Office Action 1: Non-Final Rejection (2024-09-16)
Rejection types: 102 - Lack of Novelty, 103 - Obviousness, 112 - Written Description/Enablement

CLAIMS:
AMENDMENTS TO THE CLAIMS

This listing of claims will replace all prior versions, and listing, of claims in the application.

LISTING OF CLAIMS:

1-98. (Cancelled)297

99\. (New) A method of analyzing a biological sample, comprising:a) hybridizing a nucleic acid oligonucleotide to an oligonucleotide hybridization region in a target ribonucleic acid (RNA) in the biological sample;
b) cleaving the target RNA with an RNase H in the oligonucleotide hybridization region to generate a cleaved target RNA;
c) hybridizing a circular probe or a circularizable probe or probe set comprising a target recognition sequence complementary to a target sequence in the target RNA;
wherein the oligonucleotide hybridization region is adjacent to the 3' end of the target sequence or is overlapping with the 3' end of the target sequence;d) performing rolling circle amplification of the circular probe or of a circularized probe generated from the circularizable probe or probe set to generate a rolling circle amplification product (RCP) using the cleaved target RNA as a primer; and
e) detecting the RCP in the biological sample.

100\. (New) The method of claim 99, wherein the biological sample is contacted with the nucleic acid oligonucleotide and with the RNase H simultaneously before contacting the biological sample with the circular probe or the circularizable probe or probe set.

101\. (New) The method of claim 99, wherein the method comprises washing the biological sample after cleaving the target RNA with the RNase H and before contacting the biological sample with the circular probe or the circularizable probe or probe set.

102\. (New) The method of claim 99, wherein the oligonucleotide hybridization region and the target sequence overlap by between 1 and 20 nucleotides.

103\. (New) The method of claim 99, wherein the method does not comprise contacting the biological sample with a DNA primer that hybridizes to the circular probe or the circularized probe.

104\. (New) The method of claim 99, wherein the target recognition sequence of the circularizable probe or probe set is a split recognition sequence comprising a first hybridization region having a first ligatable end and a second hybridization region having a second ligatable end,wherein the first hybridization region hybridizes to a 5' portion of the target sequence, and the second hybridization region hybridizes to a 3' portion of the target sequence, and the method comprises ligating the first ligatable end to the second ligatable end to generate the circularized probe.

105\. (New) The method of claim 99, wherein the RNase H comprises an RNase H1 and/or an RNase H2.

106\. (New) The method of claim 99, wherein contacting the biological sample with the RNase H comprises contacting the biological sample with between 0.5 enzyme units (U) and 50 U of the RNase H.

107\. (New) The method of claim 99, wherein the method comprises imaging the biological sample to detect the RCP.

108\. (New) The method of claim 107, wherein the imaging comprises detecting a signal associated with a fluorescently labeled probe that directly or indirectly binds to the RCP.

109\. (New) The method of claim 108, wherein a sequence of the RCP is analyzed by sequential hybridization, sequencing by hybridization, sequencing by ligation, sequencing by synthesis, sequencing by binding, or a combination thereof.

110\. (New) The method of claim 99, wherein the oligonucleotide hybridization region and the 3' portion of the target sequence overlap by about 6 to about 10 nucleotides.

111\. (New) The method of claim 99, wherein the method comprises contacting the biological sample with a polymerase in a first reaction mixture comprising a non-catalytic cofactor of the polymerase, and then contacting the biological sample with a second reaction mixture comprising a catalytic cofactor of the polymerase to perform the rolling circle amplification.

112\. (New) The method of claim 111, wherein the di-cation that is not a co-factor of the polymerase is barium, strontium, iron, cobalt, nickel, tin, zinc, or europium.

113\. (New) The method of claim 111, wherein the non-catalytic cofactor of the polymerase is calcium or strontium.

114\. (New) The method of claim 111, wherein the non-catalytic cofactor of the polymerase is strontium.

115\. (New) The method of claim 99, wherein a sequence of the RCP is analyzed at a location in the biological sample or a matrix embedding the biological sample.

116\. (New) The method of claim 99, comprising reacting at least one RNA in the biological sample with a polynucleotide kinase (PNK).

117\. (New) The method of claim 116, wherein the method comprises reacting at least one RNA in the biological sample with the PNK after contacting the biological sample with the nucleic acid oligonucleotide and the RNase H.

