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 .
Claims 1, 6, 12, 14-15, 18, 26, 37-38, 40, 43-45, 56-57, 59, and 89-92 are pending and under examination. Claims 1, 18, 37, 40, 57, 59, and 90 are amended. Claims 2-5, 7-11, 13, 16-17, 19-25, 27-36, 39, 41-42, 46-55, 58, and 60-88 are cancelled.
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 1/15/26 has been entered.
Priority
The instant application 17/738,538 filed on 5/6/22 claims domestic priority to provisional application 63/185,944 filed 5/7/21. The priority date is determined to be 5/7/21.
Response to Arguments
Applicant's arguments, see pages 6-12, filed 1/15/26, with respect to the rejections of claims 1, 6, 12, 14-15, 18, 26, 37-38, 40, 43-45, 56-57, 59, and 89-92 under 35 USC 103 have been fully considered and are found persuasive. Therefore, the 35 USC 103 rejections documented in the Final mailed on 10/15/25 have been withdrawn. However, upon further consideration, new grounds of rejections necessitated by claim amendments filed 1/15/26 are made in this Non-Final Office Action.
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.
Claims 1, 6, 12, 14-15, 18, 26, 37-38, 40, 43-45, 56-57, 59, and 89-92 are rejected under 35 U.S.C. 103 as being unpatentable over Samusik et al. (2017; WO 2017/147483 A1) in view of Fujimoto et al. (2019; NPL citation 227 in IDS filed 7/5/22; “DNA photo-cross-linking using a pyranocarbazole-modified oligodeoxynucleotide with a D-threoninol Linker”; RSC Adv, 2019, 9, 30693; DOI: 10.1039/c9ra06145b) and Ohtsuka et al. (1985; NPL citation 252 on IDS filed 7/25/22; “An alternative approach to deoxyoligonucleotides as hybridization probes by insertion of deoxyinosine at ambiguous codon positions”; J Biol Chem. 1985 Mar 10;260(5):2605-8; https://doi.org/10.1016/S0021-9258(18)89400-5).
This rejection is necessitated by claim amendments filed 1/15/26.
(i) Samusik et al. teaches limitations relevant to claims 1, 26, 44-45, 56-57, 59, and 89-91.
Relevant to claim 1 (a), Samusik et al. paragraphs 0012-0014 teach their Splint Nucleotide Assisted Intramolecular Ligation followed by Rolling Circle Amplification (SNAIL-RCA), which requires two Padlock Oligonucleotides (Pos; first/second circular probe or circularizable probe) and two Splint Primer Oligonucleotides (SPOs; first/second oligonucleotide) that hybridize to target mRNA sequences. Relevant excerpts include: “In the methods of the invention, mRNA present in a cell of interest serves as a scaffold for an assembly of a complex that comprises two oligonucleotides, referred to herein as Splint Primer Oligonucleotide (SPO) and Padlock Oligonucleotide (PO)… Each of SPO and PO comprise a first complementarity region (CR1 and CR1', respectively) that are complementary to adjacent sequences on the target mRNA… Each of SPO and PO further comprise a second complementarity region (CR2 and CR2') located adjacent to CR1 or CR1’. CR2', which is present on PO, is a split region, where the 5' and the 3' ends of PO hybridize to CR2 in a butt head-to-end fashion, such that after the hybridization the 5' and the 3' ends of PO are positioned directly adjacent to one another. A schematic is shown in Figure 1.”
Further relevant to claim 1 (a), Samusik et al. paragraphs 0023 and 0043 teach limitations for common primer region for the SPOs and POs. Relevant excerpts include: “For example, in the detection of a fusion event, if a first gene is fused to a second gene the SNAIL method can be adapted, where primers can be targeted to gene 1, with the SPO sequence; and a PO probe targeted to gene 2. A signal is obtained only when the fusion transcript is present, as the individual probes do not give rise to an amplification product. A plurality of individual primers may be designed for each of gene 1 and gene 2, e.g. 2, 3, 4, 5, 6 or more… A plurality of oligonucleotide pairs can be used in a reaction, where one or more pairs specifically bind to each target nucleic acid. For example, two primer pairs can be used for one target nucleic acid in order to improve sensitivity and reduce variability.”
Further relevant to claim 1 (a), Samusik et al. paragraphs 0042 and 0044 teach common hybridization region limitations for hybridization to target-adjacent sequences. Relevant excerpts include: “Each of SPO and PO comprise a first complementarity region (CR1 and CR1 ', respectively) that are complementary to adjacent sequences on the target mRNA. Each of SPO and PO further comprise a second complementarity region (CR2 and CR2') located adjacent to CR1 or CR1 '… The target binding site binds to a region of the target nucleic acid. In a pair, each target site is different, and the pair are complementary adjacent sites on the target nucleic acid… and may be contiguous sites. Target sites are typically present on the same strand of the target nucleic acid in the same orientation.”
