by Ju et al. in view of Peris et al. The ordinary artisan would have expected aggregates of diverse sequencing targets associated with a single nanoparticle (as with non-monovalent particle:DNA complexes) to result in colocalization of fluorescent signals from non-identical templates on a flow cell.
Claims 9-13, 15-16, and 18-19 remain/are rejected under 35 U.S.C. 103 as being unpatentable over Ju et al., WO 2017/176679 A1 (published on October 12, 2017) in view of Peris et al., US 2010/0311144 A1 (Published December 9, 2010) as applied to claims 1-4 and 6-7 above, and further in view of Drmanac et al., US 2017/0240961 A1 (Published August 24, 2017).
Regarding claim 9, as discussed in the 103 rejections above, Ju et al. in view of Peris et al. teaches an incorporation mix comprising a liquid carrier, a complex including a polymerase linked to a plasmonic nanostructure, and a labeled nucleotide including a 3’ OH blocking group and a dye label attached to the base of the nucleotide. (See 103 rejection of claim 1 in section 12 of this action).
Ju et al. in view of Peris et al. does not teach that the plasmonic nanostructure is supplied in a second mixture comprising a second liquid carrier and that the nanostructure is functionalized to associate within proximity of the labeled nucleotide after an incorporation event. However, Drmanac et al. teaches reagents for DNA sequencing comprising reagents for an incorporation step (i.e. an incorporation mix) wherein a 3-OH blocked nucleotide comprising an affinity label is incorporated into the primer according to the sequence of a template nucleic acid (Drmanac et al., paragraph 0048-0050). Drmanac further teaches reagents for a detection (i.e. imaging) step wherein a detectably-labeled affinity agent binds to the incorporated nucleotide (i.e. the detection reagent is functionalized to associate itself within proximity (i.e. by binding to) the labeled nucleotide after an incorporation event (Drmanac et al., paragraphs 0056-0058). Drmanac et al. further teaches that use of affinity agents to detect incorporated labeled nucleotides provides for localization of multiple copies of a detectable label to the incorporation site (Drmanac et al., paragraph 0057). Finally, Drmanac et al. teaches that the affinity agent can comprise a detectable label including metals or magnetic particles (Drmanac et al., paragraph 0037).
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 incorporation mix taught by Ju et al. in view of Peris et al. comprising a pre-formed polymerase-nanoparticle complex for signal amplification following incorporation of a detectably labeled nucleotide to supply the nanoparticle as an uncoupled “affinity” reagent in a second “imaging” step, as taught by Drmanac et al. The ordinary artisan would have been motivated to couple the nanoparticle to the polymerase:DNA complex after incorporation of a labeled nucleotide, as taught by Drmanac et al., rather than before the incorporation step, because of the teaching of Drmanac et al. that the use of such affinity agents to couple detectable labels to incorporated nucleotides provides for the localization of multiple labels to a site (Drmanac et al., paragraph 0057). The ordinary artisan would have recognized that such a modification would have allowed for increased localized signal independent of the constraints of polymerase:nucleotide analog steric hindrance that would be expected to limit direct labeling of a nucleotide with multiple detectable labels. The ordinary artisan would have had a reasonable expectation of success when delaying the coupling of the nanoparticle to the incorporation complex until after incorporation of the labeled nucleotide because Drmanac et al. teach interaction pairs wherein the detectable label is attached to an affinity reagent comprising a thiol-containing molecule (or biotin or streptavidin) that successfully couples to its interaction partner (a cysteine or streptavidin or biotin, respectively) after incorporation of a labeled nucleotide (Drmanac et al., paragraph 0029).
Regarding claim 10, Ju et al. in view of Peris et al. teaches plasmonic nanoparticles (i.e. nanostructures) can be composed of any suitable metal and/or non-metal including: Groups II-V1 or the periodic table including GaAs (Gallium Arsenide) and Silicon (Peris et al., paragraphs 0142-0144), or of various noble metals, most commonly gold or silver (Ju et al., paragraph 0010).
Regarding claim 11, Ju et al. in view of Peris et al. teaches that a single nanoparticle can be functionalized with a plurality (i.e. a second) of polymerases (Peris et al., paragraph 0082).
