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 .
Claim Interpretation
Claim 3 recites that “each primary probe hybridizes to a target”. “Hybridization” is a term referring to a nucleic acid forming a duplex with another, complementary, nucleic acid. Claim 11, however, lists several types are target that are not nucleic acid (e.g., proteins, lipids, glycans, etc.). Claim 12, depending from claim 11, recites that the targets are “conjugated to one or more oligonucleotide sequences”, thus providing a mechanism for how a primary probe could “hybridize” to such non-nucleic acid targets. Therefore, the language “each primary probe hybridizes to a target” in claim 3 will be construed to mean that each primary probe hybridizes to a target or (in the case of non-nucleic acid targets) to an oligonucleotide conjugated to a target.
In addition, the step (viii) in claim 60 will be construed as the same exonuclease treatment as step (vi) of claim 3, and not a second, separate, exonuclease treatment.
In claim 61, which depends from claim 60, the limitation “the interaction” will be construed as the interaction between a probe and its target, as that is the only “interacting” that is recited in claim 60.
Claim Objections
Claim 3 is objected to because of the following informalities: in step (vi), the claim should be amended to read “contacting the sample with one or more exonucleases”. Appropriate correction is required.
Claims 44 and 45 are objected to because of the following informalities: the language “…wherein the method further comprises a step of removing comprises contacting…” in claim 45 should be written as “…wherein the removing comprises contacting…”, since claim 45 depends from claim 44, which already indicates the method further comprises “removing”, and claim 45 only further specifies how the “removing” is carried out.
In addition, the term “detectably labeled oligonucleotides” in claims 44 and 45 should be replaced with the term “detectably labeled readout probes” to match the language of claim 36.
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.
Claim 4 is 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 “wherein any of the steps (i)-(vii) are repeated”. Claim 4 depends from claim 3, which recites only steps (i)-(vi).
Claims 5-9 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.
Each of these claims recites “stabilizing” the probes. While the specification at paragraph [0062] provides examples of what “stabilizing” can be, the specification does not define what “stabilizing” means (i.e., what the probes are stabilized against, such as against exonuclease or other degradation, movement, de-hybridization, etc.). Therefore, the scope of claims 5-9 is indefinite. Claim 10 is not subjected to this rejection as it goes on to explicitly recite what the stabilizing entails.
Claims 36-39, 44 and 45 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 36 recites the limitations "the cell" and “the nucleic acids” in step (vii). There is insufficient antecedent basis for this limitation in the claim. Claims 37-39, 44 and 45 depend from claim 36 and are rejected for the same reason.
Claim 43 is 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.
Among the list of possible exonucleases are two terms: “truncated” and “Micrococcal”. Regarding “truncated”, it is not understood whether this was intended to refer to the preceding alternative, Exonuclease VIII, indicating that the alternative is “truncated Exonuclease VIII”, or instead was meant to refer to a particular exonuclease known as “truncated exonuclease”. The specification does not further elaborate. Assuming this was meant to refer to “truncated Exonuclease VIII” (which is apparently known in the art), the language “truncated Exonuclease VIII”, rather than “Exonuclease VIII, truncated” would be more favorably considered. Likewise, the term “Micrococcal nuclease” would be more favorably considered over “Micrococcal”.
Claims 60 and 61 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.
The term “close proximity” in claim 60 is a relative term which renders the claim indefinite. The term “close proximity” is not defined by the claim, 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. For purpose of examination over the prior art, any distance will be considered “close proximity”. Claim 61 depends from claim 60 and is rejected for the same reason.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claim(s) 3, 5, 6, 10-12, 14, 41, 43, 59 and 60 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Feaver (US 2004/0248103).