118\. (New) The method of claim 117, wherein the PNK is a T4 Polynucleotide Kinase (T4 PNK) or a T7 Polynucleotide Kinase (T7-PNK).

119\. (New) The method of claim 99, wherein the nucleic acid oligonucleotide is 20 to 35 nucleotides in length.

120\. (New) A method of analyzing a biological sample, comprising:a) contacting the biological sample with a plurality of nucleic acid oligonucleotides, wherein a first oligonucleotide of the plurality hybridizes to a first oligonucleotide hybridization region in a first target ribonucleic acid (RNA) in the biological sample, and a second

oligonucleotide of the plurality hybridizes to a second oligonucleotide hybridization region in a second target RNA in the biological sample;b) cleaving the first and second target RNAs with an RNase H in their respective oligonucleotide hybridization regions to generate a first cleaved target RNA and a second cleaved target RNA;c) contacting the biological sample with a plurality of circular probes or circularizable probes or probe sets,
wherein a first circular probe or first circularizable probe or probe set of the plurality comprises a first target recognition sequence complementary to a first target sequence in the first target RNA,
wherein a second circular probe or second circularizable probe or probe set of the plurality comprises a second target recognition sequence complementary to a second target sequence in the second target RNA,
wherein the first and second circular probe or the first and second circularizable probe or probe set hybridize to their respective target RNAs,
wherein the first oligonucleotide hybridization region is overlapping with the 3' end of the first target sequence, and
wherein the second oligonucleotide hybridization region is overlapping with the 3' end of the second target sequence;
d) performing rolling circle amplification of the first and second circular probe or of a first and second circularized probe generated from the first and second circularizable probes or probe sets to generate a first and second rolling circle amplification product (RCP) using the first cleaved target RNA and the second cleaved target RNA as primers; ande) detecting the first and second RCPs in the biological sample.

121\. (New) The method of claim 120, wherein the method comprises imaging the biological sample to detect the first and second RCPs.

122\. (New) The method of claim 120, wherein a sequence of the first and second RCPs is analyzed by sequential hybridization, sequencing by hybridization, sequencing by ligation, sequencing by synthesis, sequencing by binding, or a combination thereof.

123\. (New) The method of claim 120, wherein the first oligonucleotide hybridization region and the 3' portion of the first target RNA overlap by about 6 to about 10 nucleotides and the second oligonucleotide hybridization region and the 3' portion of the second target RNA overlap by about 6 to about 10 nucleotides.

124\. (New) The method of claim 120, wherein the first oligonucleotide and the second oligonucleotide are each 20 to 35 nucleotides in length.

125\. (New) The method of claim 120, wherein the method comprises contacting the biological sample with a polymerase in a first reaction mixture comprising a non-catalytic cofactor of the polymerase, and then contacting the biological sample with a second reaction mixture comprising a catalytic cofactor of the polymerase to perform the rolling circle amplification.

