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 .
This Office Action is in reply to Applicants' correspondence of 09/12/2025. Applicants' remarks and amendments have been fully and carefully considered but are not found to be sufficient to put the application in condition for allowance. Any new grounds of rejection presented in this Office Action are necessitated by Applicants' amendments. Any rejections or objections not reiterated herein have been withdrawn in light of the amendments to the claims or as discussed in this Office Action. This Action is made FINAL.
Please Note: The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claim Status
Claims 60 and 77 have been cancelled. Claims 59, 61-62, 69, 71-73 have been amended. Claims 79-80 have been added. Claims 59, 61-76, 78-80 are pending and under examination and discussed in this Office Action.
Specification – Withdrawn
The objections to the specification as forth of the Office Action of 02/13/2026 are withdrawn in light of the amendment to the specification provided by applicant in the reply of 05/12/2026, which are entered.
Claim Objection – Withdrawn
The objection to claim 71, as set forth in the Office Action of 02/13/2026 is withdrawn in light of the amendments to the claims.
Claim Rejections - 35 USC § 112(b) - Withdrawn
The rejections of claims 72 and 73 under 35 USC § 112(b), as set forth in the Office Action of 02/13/2026 are withdrawn in light of the amendments to the claims.
Claim Rejections - 35 USC § 112(b) – Newly Applied as Necessitated by Claim Amendments
Claim 71 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 59 introduces two distinct entities:
“a first probe comprising a first overhang region and a first recognition sequence” and
“the first overhang region is bound to a first detectably labeled probe”
Claim 59 recites “detecting a first signal associated with the first detectably labeled probe” attributing the first signal to entity (2), the first detectably labeled probe.
However, claim 71 recites “the first probe is covalently or non-covalently bound to a first detectable label for producing the first signal” attributing the first signal to entity (1), the first probe. it is unclear which entity produces the first signal, as claim 59 and claim 71 assign signal production to different entities. The scope of claim 71 is therefore indeterminate.
Claim Rejections - 35 USC § 103 – Modified as Necessitated by Amendment
Claims 59, 61-62, 65-66, 68, 70, 72-74, 76, 78-80 are rejected under 35 U.S.C. 103 as being unpatentable over Gyllborg et al. (2020) in view of Fehr (US20100092960A1).
Regarding instant Claim 59, Gyllborg teaches a method for analyzing a biological sample comprising sequential rounds of probe hybridization, detection, and removal in mouse brain tissue sections and human brain tissue sections (p. 2, right column, para 2; p. 1, left column, para 1).
Gyllborg teaches a bridge-probe comprising a 17 base pair recognition sequence complementary to the ID sequence of the rolling circle product (RCA) in the tissue section (first recognition sequence bound to target nucleic acid in the biological sample) and a 20 nucleotide tail sequence (first overhang region) bound to a readout detection probe conjugated in a fluorophore (first detectably labeled probe) (p. 4, left column, para 3; Fig. 1B).
Gyllborg teaches that after hybridization of bridge-probes and readout detection probes, the tissue sections are imaged and fluorescent signals from the readout detection probes are detected at the spatial locations of the RCPs within the tissue section (p. 3, left column, para 2). Following imaging, the bridge-probes and readout detection probes are removed from the RCPs (p. 3, left column, para 3).
Gyllborg does not teach contacting the biological sample with a helicase to remove the first probe. Gyllborg uses a chemical stripping solution (65% formamide, 2XSSC for 30 min at 30oC) to remove bridge-probes and readout detection probes. This deficiency is made up in the teachings of Fehr.
Fehr teaches methods in which a helicase dissociates hybridized probes from a nucleic acid template (Abstract; [0009; 0026; 0032]). Fehr teaches that helicases are NTP-dependent motor proteins that use the energy produced by nucleic acid dependent NTP hydrolysis to catalyze the separation of two strands of a complementary nucleic acid duplex, thereby removing hybridized probes from a nucleic acid template [0056].
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to substitute the formamide based chemical stripping of Gyllborg with helicase-mediated probe removal as taught by Fehr, in order to achieve gentler enzymatic probe removal that preserves tissue integrity across multiple imaging cycles. This motivation is supported by Gyllborg’s discussion, which notes that “the harsher stripping conditions of SBL could be affecting tissue integrity” (p. 9, left column, para 2), providing a direct reason for a person of ordinary skill in the art to seek a less chemically harsh probe removal method. Fehr directly provides helicase- mediated probe displacement as such an alternative enzymatic approach.