Regarding claims 3 and 41, Feaver disclosed a method comprising:
(i) contacting a sample with one or more primary probes that bind one or more targets, wherein each primary probe hybridizes to a target,
See Fig. 1; half circle probes (primary probes) HCP1 and HCP2 hybridize to oligos G1 and G2, which in turn are conjugated to a target analyte via specific binding molecules SBM1 and SBM2. Due to the proximity of the two SBMs and their associated oligonucleotides, probes HCP1 and HCP2 are able to ligate to form a closed, circular molecule. Following ligation, rolling circle amplification (RCA) is carried out to produce a long tandem sequence DNA (TS-DNA). See paragraph [0004]. Although Fig. 1 shows the case of two antibodies binding to different sites on a single analyte, Feaver also disclosed the scenario in which the two antibodies bind to two analytes in close proximity (paragraph [0005]).
wherein each primary probe comprises one or more secondary probe binding sites and/or one or more readout probe binding sites;
See paragraph [0163]: “Detection probes are labeled oligonucleotides or oligomers having sequence complementary to detection tags on TS-DNA or transcripts of TS-DNA.” That is, the detection probes (corresponding to the claimed readout probes) bind to sites in the product of the RCA, which means that either one or both of primary probes HCP1 and HCP2 contain one or more binding sites for such probes.
(v) hybridizing readout probes capable of detection to the one or more readout probe binding sites;
See paragraph [0163]: “Detection probes are labeled oligonucleotides or oligomers having sequence complementary to detection tags on TS-DNA or transcripts of TS-DNA.”
(vi) contacting the sample one or more exonucleases each capable of removing probes that bind to targets, primary, secondary, tertiary, or quaternary probes after any steps (i)-(v) to reduce the background signal of probes bound to targets.
See paragraph [0125]: “Rolling circle replication primers can also include modified nucleotides to make it resistant to exonuclease digestion. For example, the primer can have three or four phosphorothioate linkages between nucleotides at the 3' and/or 5' end of the primer. Such nuclease resistant primers allow selective degradation of excess unligated OCP or HCPs and gap oligonucleotides that might otherwise interfere with hybridization of detection probes, address probes, and secondary OCPs to the amplified nucleic acid.”
Regarding claims 5, 6, 10 and 14, Feaver ligated the primary probes (HCP1 and HCP2) using T4 DNA ligase; see paragraph [0404].
Regarding claim 11, Feaver disclosed the target analyte could be “peptides, proteins, and other macromolecules such as lipids, complex carbohydrates, proteolipids, membrane fragments, and nucleic acids” (paragraph [0151]).
Regarding claim 12, as noted above, Feaver disclosed a scenario (Fig. 1) in which the targets were conjugated to oligonucleotides.
Regarding claim 43, Feaver disclosed the use of Exonuclease I and Exonuclease III; see paragraph [0405].
Regarding claim 59, the exonuclease step would inherently remove any non-specifically bound probes in which proper alignment of the 5’ and 3’ ends of a probe did not occur, thereby preventing ligation.
Regarding claim 60, Feaver enhanced the signals of the primary probes by RCA. Regarding “determining the proximity of the probes interacting with their targets to each other”, this would necessarily occur in Feaver’s scenario, discussed in paragraph [0005], in which two target analytes were in close proximity.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 1 is/are rejected under 35 U.S.C. 103 as being unpatentable over Feaver (US 2004/0248103) as applied to claims 3, 5, 6, 10-12, 14, 41, 43, 59 and 60 above, and further in view of Polansky (US 2004/0023207).
The disclosure of Feaver has been discussed. Feaver also disclosed (paragraph [0251]): “The materials described above as well as other materials can be packaged together in any suitable combination as a kit useful for performing, or aiding in the performance of, the disclosed method. It is useful if the kit components in a given kit are designed and adapted for use together in the disclosed method. For example, disclosed are kits for proximity-mediated rolling circle amplification, the kit comprising a plurality of binding guide conjugates and a plurality of half circle probes. The kits can also contain one or more rolling circle replication primers and one or more fluorescent change probes. The kits also can contain DNA polymerase, amplification target circles, nucleotides, buffers, ligase, open circle probes, linkers, circularization sequences, or a combination.”
Feaver did not explicitly disclose including the exonuclease in the kit.
Polansky disclosed (paragraph [0919]): “Well known advantages of commercial kits include convenience and reproducibility due to manufacturing standardization, quality control and validation procedures.”
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the application to package the reagents for performing the method of Feaver into a kit to obtain the advantages of kits described by Polansky.