Regarding claim 12, Ju et al. in view of Peris et al. teaches gold nanoparticles (i.e. plasmonic nanostructures) are conjugated to the polymerase through a cysteine of the polymerase (Ju et al., paragraph 0180). Furthermore, Ju et al. in view of Peris et al. teaches polymerases are linked (i.e. chemical conjugated) to the nanoparticles through thiol bonding between the cysteine residues on the polymerase and sulfur atoms on the nanoparticle surface (Peris et al., paragraph 0076). Ju et al. in view of Peris et al. further teaches polymerases may be linked to nanoparticles through condensation reactions between the amines on the polymerase and carboxy groups on the nanoparticles (i.e. may be chemically conjugated through an amine of the polymerase) (Peris et al., paragraph 0076).
Regarding claim 13, Ju et al. in view of Peris et al. teaches an oligonucleotide may be attached to the polymerase, wherein the oligonucleotide is hybridized to a complementary oligonucleotide that is attached to a gold nanoparticle (i.e. a plasmonic nanostructure) (Ju et al., Figure 7, see below).
Regarding claims 15-16, Ju et al. in view of Peris et al. teaches plasmonic nanoparticles can be functionalized with a variety of binding pair members including carboxyl-, amino-PEG, biotin, metal cations (i.e. Ni2+) etc. (Peris et al., paragraph 0150) and polymerases can be functionalized with corresponding binding pair members including avidin, His-tags, cysteine, etc. (Peris et al., paragraph 0069).
Regarding claim 18, Ju et al. in view of Peris et al. teaches the polymerase further comprises a surface tether attached to a flow cell binding agent (Ju et al., figure 5).
Regarding claim 19, Ju et al. in view of Peris et al. teaches plasmonic nanoparticles can be functionalized with biotin (Peris et al., paragraph 0081). Drmanac et al. teaches that a nucleotide can be labeled with biotin and an affinity agent directed thereto can be labeled with streptavidin (Drmanac et al., paragraph 0029).
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 labeled nucleotide with biotin, as taught by Drmanac et al., and modified the plasmonic nanoparticle (i.e. the detectable affinity agent) with streptavidin. The ordinary artisan would have been motivated to couple the plasmonic nanoparticle to streptavidin because Drmanac et al. teaches that biotinylated incorporated nucleotides can be readily detected using detectable labels coupled to streptavidin. The ordinary artisan would have been reasonably confident that nanoparticle coupling to streptavidin would have successfully detected biotinylated nucleotides because Peris teaches that nanoparticles can be directly attached to proteins (i.e. avidin) through thiol bonding or through amino- or carboxy- derivatized ligands (Peris et al., paragraphs 0078-0081).
Claim 14 remains/is rejected under 35 U.S.C. 103 as being unpatentable over Ju et al., WO 2017/176679 A1 (published on October 12, 2017) in view of Peris et al., US 2010/0311144 A1 (Published December 9, 2010) and Drmanac et al., US 2017/0240961 A1 (Published August 24, 2017) as applied to claims 9-13, 15-16, and 18-19 above, and further in view of Yao et al., “Clicking DNA to gold nanoparticles: poly-adenine-mediated formation of monovalent DNA-gold nanoparticle conjugates with nearly quantitative yield”, NPG Asia Materials (2015) 7, e159, (Published January 30, 2015).
Ju et al. in view of Peris et al. and Drmanac et al. do not teach that the oligonucleotide tether includes an additional portion that is wrapped around the plasmonic nanostructure. However, Yao et al. teach regulating the stoichiometry of the interaction between plasmonic nanostructures and oligonucleotides based on the length of a poly-adenine portion on the oligonucleotide including: monovalent (i.e. 1:1), divalent (2:1), and trivalent (3:1) conjugates between polyadenylated DNA and gold nanoparticles (i.e. plasmonic nanostructures) wherein the polyadenylated portion of the DNA is wrapped around the nanoparticle. Furthermore, Yao et al. teaches that a non-poly-A portion of the nanoparticle-wrapping oligonucleotide is capable of specifically hybridizing to a complementary DNA sequence (Yao et al., figure 6, see below). Finally, Yao et al. teaches that the tailored complexes formed between a polyadenylated oligonucleotide and a gold nanoparticle is
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useful in DNA-regulated plasmonic applications and biomolecular imaging.