Gyllborg further teaches that the next cycle of bridge-probes (second probe) is hybridized to the RCPs following the stripping step (p. 3, left column, para 3). Each cycle’s bridge-probe comprises the same 17bp recognition sequence binding the ID sequence on the RCP (second recognition sequence complementary to a second target sequence of the plurality) and a different 20 nucleotide tail sequence specific to that cycle (second overhang region), bound to a second readout detection probe conjugated to a fluorophore (second detectably labeled probe) (p. 4, left column, para 3 – right column, para 1; Fig. 1B; Supplementary Figure S1B).
Gyllborg teaches detecting a second signal associated with the second detectably labeled probe at the same location in the biological sample. The second cycle readout detection probe generates a fluorescent signal at the same RCP locations imaged in the first cycle (p. 3, left column, para 2; Fig. 1C).
The target nucleic acid is the RCP, which contains multiple repetitive ID sequence targets constituting the “plurality of target sequences” (p. 4, left column, para 3).
Therefore, the combination of Gyllborg and Fehr teaches each limitation of claim 59.
Regarding instant Claim 61, Gyllborg teaches that bridge-probes in successive cycles use the same 17 base pair recognition sequence complementary to the same ID sequence on the RCP (p. 4, left column, para 3). Since the first and second recognition sequences are each complementary to the same ID sequence, the first and second target sequences are identical in sequence and position, constituting complete overlap.
Regarding instant Claim 62, Gyllborg teaches that the target nucleic acid is an RCA product of padlock probes (PLPs), which are circularizable probes that ligate to and bind cDNA derived from mRNA in the biological sample (Fig. 1; p. 2, right column, para 4 – p. 3, left column, para 1). Gyllborg further teaches that the RCP contains multiple ID sequences constituting a combinatorial barcode (p. 4, left column, para 3), which are the plurality of barcode sequences.
Regarding instant Claim 65, Fehr teaches that the helicases used in the probe displacement methods includes UvrD, Rep, and RecQ (claim 13; [0056]). Rep-X is an engineered variant of Rep, and Tte UvD is a thermophilic variant of UvrD, each of which falls within the “homolog or variant thereof”. RecQ is explicitly taught by Fehr.
It would have been obvious to one of ordinary skill in the art to use any of the helicases recited in claim 65, or homologs or variants thereof, in the methods of Gyllborg for the purpose of helicase-mediated probe removal as described above.
Regarding instant Claim 66, Fehr teaches that the helicase compositions and methods include ATP (claim 18; [0013]).
It would have been obvious to one of ordinary skill in the art to contact the helicase with the biological sample in a buffer comprising ATP, as ATP is required for helicase activity as taught by Fehr.
Regarding instant Claim 68, Fehr teaches that helicase are NTP-dependent motor proteins that use the energy produced by nucleic acid dependent NTP hydrolysis to catalyze strand separation [0056]. ATP is a nucleoside triphosphate (NTP). Therefore, the helicase dissociates the first probe from the target nucleic acid in an ATP-dependent reaction as taught by Fehr.
Regarding instant Claim 70, Gyllborg teaches that after the stripping step, tissue sections are washed with 2XSSC (five washes) to remove the stripping solution before the next cycle of bridge-probes is introduced (p. 3; left column, para 3).
A person of ordinary skill in the art would recognize that the helicase must be removed from the biological sample prior to introduction of the second probe to prevent the active helicase from immediately dissociating the newly hybridized second probe before the second signal can be detected.
Regarding instant Claim 71, Gyllborg teaches bridge-probes and readout detection probes that generate fluorescent signals at RCP locations in tissue. The detectable labels (fluorophores) are conjugated to the readout detection probes (first and second detectably labeled probes of claim 59) and not directly to the bridge probe (first and second probes of claim 59).
It is noted that claim the limitations of claim 71 are rejected earlier in this Office Action as unclear under 35 USC § 112(b) as set forth above. Is so far as the teachings of Gyllborg are relevant to labeled probes (as required by the claim), the claim is also addressed here as being met by the teachings of the cited prior art.
Regarding instant Claim 72, Gyllborg teaches that each imaging cycle uses a cycle dependent bridge-probe library in which the 20 nucleotide tail sequence of the bridge probe differs between cycles, with each cycle’s tail sequence designed to bind a specific readout detection probe bearing a specific fluorophore (p. 4, left column, para 3 – right column, para 1; Fig. 1B; Supplementary Figure S1B). The first cycle bridge probe tail (first overhang region) and the second cycle bridge probe tail (second overhang region) are therefore different sequences.