OFFICE ACTION:
Detailed Action
►	The applicant's Preliminary Amendment filed 30 JUL 2024 has been entered.  Following the entry of the Preliminary Amendment, Claim(s) 99-125 is/are pending.
►  	The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA . In the event the determination of the status of the application as subject to AIA  35 U.S.C. 102 and 103 (or as subject to pre-AIA  35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
35 U.S.C. 112(b)/ 112 (pre-AIA ), second paragraph
►	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 Rejection(s) under 35 U.S.C. 112(b)
►	Claim(s) 112 is/are rejected under 35 U.S.C. 112(b), as being indefinite in that it fails to point out what is included or excluded by the claim language.
Claim 112 is indefinite because the phrase “the di-cation” on line 1 lacks proper antecedent basis in Claim 99 and/or Claim 111.
35 U.S.C. 102
►	The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that may 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.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
35 U.S.C. 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.
Claim Rejection(s) under 35 U.S.C. 102
►	Claim 99, 103-106 is/are rejected under 35 U.S.C. 102(a)(1)  as being anticipated by Takahashi et al. [Science Reports 8:7770(2018) – hereinafter “Takahashi”].
Claim 99 is drawn to a method of analyzing a biological sample, comprising: a) hybridizing a nucleic acid oligonucleotide to an oligonucleotide hybridization region in a target ribonucleic acid (RNA) in the biological sample; b) cleaving the target RNA with an RNase H in the oligonucleotide hybridization region to generate a cleaved target RNA; c) hybridizing a circular probe or a circularizable probe or probe set comprising a target recognition sequence complementary to a target sequence in the target RNA; wherein the oligonucleotide hybridization region is adjacent to the 3’ end of the target sequence or is overlapping with the 3’ end of the target sequence; d) performing rolling circle amplification of the circular probe or of a circularized probe generated from the circularizable probe or probe set to generate a rolling circle amplification product (RCP) using the cleaved target RNA as a primer; and e) detecting the RCP in the biological sample.
Regarding Instant Claim 99, Takahashi teach a method of detecting  a target RNA (e.g. mRNA) they term “RNaseH-assisted RNA-primed rolling circle amplification (hereinafter - RNase Assisted RPRCA) which comprises all of the limitations of Claim 99. For example consider Fig. 1 and Fig. 3A. There, Takahashi teach “hybridizing a circular probe or a circularizable probe or probe set comprising a target recognition sequence complementary to a target sequence in the target RNA; wherein the oligonucleotide hybridization region is adjacent to the 3’ end of the target sequence or is overlapping with the 3’ end of the target sequence (i.e. step “c” of Claim 99); Takahashi further teach teach d) performing rolling circle amplification of the circular probe or of a circularized probe generated from the circularizable probe or probe set to generate a rolling circle amplification product (RCP) using the cleaved target RNA as a primer; and e) detecting the RCP in the biological sample.  As regards step “a)”, note that Takahashi teach using multiple different probes  targeting different regions of target mRNA (e.g. P1 and P5 of Fig. 3A) wherein P5 targets the 3’ end while P1 hybridizes upstream (i.e. torwards the 5’ end of the target RNA. P1 constitutes the nucleic acid oligonucleotide of step “a)”
Claim 103 is drawn to an embodiment of the method of Claim 99,  wherein the method does not comprise contacting the biological sample with a DNA primer that hybridizes to the circular probe or the circularized probe.
No DNA primer is used in Fig. 1 of Takahashi.
Regarding Claim 104 Takahashi teaches the limitation(s) recited by Claim 104, see especially Fig.1.
Regarding Claim 106.  Takahashi teaches the use of RNase H are very low concentrations e.g. 0.6 units/reaction, the para bridging pp. 4-5, thereby meeting the limitation of Claim 106 which reads  “between 0.5 U -50U”.
Claim Rejection(s) under 35 U.S.C. 103
►	Claim(s_100-101, 105-106 and 119 is/are  rejected under 35 U.S.C. 103 as being unpatentable over Takahashi.
Regarding Claims 100-101, the selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results.
Regarding Claim 105 Takahashi teaches the use of RNaseH (presumably RNase H1). Regardless, several RNase H molecules  were known including RNase H1 and RNase H2 (?Official Notice – see for example Hyjek et al. [DNA Repair 84 (2019)102672].
Regarding Claim 106.  Takahashi teaches the use of RNase H are very low concentrations e.g. 0.6 units, the para bridging pp. 4-5. Regardless,  where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
As regards Claim 119, Takahashi teach 70-mer circularizable probes but do not teach  don’t teach that P1, for example, (i.e. the oligo nucleotide of Claim 99) should be between 20-35 nucleotides in length. However, where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
►	Claim(s) 107-109 and 115 is/are  rejected under 35 U.S.C. 103 as being unpatentable over Takahashi as applied above against Claim 99 and further in view of Kűhnmund et al.[ US 2021/0198727 – hereinafter “Kűhnmund”].
Claim 107 is drawn to an embodiment of the method of Claim 99,  wherein the method further comprises imaging the biological same  to detect the RCP.
Takahashi teach detection  of their RCPs by fluorescence detection, see especially Fig. 1, but fail to teach detecting RCP by imaging their biological sample. However,  it was known to perform RCA-type reaction in situ (i.e. within biological sample/tissue) with subsequent detection of the RCP using fluorescently labeled probes which detection comprises imaging of said sample. Consider for example Fig.1 and para 34-37 and 141 in  Kűhnmund.
As regards Claims 108-109 and 115 see Kűhnmund  and the para(s) cited above.
►	Claim(s) 116-118 is/are  rejected under 35 U.S.C. 103 as being unpatentable over Takahashi as applied above against Claim 99 and further in view of Wang et al. [EMBO J 14:2873-3880(2002) – hereinafter “Wang”].
Regarding Claims 116-117, Takahashi  does not teach the use of PNKs However, PNKs were known as was the use PNKs in nucleic acid based assays, see at least the abstract in Wang. Absent an unexpected result, it would have been prima facie obvious to the PHOSITA to modify the method of  Takahashi  wherein a PNK treatment steps is included in said assay following the RNase H step. The   PHOSITA would have been motivated to make this modification in order to repair any damaged ends resulting from the RNaseH nicking step.
As regards Claim 118, Wang teach T4 PNK. See at least the abstract.
Claim Objections
►	Claim(s) 102 and 110-114 is /are objected to as being dependent upon a rejected base claim, but would be allowable over the prior art of record if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
Allowable Subject Matter
►	Claim(s) 120-125 are novel and unobvious over the prior art now of record. In particular Takahashi does not teach the limitation recited in “c) of Claim 120 which reads  “wherein the first oligonucleotide hybridization region is overlapping with the 3’ end of the first target sequence, and wherein the second oligonucleotide hybridization region is overlapping with the 3’ end of the second target sequence”.
Conclusion
C.