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 gold-nanoparticle-tethering primer, taught by Ju et al. in view of Peris et al. and Drmanac et al. to further comprise a poly-A “additional portion” at the 5’ end of the tether to control the stoichiometry (and therefore, geometry) of the nanoparticle:oligonucleotide:polymerase complexes assembled as taught by Yao et al. The ordinary artisan would have been motivated to modify the tethering primer with a 5’ poly-A portion capable of wrapping around a gold nanoparticle because of the teaching of Yao et al. that tailored stoichiometric DNA:nanoparticle interactions allow for increased control and uniformity of the resulting complexes (Yao et al., abstract and figure 1). The ordinary artisan would have been reasonably confident that a length-tailored poly-A modification to the tethering primer would have advantageously produced regular, predictably assembled DNA:nanoparticle complexes in the sequencing by synthesis reactions taught by Ju et al. in view of Peris et al. and Drmanac et al.
Claim 17 remains/is rejected under 35 U.S.C. 103 as being unpatentable over Ju et al., WO 2017/176679 A1 (published on October 12, 2017) in view of Peris et al., US 2010/0311144 A1 (Published December 9, 2010) and Drmanac et al., US 2017/0240961 A1 (Published August 24, 2017) as applied to claims 9-13, 15-16, and 18-19 above, and further in view of Sun et al., “Structure and Enzymatic Properties of a Chimeric Bacteriophage RB69 DNA Polymerase and Single-Stranded DNA Binding Protein With Increased Processivity” PROTEINS: Structure, Function, and Bioinformatics 65:231-238 (2006).
Regarding claim 17, Ju et al. in view of Peris et al. and Drmanac et al. do not teach that the polymerase further comprises a DNA binding domain attached to a surface thereof. This limitation has been interpreted as requiring modifying the polymerase to comprise a DNA binding domain in addition to the DNA binding domain intrinsic to DNA polymerases. Peris et al. teaches modified DNA polymerases optimized for binding to nanoparticles (i.e. plasmonic nanostructures) based on the bacteriophage RB69 DNA polymerase (Peris et al., paragraph 0002).
However, Sun et al. teaches a functional chimeric enzyme of the bacteriophage RB69 DNA polymerase with its cognate single strand binding protein (i.e. a DNA binding domain) on a surface of the DNA polymerase. The resulting fusion protein exhibits increased template DNA affinity and processivity while maintaining fidelity (Sun et al., abstract).
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 DNA polymerases taught by Ju et al. in view of Peris et al. and Drmanac et al. to further comprise an additional DNA binding domain attached to a surface thereof, as taught by Sun et al. The ordinary artisan would have been motivated to modify said polymerases because of the teaching of Sun et al. that addition of a DNA binding domain to the surface of the RB69 DNA polymerase improved several key properties of the DNA polymerase that are useful in PCR and DNA sequencing including processivity and affinity for template DNA (Sun et al., abstract). The ordinary artisan would have been reasonably confident that the fusion protein described by Sun et al. would have been compatible with the modifications for nanoparticle binding taught by Peris et al. because they are both based on wild type RB69 polymerase, and the fusion protein described by Sun et al. is a relatively simple in-frame fusion of the two proteins at the N-terminus of the polymerase comprising a short 6 amino acid linker domain (Sun et al., abstract).
Response to arguments
The response addresses all of the above 103 rejections “together, as each is based on the combination of at least Ju and Peris”. The response asserts that the ordinary artisan would not have been led to utilize an optically detectable dye label to replace a Raman cluster tag of the Raman-cluster-tagged nucleotide because Ju teaches a “non-fluorescent detection approach” and further asserts that Raman spectroscopy is known in the art as a label-free technique. The response asserts that the “proffered modification would destroy the stated purpose of Ju” (Remarks, page 3). This line of argument has been thoroughly considered and is not persuasive.