Claim(s) 3-11, 19-21, 36, 41, 43 and 59-61 is/are rejected under 35 U.S.C. 103 as being unpatentable over Tavakoli (Methods in Enzymology 641:459-476 (2020)) in view of Zhang (US 5,876,924).
Regarding claim 3, Zhang disclosed a method comprising:
(i) contacting a sample with one or more primary probes that bind one or more targets, wherein each primary probe hybridizes to a target, wherein each primary probe comprises one or more secondary probe binding sites and/or one or more readout probe binding sites;
See Fig. 3 and section 4.3, page 467:
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(ii) optionally, hybridizing one or more secondary probes to the primary probes or to one or more targets; wherein each secondary probe comprises one or more tertiary probe binding sites or one or more readout probe binding sites;
See Fig. 3 and section 4.3, page 467:
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(iii) optionally, hybridizing one or more tertiary probes to at least one secondary probe or one or more targets, wherein each tertiary probe comprises one or more quaternary probe binding sites or one or more readout probe binding sites;
See Fig. 3 and section 4.3, page 467:
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(v) hybridizing readout probes capable of detection to the one or more readout probe binding sites;
See Fig. 3 and section 4.3, page 467:
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Regarding claim 4, Tavakoli disclosed (section 4.3, step 4.3.3, page 467): “Perform subsequent rounds of clamFISH [sic, clampFISH] for all the targets at the same time, cycling secondary and tertiary clampFISH probes using the correct series.” Thus, Tavakoli disclosed repeating the claimed steps (ii) and (iii).
Regarding claims 5-8, 10, 19, 20, and 41, Tavakoli disclosed ligating the 5’ and 3’ ends of each primary, secondary and tertiary probe in cis using Click chemistry, which is a chemical ligation between a 5’ alkyne group and a 3’ azide group on each probe; see page 462, first full paragraph and Fig. 1.
Regarding claim 11, Tavakoli’s targets were RNA (which are also transcripts); see Fig. 3.
Regarding claim 21, Tavakoli’s readout probes were oligonucleotides with fluorophores; see page 467, section 4.3, step 4.3.4, “smFISH probes” and see Fig. 3, “fluorescently labeled smFISH probes”. Page 461, second full paragraph, describes smFISH probes as “short DNA oligonucleotide probes” that are “fluorescently labeled”.
Regarding claim 36, Tavakoli imaged the cells; see Fig. 3.
Regarding claim 60, Tavakoli ligated the probes; see Fig. 3, which illustrates multiple primary probes hybridized to the same target RNA; these probes were in “close proximity”. Tavakoli imaged the cells following the addition of the readout probes, thereby determining the proximity of the probes interacting with their targets. Note the limitation of step (ix) is optional.
Regarding claim 61, Tavakoli’s target was RNA, the probes were DNA. Hence a RNA-DNA interaction.
Tavakoli did not disclose (vi) contacting the sample one or more exonucleases each capable of removing probes that bind to targets, primary, secondary, tertiary, or quaternary probes after any steps (i)-(v) to reduce the background signal of probes bound to targets, as recited in claim 3, or the particular exonucleases recited in claim 43. Consequently, Tavakoli did not remove probes bound non-specifically to targets, primary, secondary or tertiary probes using an exonuclease as recited in claim 59, or treat the ligated probes with an exonuclease as recited in claim 60. Tavakoli also did not disclose a “quaternary” probe as recited in claim 9.1
Zhang disclosed a similar method of detecting a target, the method comprising the steps of:
(i) contacting a sample with one or more primary probes that bind one or more targets, wherein each primary probe hybridizes to a target, wherein each primary probe comprises one or more secondary probe binding sites and/or one or more readout probe binding sites;
Fig. 14 and col 20, lines 2-18, italicized comments provided: “In this method, depicted in FIG. 14, the circularizable amplification probe [corresponding to the claimed “primary probe”] contains, as described hereinabove, 3'- and 5' regions that are complementary to adjacent regions of the target nucleic acid [corresponding to the claimed “target”]. The circularizable probes further contain a non-complementary, or generic linker region. In the present signal amplification method, the linker region of the circularizable probe contains at least one pair of adjacent regions that are complementary to the 3' and 5' regions of a first generic circularizable signal probe (CS-probe) [corresponding to the claimed “secondary probe”]. The first CS-probe contains, in its 3' and 5' regions, sequences that are complementary to the adjacent regions of the linker region of the circularizable amplification probe. Binding of the circularizable amplification probe to the target nucleic acid [corresponding to “primary probe hybridizes to a target”], followed by ligation, results in a covalently linked circular probe having a region in the linker available for binding to the 3' and 5' ends of a first CS-probe.”