APPLICANT REBUTTAL: Claims 99-125 are pending in the present application. By virtue of this response,

Claims 99, 111, and 112 have been amended, no claims have been canceled, and no new claims are added. Accordingly, Claims 99-125 are currently under consideration. Amendment and cancellation of certain claims is not to be construed as a dedication to the public of any of the subject matter of the claims as previously presented.

Support for the amendments to Claims 99, 111, and 112 is found throughout the application as originally filed, including in the original claims. For example, support for the amendments to Claim 99 is found, without limitation, in Claim 99 as previously presented, at [0046], and Figures 1 and 2. For example, support for the amendments to Claims 111 and 112 is found, without limitation, at [0028].

No new matter has been added by way of the amendment. Entry of the amendment is respectfully requested.

Allowable Subject Matter

Applicant thanks Examiner Whisenant for acknowledging the inventive subject matter of Claims 120-125. For at least the reasons discussed below, Applicant respectfully submits that pending Claims 99-125 are allowable.

Interview Summary

Applicant thanks Examiner Whisenant for the courtesy extended during the video conference interview with Applicant's representatives Corey Garyn (undersigned) and Kelsey Roberts Kingman (Reg. No. 79,382) on October 11, 2024.

The parties discussed the rejections under 35 U.S.C. § 102, 35 U.S.C. § 103, and 35 U.S.C. § 112, and discussed potential claim amendments for clarity. No agreement was reached.

Applicant believes this statement of the substance of the interview complies with

MPEP § 713.04. However, should the Examiner feel any additional action is required on the part of the Applicant, the Examiner is requested to contact the undersigned.

Claim Rejections - 35 U.S.C. § 112

Claim 112 is rejected under 35 U.S.C. §112(b) as allegedly being indefinite.

Specifically, the Office states that Claim 112 is indefinite because "the phrase 'the di-cation' on line 1 lacks proper antecedent basis in Claim 99 and/or Claim 111." Office Action at page 2.

Without acquiescing to the rejection and solely to expedite prosecution, Applicant has amended Claim 111 to recite "wherein the method comprises contacting the biological sample with a polymerase in a first reaction mixture comprising a non-catalytic metal of the polymerase," and Claim 112 to recite "wherein the non-catalytic metal of the polymerase is barium, strontium, iron, cobalt, nickel, tin, zinc, or europium." These amendments remove the term 'the di-cation' from pending Claim 112, thereby rendering the rejection moot.

Therefore, Applicant respectfully requests that the metes and bounds of Claim 112 is clear. The rejection under 35 U.S.C. § 112(b) should be withdrawn.

Claim Rejections - 35 U.S.C. § 102

Claims 99, 103-106 are rejected under 35 U.S.C. §102(a)(1) as allegedly being anticipated by Takahashi et al. (Science Reports 8:7770(2018); hereinafter "Takahashi"). Specifically, the Office states that "Takahashi teach a method of detecting a target RNA (e.g. mRNA) they term 'RNAseH-assisted RNA-primed rolling circle amplification (hereinafter - RNase Assisted RPRCA) which comprises all of the limitations of Claim 99." Office Action at page 4. Applicant respectfully requests reconsideration.