Regarding instant Claim 73, Gyllborg teaches that bridge-probes comprise a recognition sequence, a non-complementary linker, and a tail sequence for binding a fluorophore conjugated readout detection probe (p. 4, left column, para 3). No quenching moiety is present in either the bridge probe or the readout detection probe as described in Gyllborg. The system relies on direct fluorophore conjugation without a quencher fluorophore, satisfying the limitation of claim 73 that the first and second probes do not comprise a moiety that quenches the first and second signals.
Regarding instant Claim 74, Gyllborg teaches sequential rounds of hybridization, detection, and stripping across multiple cycles (p. 4, right column, para 2; Fig. 1C). After detecting the second signal, the same stripping procedure is applied to remove the second bridge probe before the third cycle (p. 3, left column, para 3).
Under the combination with Fehr, this stripping step constitutes contacting the biological sample with a helicase after detecting the second signal to dissociate the second probe from the target nucleic acid.
Regarding instant Claim 76, as discussed regarding claim 62 above, Gyllborg teaches that the target nucleic acid is a rolling circle amplification product of padlock probes (circularizable probes) that bind to cDNA (nucleic acid molecule) in the biological sample (Fig. 1; p. 2, right column, para 4 – p. 3, left column, para 1).
Regarding instant Claim 78, Gyllborg teaches that the biological sample is a tissue sample, specifically mouse brain tissue sections and human middle temporal gyrus tissue sections (p. 2, right column, para 2; p. 1, left column, para 1).
Regarding instant Claim 79, Gyllborg teaches that PLPs are designed with two 15 nucleotide target complementary arm sequences flanking a backbone comprising a 20 nucleotide ID sequence and a 20 nucleotide anchor sequence, resulting in a final 70 nucleotide PLP (p. 4, left column, para 3). In the RCP, successive copies of the ID sequence (first and second target sequences to which bridge probe recognition sequences are complementary) are therefore separated by approximately 50 nucleotides of arm and anchor sequences, constituting the intervening sequences between consecutive ID sequence copies in each tandem repeat unit of the RCP, such that the first and second target sequences are interspersed with other sequences in the target nucleic acid (p. 4, left column, para 3).
Regarding instant Claim 80, depends from claim 59 and is rejected for the same reasons as claim 59. The examiner notes that the “immediately adjacent” limitation of claim 80 is not explicitly taught by Gyllborg, wherein successive ID sequence copies in the RCP are separated by intervening arm and anchor sequences of approximately 50 nucleotides rather than being immediately adjacent.
Claims 63-64, 67, and 69 are rejected under 35 U.S.C. 103 as being unpatentable over Gyllborg at al. (2020) in view of Fehr (US20100092960A1), as applied to claim 59 above, and further in view of Rajagopal and Patel (2007).
Regarding instant Claim 63, Gyllborg and Fehr, as applied to claim 59, do not teach contacting the biological sample with a single-stranded binding protein simultaneously with or after contacting the helicase.
Rajagopal and Patel teach that single-strand binding proteins enhance the unwinding processivity of helicase-mediated strand separation by stabilizing the helicase at the unwinding junction and preventing its dissociation (p. 3, para 2).
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to contact the biological sample with a single-stranded binding protein simultaneously with or after contacting with the helicase, in order to enhance helicase-mediated probe dissociation and improve the efficiency and completeness of probe removal.
Regarding instant Claim 64, Rajagopal and Patel teaches that single-strand binding proteins bind to single-stranded nucleic acid generated during helicase-mediated strand separation, including displaced strands produced during unwinding, thereby preventing reannealing of complementary strands (p. 3, para 3; p. 4, para 2; Fig. 4).
In the context of the combination with Gyllborg and Fehr, the single-stranded nucleic acid generated by helicase-mediated probe dissociation would include the target nucleic acid (RCP) and/or the displaced first probe. It would have been obvious to one of ordinary skill in the art that the single-stranded binding protein would bind to the target nucleic acid and/or the first probe following helicase-mediated dissociation.
Regarding instant Claim 67, Rajagopal and Patel teaches helicase-mediated nucleic acid unwinding reactions performed in buffer containing 10 mM ATP (p. 7; para 4). The claimed range of 0.1 mM to 10 mM overlaps with the 10mM concentration taught by Rajagopal and Patel. It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to select an ATP concentration within the range of 0.1 mM to 10 mM when performing helicase-mediated probe dissociation, as optimization of ATP concentration within a known range is routine experimentation (MPEP 2144.05).