(ii) optionally, hybridizing one or more secondary probes to the primary probes or to one or more targets; wherein each secondary probe comprises one or more tertiary probe binding sites or one or more readout probe binding sites;
Fig. 14 and col 20, lines 18-26, italicized comments provided: “The addition of the first CS-probe [corresponding to the claimed “secondary probe”] results in binding of its 3' and 5' regions to the complementary regions of the linker of the circular amplification probe [corresponding to “hybridizing one or more secondary probes to the primary probes”]. The 3' and 5' regions of the CS-probe are joined by the ligating agent to form a closed circular CS-probe bound to the closed circular amplification probe. The first CS-probe further contains a linker region containing at least one pair of adjacent contiguous regions designed to be complementary to the 3' and 5' regions of a second CS-probe [corresponding to the claimed “tertiary probe”].”
(iii) optionally, hybridizing one or more tertiary probes to at least one secondary probe or one or more targets, wherein each tertiary probe comprises one or more quaternary probe binding sites or one or more readout probe binding sites;
Fig. 14 and col 20, lines 27-35, italicized comments provided: “The second CS-probe [corresponding to the claimed “tertiary probe”] contains, in its 3' and 5' regions, sequences that are complementary to the adjacent regions of the linker region of the first CS-probe. The addition of the second CS-probe results in binding of its 3' and 5' regions to the complementary regions of the linker of the first CS-probe [corresponding to “hybridizing one or more tertiary probes to at least one secondary probe”]. The 3' and 5' regions of the second CS-probe are joined by the ligating agent to form a closed circular CS-probe, which is in turn bound to the closed circular amplification probe.”
Regarding claim 9, Zhang further disclosed:
(iv) optionally, hybridizing one or more quaternary probes to at least one tertiary probe or one or more targets, wherein each quaternary probe comprises one or more readout probe binding sites;
Fig. 14 and col 20, lines 36-40:
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Regarding claims 3, 59 and 60 Zhang further disclosed:
(vi) contacting the sample one or more exonucleases each capable of removing probes that bind to targets, primary, secondary, tertiary, or quaternary probes after any steps (i)-(v) to reduce the background signal of probes bound to targets.
Col 19, line 28: “After probe ligation, i.e. circularization, treatment of the reaction mixture with an exonuclease provides a "clean-up" step and thus reduces background and carryover contamination by digesting unligated probes or linear DNA fragments but not closed circular molecules. The covalently linked circular molecules remain intact for subsequent amplification and detection.”
Regarding claim 43, Zhang disclosed (col 19, line 38): “In a preferred embodiment, the exonuclease is exonuclease III, exonuclease VII, mung bean nuclease or nuclease BAL-31.”
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the application to modify the method of Tavakoli by incorporating a step of treating with exonuclease as taught by Zhang after the ligation steps and prior to the addition of the readout probes in order to obtain the benefit of reducing “background and carryover contamination by digesting unligated probes or linear DNA fragments but not closed circular molecules”.
It would also have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the application to modify the method of Tavakoli by incorporating quaternary probes as in the method of Zhang, as this represents nothing more than the combination of prior art elements according to known techniques to yield predictable results (MPEP 2143(I)(A)). In this case, the addition of quaternary probes would have provided for increased signal by incorporating yet another level of signal amplification.
In making such modifications, one would have arrived at the claimed invention. Regarding claim 59, the addition of an exonuclease step would inherently remove any non-specifically bound probes in which proper alignment of the 5’ and 3’ ends of a probe did not occur, thereby preventing ligation.