Without acquiescing to the rejection and solely to expedite prosecution, Applicant has amended claim 99 to recite "c) subsequent to b), hybridizing a circular probe or a circularizable probe or probe set to the cleaved target RNA, wherein the circular probe or circularizable probe or probe set comprises a target recognition sequence complementary to the target sequence."

As an initial matter, the Office alleges that Takahashi discloses all features of Claim 99 of the instant application at both Fig. 1 and Fig. 3A. Office Action at page 4.

Applicant respectfully submits that the Office has not identified any teaching of using

both a nucleic acid oligonucleotide and a circular probe or a circularizable probe or probe set as required by instant Claim 99. As shown in Figure 1 reproduced below, Takahashi does not disclose "a method of analyzing a biological sample, comprising: a) hybridizing a nucleic acid oligonucleotide to an oligonucleotide hybridization region in a target ribonucleic acid (RNA) in the biological sample... b) cleaving the target RNA with an RNase H in the oligonucleotide hybridization region to generate a cleaved target RNA... [and] c) subsequent to b), hybridizing a circular probe or a circularizable probe or probe set to the cleaved target RNA, wherein the circular probe or circularizable probe or probe set comprises a target recognition sequence complementary to the target sequence" as recited by instant Claim 99.

Non-specific hybridization Specific hybridization 0.822.903 RNA

ck probe

RNaso H 0.821.043 0.411.127

Not 2.333.10 0.832.383Figure 1. A schematic representation of targeted RNA detection by RNase H-assisted RNA-prtmed rolling circle amplification using a padlock probe.

Figure 1 of Takahashi Specifically, in Figure 1 of Takahashi, the padlock probe is used to guide target mRNA cleavage by RNase H. In contrast, pending Claim 99 requires a separate nucleic acid oligonucleotide from the circular probe or circularizable probe or probe set for generating the cleaved target RNA. Takahashi does not disclose the use of both a nucleic acid oligonucleotide and a circular probe or a circularizable probe or probe set as recited in instant Claim 99. For these reasons alone, Takahashi fails to teach each and every element of instant claim 99.

Furthermore, amended Claim 99 recites generation of the cleaved target RNA prior to hybridization of a circular probe or a circularizable probe or probe set. Takahashi fails to disclose this limitation, as Takahashi's padlock probe is used for both RCA and to guide target RNA cleavage, and therefore cannot be hybridized subsequent to cleavage of the target RNA.

For at least these reasons alone, Takahashi does not anticipate instant Claim 99 and all dependent claims therefrom.

The Office also alleges that Takahashi discloses the limitations of Claim 99 at Fig. 3A, stating that "Takahashi teach[es] using multiple different probes targeting different regions of target mRNA (e.g., P1 and P5 of Fig. 3A) wherein P5 targets the 3' end while P1 hybridizes upstream (i.e. towards [sic] the 5' end of the target RNA. P1 constitutes the nucleic acid oligonucleotide of step 'a)'." Office Action at page 5.

Figure 3A of Takahashi is reproduced below:

 0.7834.213Figure 3A of Takahashi

Applicant respectfully notes that Figure 3 of Takahashi is directed towards examining "whether the padlock probe position and arm sequence on the target mRNA affected the sealing efficiency [i.e. ligation efficiency] of SplintR ligase," noting that "the arm positions and nucleotide pair at both arm ends of each padlock probe on the target GFP mRNA are illustrated in Fig. 3A." Takahashi at page 3. Put another way, Figure 3A of Takahashi provides a schematic of the different hybridization positions of tested padlock probes on the target mRNA, but these tests only included one of these padlock probes at a time.

This is further clarified in Figure 3B, which is reproduced below:

 2.493.44Figure 3B of Takahashi

Figure 3B of Takahashi shows "denatured polyacrylamide gel analysis of ligation products using padlock probes with different hybridizing sequence on the mRNA sequence." Takahashi at Fig. 3B legend (page 5). Each individual column of the polyacrylamide gel of Figure 3B shows the ligation product of a single padlock probe (per reaction of P1-P5). As such, Takahashi does not disclose use of multiple padlock probes within a single reaction.