Regarding instant Claim 69, Rajagopal and Patel teaches that single-strand binding proteins facilitate continued helicase activity and strand dissociation by stabilizing the helicase at the unwinding junction and preventing reannealing of the separated strands (p. 4, para 3). Under the combination, the single-stranded binding protein would facilitate dissociation of the first probe by maintaining helicase engagement at the bridge probe-RCP duplex and preventing reannealing of the displaced probe.
Claim(s) 75 are rejected under 35 U.S.C. 103 as being unpatentable over Gyllborg at al. (2020) in view of Fehr (US20100092960A1), as applied to claim 59 above, and further in view of Kishi et al (2019).
Regarding instant Claim 75, claim 75 depends from claim 59, which is rejected over Gyllborg in view of Fehr as set forth above. The additional limitation of claim 75, that the target nucleic acid in the biological sample is an endogenous nucleic acid analyte in the biological sample, is not taught by Gyllborg, wherein the target nucleic acid is a rolling circle amplification product (RCP) rather than an endogenous nucleic acid. This deficiency is made up in the teachings of Kishi.
Kishi teaches a method for analyzing a biological sample in which FISH probes comprising a target recognition sequence and a PER concatemer extension on the probe’s 3’ end (first overhang region) are hybridized to endogenous RNA and DNA targets (endogenous nucleic acid analytes) in fixed cell and tissue sections (Abstract; p. 535, right column, para 2). Kishi further teaches that fluorescent imager strands (first detectably labeled probes) hybridize to the concatemer tail of the primary probe, generating a detectable signal at the location of the endogenous target within the tissue (p. 534, left column, para 2).
Kishi teaches that DNase I and Exonuclease I enzymes can be used to strip both primary probes and imagers in tissue while preserving mRNA integrity, enabling a second round of mRNA detection (p. 540, right column, para 4). However, Kishi uses enzymatic digestion rather than helicase-mediated removal to strip the primary probe. This deficiency is made up in the teachings of Fehr as described above.
It would have been obvious to one of ordinary skill in the art, before the effective filling date of the claimed invention, to apply the sequential probe cycling method of Gyllborg with helicase-mediated probe removal as taught by Fehr to detect endogenous nucleic acid analytes directly, as taught by Kishi. A POSITA would have been motivated to do so because Kishi demonstrates that the same general probe method (recognition sequence, overhang extension, secondary fluorescent labeled probe) used in Gyllborg’s sequential cycling method can be applied directly to endogenous RNA and DNA targets in tissue without requiring the padlock probe and RCA amplification steps of Gyllborg. The skilled artisan would have had a reasonable expectation of success because Kishi explicitly demonstrates high detection efficiency of endogenous mRNA and DNA in thick tissue sections using this approach.
Response to Applicant’s Remarks
Applicant’s remarks dated 05/12/2026 have been fully considered. The rejections of claims 59-78 over Fehr in view of Severins, Fehr in view of Severins and Nilsson, and Fehr in view of Severins and Rajagopal, as set forth in the Office Action 02/13/2026, have been withdrawn and replaced by the new rejections set forth above, which are necessitated by the amendments to the claims. Applicant’s arguments directed to the teachings of Severins and Nelsson are therefore moot with respect to the pending rejections.
Applicant observed that Fehr does not teach contacting the biological sample with probes in discrete cycles with immediate detection and dissociation steps, and that Severins does not teach subsequent probe binding and detection after a preceding probe is detected and removed. These arguments are not disputed. In the new rejections, Fehr is not relied upon for these teachings. Gyllborg teaches the discrete sequential probe cycling method, the probe design comprising a recognition sequence and overhang region bound to a detectablly labeled probe, and detection at a spatial location in a biological sample. Fehr is relied upon solely for the teaching of helicase-mediated probe removal, which fills the single gap in Gyllborg’s.
Applicant observed that Severins does not teach sequential probe introduction after detection. This argument is noted and is not disputed. Severins is no longer cited in the pending rejections.
The cancellation of claim 77 and 60 have been acknowledged.
The amendment to claim 59 incorporating the first and second overhang region and first and second detectably labeled probe method has been entered. As discussed in 103 rejection above, this specific probe method is directly taught by Gyllborg’s bridge probe and readout detection probe system. The combination of Gyllborg’s sequential probe cycling in tissue with Fehr’s helicase-mediated probe removal renders the amended claims unpatentable.
Conclusion
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). 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.
/NURA M. CHOUDHURY/ Examiner, Art Unit 1683
/STEPHEN T KAPUSHOC/ Primary Examiner, Art Unit 1683
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