Relevant to claim 1 (c), Samusik et al. paragraph 0046 and claim 1 teach rolling circle amplification using the PO as a template and the SPO as a primer. Relevant excerpts include: “A single-stranded, circular polynucleotide template is formed by ligation of the PO, which circular polynucleotide comprises a region that is complementary to the SPO probe. Upon addition of a DNA polymerase in the presence of appropriate dNTP precursors and other cofactors, the SPO probe is elongated by replication of multiple copies of the template… [claim 1] … performing rolling circle amplification using the PO as a template and SPO as a primer for a polymerase…”
Relevant to claims 26 and 44, Samusik et al. paragraphs 0018-0019, 00130, and 00138 teach simultaneous analysis of multiple targets in single cells. Relevant excerpts include: “The methods of the invention enable cost-efficient detection of specific nucleic acids in single cells, and may be combined with flow cytometry or mass cytometry to simultaneously analyze large numbers of cells for a plurality of nucleic acids… An advantage of SNAIL includes the ability to simultaneously analyze multiple nucleic acids and proteins in single cells… An advantage of SNAIL includes the ability to simultaneously analyze multiple nucleic acids and proteins in single cells… We created a simple two-probe proximity ligation system termed SNAIL-RCA that enables in situ amplification, detection and visualization of genes… This simplified design allows utilizing the same complementarity/ligation sequence for many different mRNAs, only varying the target hybridization sequence. We implemented multiplexed imaging by iterative reannealing of fluorescent detection probes onto the same specimen. With this approach we have detected the expression of 24 genes in OVCAR4 cells (Figure 3).”
Relevant to claim 45, Samusik et al. paragraphs 0014, 0019, 0042, and 0046 teach PO circularization through ligation. Relevant excerpts include; “In the linear form of PO, the 5' terminus is phosphorylated, so that upon annealing of both ends to CR2, the oligonucleotide can be circularized by ligation, using any suitable DNA ligase enzyme, e.g. T4 DNA ligase… performing a ligation reaction, in which PO probes, is suitably hybridized to the splint (SPO) are ligated to generate a circle… A single-stranded, circular polynucleotide template is formed by ligation of the PO, which circular polynucleotide comprises a region that is complementary to the SPO probe.”
Relevant to claim 56, Samusik et al. paragraphs 0048 and 00130 teach in situ detection. Relevant excerpts include: “The presence and quantitation of an amplified SNAIL padlock sequence in a cell may be determined by contacting the cell with an oligonucleotide probe under conditions in which the probe binds to the amplified product…We created a simple two-probe proximity ligation system termed SNAIL-RCA that enables in situ amplification, detection and visualization of genes.”
Relevant to claim 57, Samusik et al. paragraph 0017 teaches hybridization and dehybridization of detectably-labeled probes bound to the RCA products. Relevant excerpts include: “RCA product can be detected by various methods, which include, without limitation, hybridization to a sequence specific detection oligonucleotide (DO), also referred to as a detection probe. In some embodiments the DO is conjugated to a detectable label, e.g. fluorophore, lanthanide, biotin, radionuclide, etc., where the label may be detectable by optical microscopy, SIMS ion beam imaging, etc. In some embodiments the DO is unlabeled, where the presence of the DO can be detected in a polymerization reaction primed by the DO, and where the polymerization reaction may comprise one or more dNTP comprising a detectable label. Such polymerization products may further comprise a step of adding a label, detecting a label, and removing the label for sequential detection of different products.”
Relevant to claim 59, Samusik et al. paragraphs 0017 and 0050-0051 teach intermediate probes and hybridization and dehybridization of detectably-labeled probes bound to the RCA products. Relevant excerpts include: “Such polymerization products may further comprise a step of adding a label, detecting a label, and removing the label for sequential detection of different products… A ‘label’ or ‘label moiety’ for a nucleic acid probe is any moiety that provides for signal detection and may vary widely depending on the particular nature of the assay. Label moieties of interest include both directly and indirectly detectable labels… An antibody that specifically binds to an antigenic label can be directly or indirectly detectable. For example, the antibody can be conjugated to a label moiety (e.g., a fluorophore) that provides the signal (e.g., fluorescence); the antibody can be conjugated to an enzyme (e.g., peroxidase, alkaline phosphatase, etc.) that produces a detectable product (e.g., fluorescent product) when provided with an appropriate substrate…”
Relevant to claims 89 and 91, Samusik et al. paragraph 0043 teaches two primer pairs designed towards the same target. Relevant excerpts include: “For example, two primer pairs can be used for one target nucleic acid in order to improve sensitivity and reduce variability.”