Claim(s) 1 is/are rejected under 35 U.S.C. 103 as being unpatentable over Tavakoli (Methods in Enzymology 641:459-476 (2020)) in view of Zhang (US 5,876,924) as applied to claims 3-11, 19-21, 36, 41, 43 and 59-61 above, and further in view of Polansky (US 2004/0023207).
The disclosures of Tavakoli and Zhang have been discussed. Tavakoli did not mention putting the materials for performing the method into a “kit”. Zhang did disclose kits; col 25, line 7: “Reagents for use in practicing the present invention may be provided individually or may be packaged in kit form.”
Polansky disclosed (paragraph [0919]): “Well known advantages of commercial kits include convenience and reproducibility due to manufacturing standardization, quality control and validation procedures.”
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the application to package the reagents for performing the method suggested by the combined disclosures of Tavakoli and Zhang into a kit to obtain the advantages of kits described by Polansky.
Claim(s) 36-39 and 44-45 is/are rejected under 35 U.S.C. 103 as being unpatentable over Tavakoli (Methods in Enzymology 641:459-476 (2020)) in view of Zhang (US 5,876,924) as applied to claims 3-11, 19-21, 36, 41, 43 and 59-61 above, and further in view of Cai (US 2015/0267251).
The disclosures of Tavakoli and Zhang have been discussed. Tavakoli did not disclose the optional limitation of claim 36: repeating the contacting and imaging steps, each time with a new plurality of detectably labeled readout probes, so that a target nucleic acid in the sample/cell is described by a barcode, determined by the repeated contacting and imaging steps, that can be differentiated from the barcodes of the other target nucleic acids. Likewise, Tavakoli did not disclose the limitations recited in claims 37-39 or 44-45.
Cai disclosed a method “for high-throughput profiling of a large number of targets, such as transcripts and/or DNA loci” based on “sequential barcoding” (abstract). More specifically, Cai disclosed “methodologies in which multiple rounds of hybridization (contacting steps) with labeled probes profiles nucleic acids (e.g., mRNAs) present in cells. Specifically, as depicted in FIG. 1, sets of probes that hybridize with nucleic acid targets in cells are provided, wherein probes (i.e., detectably labeled oligonucleotides that hybridize with different targets) are labeled within a single set and, furthermore, at least one probe is differently labeled in different sets.”
See Fig. 1 and paragraphs [0085]-[0091] (emphasis provided):
[0085] In some embodiments, the present invention (e.g., as represented in FIG. 1), provides methods comprising steps of:
(a) performing a first contacting step that involves contacting a cell comprising a plurality of transcripts and DNA loci with a first plurality of detectably labeled oligonucleotides, each of which targets a transcript or DNA locus and is labeled with a detectable moiety, so that the composition comprises at least:
[0086] (i) a first oligonucleotide targeting a first transcript or DNA locus and labeled with a first detectable moiety; and
[0087] (ii) a second oligonucleotide targeting a second transcript or DNA locus and labeled with a second detectable moiety; (b) imaging the cell after the first contacting step so that hybridization by oligonucleotides of the first plurality with their targets is detected; (c) performing a second contacting step that involves contacting the cell with a second plurality of detectably labeled oligonucleotides, which second plurality includes oligonucleotides targeting overlapping transcripts and/or DNA loci that are targeted by the first plurality, so that the second plurality comprises at least:
[0088] (i) a third oligonucleotide, optionally identical in sequence to the first oligonucleotide, targeting the first transcript or DNA locus; and
[0089] (ii) a fourth oligonucleotide, optionally identical in sequence to the second oligonucleotide, targeting the second transcript or DNA locus, wherein the second plurality differs from the first plurality in that at least one of the oligonucleotides present in the second plurality is labeled with a different detectable moiety than the corresponding oligonucleotide targeting the same transcript or DNA locus in the first plurality, so that, in the second plurality:
[0090] (iii) the third oligonucleotide is labeled with the first detectable moiety, the second detectable moiety or a third detectable moiety; and
[0091] (iv) the fourth oligonucleotide is labeled with the first detectable moiety, the second detectable moiety, the third detectable moiety, or a fourth detectable moiety, wherein either the third oligonucleotide is labeled with a different detectable moiety than was the first oligonucleotide, or the fourth oligonucleotide is labeled with a different detectable moiety than was the second oligonucleotide, or both; (d) imaging the cell after the second contacting step so that hybridization by oligonucleotides of the second plurality with their targets is detected; and (e) optionally repeating the contacting and imaging steps, each time with a new plurality of detectably labeled oligonucleotides comprising oligonucleotides that target overlapping transcripts or DNA loci targeted by the first and second pluralities, wherein each utilized plurality differs from each other utilized plurality, due to at least one difference in detectable moiety labeling of oligonucleotides targeting the same transcript or DNA locus.