As described in the 103 rejection of record, Ju et al. teaches that while Raman tags produce very sharp Raman spectrum peaks compared to fluorescent signals produced by fluorescent tags, the Raman tag signals are substantially weaker than fluorescent signals. Ju teaches that the spontaneous Raman signals from the Raman tag-labeled nucleotides are much weaker than fluorescent signals, but are enhanced by local surface plasmons (i.e. the plasmonic nanostructure attached to the polymerase) (Ju et al., paragraphs 0009-0010). Peris et al. teaches that energy transfer between the nanoparticle and the fluorescent label produces fluorescent signals that have improved properties (increased photostability, high quantum yield, modulated blinking, etc. (Peris et al., paragraphs 0156-0161). Ju et al. and Peris et al. both teach that the signal generated by the detectable nucleotides (comprising a Raman tag or an optical tag, respectively) is enhanced by proximity to a plasmonic nanostructure. The stated purpose of both references is improved detection of nucleic acid incorporation in the context of nucleic acid sequencing. Both Ju et al. and Peris et al. achieve this goal through similar techniques using different detectable labels. Furthermore, Peris et al. teaches that the detectable label (the “reporter moiety”) linked to the nucleotide can be “luminescent, photoluminescent, electroluminescent, bioluminescent, chemiluminescent, fluorescent, phosphorescent, chromophore, radioisotope, electrochemical, mass spectrometry, Raman, hapten, affinity tag, atom, or an enzyme”. (Peris et al., paragraphs 0096-0097). Therefore, Peris et al. explicitly teaches that fluorescent and Raman tags are interchangeable for plasmonic nanostructure-enhanced detection of nucleic acid incorporation.
For these reasons and those already of record, the rejection is maintained.
Claims 24-25 remain/are rejected under 35 U.S.C. 103 as being unpatentable over Peris et al., US 2010/0311144 A1 (Published December 9, 2010).
Regarding claim 24, Peris et al. teaches labeled terminator nucleotides (i.e. comprising a 3’ OH blocking group) comprising reporter moieties that may be attached to a base of the nucleotide. Peris additionally teaches that two or more different reporter moieties can be selected to be linked to a nucleotide, and that any suitable reporter moiety may be used, including fluorescent tags, Raman tags, affinity tags, or enzymes (Peris et al., paragraphs 0094-0103). Peris et al. does not explicitly teach a labeled nucleotide comprises a plasmonic nanostructure. However, Peris et al. does teach labeling a polymerase with such nanoparticle(s) wherein the nanoparticle is brought into within about 5-20 nm of the label on the nucleotide (Peris et al., paragraphs 0139 and 0149).
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 labeled nucleotide to further comprise a nanoparticle (i.e. a plasmonic nanostructure) such as those taught by Peris et al. The ordinary artisan would have been motivated to combine the nucleotide dye labels with the nanoparticles because of the teaching of Peris et al. that confining the two moieties (a dye label and corresponding nanoparticle) in sufficiently close proximity allows for energy transfer between the nanoparticle and the fluorescent label and produces fluorescent signals that have improved properties (increased photostability, high quantum yield, modulated blinking, etc. (Peris et al., paragraphs 0156-0161).
Regarding claim 25, Peris et al. teaches plasmonic nanoparticles (i.e. nanostructures) can be composed of any suitable metal and/or non-metal including: Groups II-V1 or the periodic table including GaAs (Gallium Arsenide) and Silicon (Peris et al., paragraphs 0142-0144).
Claim 26-28 remain/are rejected under 35 U.S.C. 103 as being unpatentable over Peris et al., US 2010/0311144 A1 (Published December 9, 2010) as applied to claims 24-25 above, and further in view of in view of Yao et al., “Clicking DNA to gold nanoparticles: poly-adenine-mediated formation of monovalent DNA-gold nanoparticle conjugates with nearly quantitative yield”, NPG Asia Materials (2015) 7, e159, (Published January 30, 2015).
Regarding claim 26, as described in the 103 rejection of claims 24-25 above, the teachings of Peris et al. render obvious the labeled nucleotide of claim 24. Peris et al. does not teach that the plasmonic nanostructure is attached to the base of the nucleotide through a double stranded deoxyribonucleic acid. However, Yao et al. teaches such nanoparticles can be linked to a first single stranded nucleic acid having a poly-A moiety that is then hybridized to a second nucleic acid having complementary sequence to the non-poly-A portion of the first nucleic acid, resulting in a labeled nucleic acid with a double stranded linker to a plasmonic nanostructure (See Yao et al., figure 1 or figure 6 above).
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 used the wrapped nucleic acid nanoparticle assemblies taught by Yao et al. to label a particular nucleotide (i.e. the single nucleotide at the most distal terminus of the second nucleic acid) with a plasmonic nanostructure. The ordinary artisan would have been motivated to combine the nucleic acid coupled nanoparticles taught by Yao et al. with the dye and/or nanoparticle labeled nucleotides taught by Peris et al. The ordinary artisan would have been reasonably confident that the DNA linker taught by Yao et al. would have successfully enhanced the fluorescent signal of the dye label on the nucleotide by confining the two moieties (a dye label and corresponding nanoparticle) in sufficiently close proximity because Yao et al. teaches that the length of the oligonucleotide coupled to the nanoparticle is tunable to permit assembly and plasmonic interaction between multiple nanoparticles, thus allowing for energy transfer between the nanoparticle and the fluorescent label required to produce the enhanced fluorescent signals taught by Peris et al.