Relevant to claim 90, Samusik et al. paragraph 0043 teaches detection of 2 different cellular RNAs. Relevant excerpts include: “It is also of interest to detect a plurality of different target nucleic acids in a cell, e.g. detecting up to 2… or more distinct target nucleic acids.”
(ii) Samusik et al. is silent to specifics regarding photoreactive nucleotides (relevant to claims 1, 6, 12, 14-15, 18, 38, 45, and 92). However, these limitations were known in the prior art and taught by Fujimoto et al.
Relevant to claim 1 (a) and (b), Fujimoto et al. Introduction teaches photo-activation of photoreactive nucleotides and crosslinking of nucleic acids. Relevant excerpts include: “Photo-cross-linking reactions between biomolecules are used for various applications such as screening antigen interactions' and improving detection sensitivity [citation] and the stability of biomolecular complexes… We report pyranocarbazole (PCX) as a photo-cross-linker that can photo-crosslink to pyrimidine in complementary DNA or RNA strand under visible light. [citation] It was anticipated that his photo-cross-linker would accelerate the intracellular application of nucleic acid photo-cross-linking such as photochemical regulation of gene expression [expression] and detection of RNA strand…”
Relevant to claims 6, 12, 14-15, 18, 38, 45, and 92, Fujimoto et al. Introduction teaches psoralen, vinylcarbazone, and 3-cyanovinylcarbazole modified D-threoninol photoreactive nucleotides and nucleic acid crosslinking. Relevant excerpts include: “In particular, the use of DNA photocross-linking in the formation of a thymine dimer induced by UV-irradiation [citation] and interstrand photo-cross-linking with psoralen [citation] has been reported… It was determined that 3-cyanovinylcarbazole modified D-threoninol (CNVD) [citation] considerably accelerated the photo-cross-linking reaction with cytosine using the D-threoninol backbone (PCXD) (Fig. 1).”
(iii) Samusik et al. and Fujimoto et al. are silent to specifics regarding universal bases (relevant to claims 1, 6, 37-38, 40, 43, and 92). However, these limitations were known in the prior art and taught by Ohtsuka et al.
Relevant to claims 1 (a), 6, 37-38, 40, 43, and 92, Ohtsuka et al. teaches probes with base analogs (universal bases) that can 'pair' with any of the four natural nucleic acid bases (page 2605). Relevant excerpts include: “use an appropriate base analog that can ‘pair’ with any of the four natural bases at the ambiguous positions, with or without hydrogen bonding. Here, we present findings resulting from the use of 2'-deoxyinosine for such purposes. Inosine is occasionally found in the first position (the wobble position) of tRNA anticodons [citation] and is known to form base pairs with A, C, and U in the decoding process. We have synthesized two oligonucleotides (23-mer and 26-mer), both containing five deoxyinosine molecules at the wobble positions, and shown that these oligonucleotides can be used as hybridization probes, and that at least one of them (the 26-mer) can be a useful probe for screening genes in human genomic libraries.”
(iv) Although Samusik et al. does not explicitly teach the Fujimoto et al. photoreactive nucleotides or Ohtsuka et al. universal bases, it would have been prima facie obvious to the skilled artisan. It is noted that Samusik et al., Fujimoto et al., and Ohtsuka et al. are all analogous disclosures to the instant nucleic acid detection.
The skilled artisan would have been motivated to combine the analogous art. Fujimoto et al. provides the motivation for photoreactive nucleotide crosslinking, as it enables improved detection sensitivity, stability of biomolecular complexes, photoreversible manipulation, and detection of RNA (Introduction). The skilled artisan would be motivated to include the Fujimoto et al. photoreactive nucleotides within the hybridization region of Samusik et al. for the above improvements.
Additionally, the skilled artisan would have been motivated to include the Ohtsuka et al. universal bases within the Samusik et al. hybridization region because Ohtsuka et al. teaches that hybridization probes containing universal bases overcome the issues of screening high complexity libraries associated with longer, cross-reactive probes (page 2605).
The skilled artisan would have a reasonable expectation of success based on the disclosure of Samusik et al. in view of Fujimoto et al. and Ohtsuka et al., as discussed in preceding paragraphs.
Conclusion
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/SARAH JANE KENNEDY/Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682