The only difference is that, the probes either hybridize directly to the target (e.g., Fig. 2a), or to a “bridging strand” hybridized to an “intermediate strand” hybridized directly to the target (in the context of hybridization chain reaction (HCR); Fig. 21a, paragraph [0310]), rather than hybridizing to circularized probes that are associated with the target (as in the method of Tavakoli).
In addition, Cai disclosed removing the detectably-labeled probes after each imaging step using DNase I; see Fig 2a.
It would have been prima facie obvious to one of ordinary skill in the art to use the same “sequential barcoding” concept disclosed by Cai in the method suggested by the combined disclosures of Tavakoli and Zhang, in order to confer on the method suggested by the combined teachings of Tavakoli and Zhang the benefit of high-throughput multiplexing of target detection. The only modification that would have been needed would have been to design the detectably-labeled probes to hybridize to the circularized “amplifiers” in the method of Tavakoli, rather than to the RNA itself. In doing so, one would have arrived at the invention of claims 36-39 and 44-45.
Claim(s) 3, 5-11, 14, 21, 41, 43 and 59 is/are rejected under 35 U.S.C. 103 as being unpatentable over Zhang (US 5,876,924) in view of Tavakoli (Methods in Enzymology 641:459-476 (2020)).
Regarding claims 3 and 41, Zhang disclosed a method of detecting a target, the method comprising the steps of:
(i) contacting a sample with one or more primary probes that bind one or more targets, wherein each primary probe hybridizes to a target, wherein each primary probe comprises one or more secondary probe binding sites and/or one or more readout probe binding sites;
Fig. 14 and col 20, lines 2-18, italicized comments provided: “In this method, depicted in FIG. 14, the circularizable amplification probe [corresponding to the claimed “primary probe”] contains, as described hereinabove, 3'- and 5' regions that are complementary to adjacent regions of the target nucleic acid [corresponding to the claimed “target”]. The circularizable probes further contain a non-complementary, or generic linker region. In the present signal amplification method, the linker region of the circularizable probe contains at least one pair of adjacent regions that are complementary to the 3' and 5' regions of a first generic circularizable signal probe (CS-probe) [corresponding to the claimed “secondary probe”]. The first CS-probe contains, in its 3' and 5' regions, sequences that are complementary to the adjacent regions of the linker region of the circularizable amplification probe. Binding of the circularizable amplification probe to the target nucleic acid [corresponding to “primary probe hybridizes to a target”], followed by ligation, results in a covalently linked circular probe having a region in the linker available for binding to the 3' and 5' ends of a first CS-probe.”
(ii) optionally, hybridizing one or more secondary probes to the primary probes or to one or more targets; wherein each secondary probe comprises one or more tertiary probe binding sites or one or more readout probe binding sites;
Fig. 14 and col 20, lines 18-26, italicized comments provided: “The addition of the first CS-probe [corresponding to the claimed “secondary probe”] results in binding of its 3' and 5' regions to the complementary regions of the linker of the circular amplification probe [corresponding to “hybridizing one or more secondary probes to the primary probes”]. The 3' and 5' regions of the CS-probe are joined by the ligating agent to form a closed circular CS-probe bound to the closed circular amplification probe. The first CS-probe further contains a linker region containing at least one pair of adjacent contiguous regions designed to be complementary to the 3' and 5' regions of a second CS-probe [corresponding to the claimed “tertiary probe”].”