Regarding claims 27-28, Peris et al. teach optimizing the distance between the two members of the electronic interaction pair (i.e. the nanoparticle and the dye label) within about 5-10 nm (i.e. within about 3 to about 12 nm) (Peris et al., paragraph 0149). Peris further teaches a variety of linking molecules known in the art that are suitable for attaching reporter moieties and/or inhibitor moieties (i.e. 3’ OH blocking groups) to nucleotides (Peris et al., paragraphs 125-0127). 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 performed routine optimization of the lengths of linking molecules for attaching the dye label and plasmonic nanostructure to any particular labeled nucleotide such that the two labels are held within sufficient proximity for electronic interaction and signal enhancement taught by Peris et al. (about 5-10 nm; “within about 3 to about 12 nm”). The ordinary artisan would have been motivated to confine the two labels within the distance limitations taught by Peris et al. because Peris et al. teaches that the requisite interaction between the dye labels and the nanoparticles is highly dependent on the distance between a dye label and a nanoparticle and decreases with increasing distance by a factor of 1/R6, where R= the distance between the molecules. The ordinary artisan would therefore have been reasonably confident that confining a dye label and nanoparticle using well-known nucleotide linkers within this distance would have resulted in the signal enhancement properties taught by Peris et al.
Response to arguments
The response addresses the 103 rejections to claims 24-28 “together, as each is based on Peris”. The response asserts that “Peris does not render claim 24 obvious” because Peris teaches nucleotides that are labeled with “a fluorescent dye or a nanoparticle… as alternatives… not their simultaneous inclusion” and “Peris does teach that two or more different reporter moieties can be selected, but never that the different moieties are linked to the same nucleotide”. The response concludes that “it would not be obvious that the resonance between a dye label and a plasmonic nanostructure would lead to improved fluorescent signals, or even functionality, when linked to a single nucleotide” (Remarks, page 6).
First, claims 24-28 as presently written are not limited to a single nucleotide that is directly linked to a plasmonic nanostructure AND a fluorophore as asserted by the response. Claim 24 requires only that the nanostructure is attached to the base of the nucleotide or to the optically detectable dye label. Claim 26 depends from claim 24 and further limits the scope of claim 24 “wherein the plasmonic nanostructure is attached to the base of the nucleotide through a double stranded deoxyribonucleic acid strand”. Therefore the claims encompass embodiments wherein the plasmonic nanostructure is covalently coupled to any moiety of a single stranded nucleic acid (i.e. any constituent base, phosphate group, hydroxyl, etc.) that is hybridized to a single stranded nucleic acid comprising the fluorophore labeled nucleotide.
Second, Peris et al. teach that the signal-enhancing resonance between the fluorophore and the nanoparticle is dependent on the proximity between these two moieties, and that confining the two moieties (a dye label and corresponding nanoparticle) in sufficiently close proximity allows for energy transfer between the nanoparticle and the fluorescent label and produces fluorescent signals that have improved properties (increased photostability, high quantum yield, modulated blinking, etc. (Peris et al., paragraphs 0156-0161).
Finally, an evidentiary reference, Pal et al., “DNA-enabled rational design of fluorescence-Raman bimodal nanoprobes for cancer imaging and therapy” Nature Communications (2019) 10:1926, is cited here only for the purpose of argument of the narrower interpretation of the claims asserted by the response. Briefly, Pal et al. teaches fluorophore labeled nucleic acids wherein the direct coupling of the bifunctional fluorophore/Raman tag to a plasmonic nanostructure enhances both the fluorescence and Raman signal of the tag (Pal et al., figure 1, see below). Therefore, it appears that the prior art does not support the assertion that the ordinary artisan would not have expected “that the resonance between a dye label and a plasmonic nanostructure would lead to improved fluorescent signals, or even functionality, when linked to a single nucleotide”.
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788
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For these reasons and those already of record, the rejections are maintained.
Conclusion
No claim is allowed.
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ZACHARY MARK TURPIN whose telephone number is (703)756-5917. The examiner can normally be reached Monday-Friday 8:00 am - 5:00 pm.
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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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