(iii) optionally, hybridizing one or more tertiary probes to at least one secondary probe or one or more targets, wherein each tertiary probe comprises one or more quaternary probe binding sites or one or more readout probe binding sites;
Fig. 14 and col 20, lines 27-35, italicized comments provided: “The second CS-probe [corresponding to the claimed “tertiary probe”] contains, in its 3' and 5' regions, sequences that are complementary to the adjacent regions of the linker region of the first CS-probe. The addition of the second CS-probe results in binding of its 3' and 5' regions to the complementary regions of the linker of the first CS-probe [corresponding to “hybridizing one or more tertiary probes to at least one secondary probe”]. The 3' and 5' regions of the second CS-probe are joined by the ligating agent to form a closed circular CS-probe, which is in turn bound to the closed circular amplification probe.”
(iv) optionally, hybridizing one or more quaternary probes to at least one tertiary probe or one or more targets, wherein each quaternary probe comprises one or more readout probe binding sites;
Fig. 14 and col 20, lines 36-40:
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(vi) contacting the sample one or more exonucleases each capable of removing probes that bind to targets, primary, secondary, tertiary, or quaternary probes after any steps (i)-(v) to reduce the background signal of probes bound to targets.
Col 19, line 28: “After probe ligation, i.e. circularization, treatment of the reaction mixture with an exonuclease provides a "clean-up" step and thus reduces background and carryover contamination by digesting unligated probes or linear DNA fragments but not closed circular molecules. The covalently linked circular molecules remain intact for subsequent amplification and detection.”
Regarding claims 5-10 and 14, Zhang ligated the probes using either DNA or RNA ligase (see claim 8), which are enzymes.
Regarding claim 11, Zhang’s target was RNA (which is also a transcript); see Fig. 14.
Regarding claim 43, Zhang disclosed (col 19, line 38): “In a preferred embodiment, the exonuclease is exonuclease III, exonuclease VII, mung bean nuclease or nuclease BAL-31.”
Regarding claim 59, the exonuclease step would inherently remove any non-specifically bound probes in which proper alignment of the 5’ and 3’ ends of a probe did not occur, thereby preventing ligation.
Zhang did not disclose addition of “readout probes” as recited in claims 3 and 21.
Tavakoli disclosed a method similar to Zhang, in which target RNA was detected by successive hybridization and ligation of primary, secondary and tertiary probes; see Fig. 3. For detection, Tavakoli hybridized fluorescently-labeled short DNA probes (readout probes) to the final set of circularized probes; see Fig. 3, “fluorescently labeled smFISH probes”. Page 461, second full paragraph, describes smFISH probes as “short DNA oligonucleotide probes” that are “fluorescently labeled”.
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the application to modify the method of Zhang by detecting the complex of circularized probes using fluorescently-labeled oligonucleotides as in the method of Tavakoli. This represents nothing more than combining prior art elements according to known techniques to yield predictable results; it represents using a known technique for detecting complexes of circularized probes bound to a target.
Claim(s) 1 is/are rejected under 35 U.S.C. 103 as being unpatentable over Zhang (US 5,876,924) in view of Tavakoli (Methods in Enzymology 641:459-476 (2020)) as applied to claims 3, 5-11, 14, 21, 41, 43 and 59 above, and further in view of Polansky (US 2004/0023207).
The disclosures of Zhang and Tavakoli have been discussed. Zhang did disclosed kits; col 25, line 7: “Reagents for use in practicing the present invention may be provided individually or may be packaged in kit form.”
Polansky disclosed (paragraph [0919]): “Well known advantages of commercial kits include convenience and reproducibility due to manufacturing standardization, quality control and validation procedures.”
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the application to package the reagents for performing the method suggested by the combined disclosures of Zhang and Tavakoli into a kit to obtain the advantages of kits described by Polansky.
Conclusion
No claims are free of the prior art.
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/SAMUEL C WOOLWINE/ Primary Examiner, Art Unit 1681
1 Tavakoli disclosed multiple rounds of alternating addition of secondary and tertiary probes. In the context of this rejection, a “quaternary” probe is construed as being of a different structure than either the primary, secondary or tertiary probes, not simply the addition of another layer of secondary or tertiary probes.
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