Therefore, Takahashi does not anticipate the limitations of amended Claim 99 or any dependent claims therefrom.

For at least these reasons, the rejections under 35 U.S.C. §102(a)(1) should be withdrawn.

Claim Rejections - 35 U.S.C. § 103

Claims 100-101, 105-106 and 119

Claims 100-101, 105-106 and 119 are rejected under 35 U.S.C. §103 as allegedly being obvious over Takahashi. Applicant respectfully traverses the rejection.

As discussed above, Takahashi does not teach or suggest a method comprising "c)

subsequent to b), hybridizing a circular probe or a circularizable probe or probe set to the cleaved

target RNA, wherein the circular probe or circularizable probe or probe set comprises a target recognition sequence complementary to the target sequence" as recited in pending Claim 99. For the same reasons discussed above, claims 100-101, 105-106 and 119 are not obvious over Takahashi.

Therefore, Takahashi fails to teach or even suggest the limitations of Claim 99 or all dependent claims therefrom. The rejections under 35 U.S.C. §103 should be withdrawn.

Claims 107-109 and 115

Claims 107-109 and 115 are rejected under 35 U.S.C. §103 as allegedly being

obvious over Takahashi as applied above against Claim 99 and further in view of Knhnemund et al. (US 2021/0198727; "Knhnemund"). Applicant respectfully traverses the rejection.

As discussed above, Takahashi does not teach or suggest a method comprising "c)

subsequent to b), hybridizing a circular probe or a circularizable probe or probe set to the cleaved target RNA, wherein the circular probe or circularizable probe or probe set comprises a target recognition sequence complementary to the target sequence" as required by claim 99.

Knhnemund does not cure the deficiencies of Takahashi. Knhnemund is cited as allegedly teaching detection of an RCP "using fluorescently-labeled probes which detection comprises imaging of said sample." Office Action at page 7. But Knhnemund is silent regarding a method comprising "c) subsequent to b), hybridizing a circular probe or a circularizable probe or probe set to the cleaved target RNA, wherein the circular probe or circularizable probe or probe set comprises a target recognition sequence complementary to the target sequence" as required by claim 99.

Therefore, claim 99 and all claims depending therefrom are not obvious over

Takahashi in view of Knhnemund. The rejection under 35 U.S.C. §103 should be withdrawn.

Claims 116-118

Claims 116-118 are rejected under 35 U.S.C. §103 as being allegedly obvious over Takahashi as applied above against Claim 99 and further in view of Wang et al. (EMBO J 14:2873-3880(2002); "Wang"). Applicant respectfully traverses the rejection.

As discussed above, Takahashi does not teach or suggest a method comprising "c)

subsequent to b), hybridizing a circular probe or a circularizable probe or probe set to the cleaved target RNA, wherein the circular probe or circularizable probe or probe set comprises a target recognition sequence complementary to the target sequence" as required by Claim 99.

Wang does not cure the deficiencies of Takahashi. Wang is cited as allegedly

teaching using of T4 PNK. Office Action at page 7. But Wang is silent regarding a method comprising "c) subsequent to b), hybridizing a circular probe or a circularizable probe or probe set to the cleaved target RNA, wherein the circular probe or circularizable probe or probe set comprises a target recognition sequence complementary to the target sequence" as required by Claim 99.

Therefore, Takahashi in view of Wang fails to teach or even suggest the limitations of Claim 99 or all claims depending therefrom. The rejections under 35 U.S.C. § 103 should be withdrawn.

Claim Rejections - Claim Objections

Claims 102 and 110-114 are objected to as being dependent upon a rejected base claim, but would be allowable over the prior art of record if rewritten in independent form including all of the limitations of the base claim and any intervening claims.

Applicant respectfully submits that, in view of the arguments above, Claim 99 and all dependent claims therefrom are both novel and non-obvious over Takahashi alone as well as Takahashi in view of Knhnemund or Wang. Therefore, the objection should be withdrawn.

CONCLUSION

In view of the above, each of the presently pending claims in this application is

believed to be in immediate condition for allowance. Accordingly, the Examiner is respectfully requested to withdraw the outstanding rejection of the claims and to pass this application to issue. If it is determined that a telephone conference would expedite the prosecution of this application, the Examiner is invited to telephone the undersigned at the number given below.

In the event the U.S. Patent and Trademark Office determines that an extension

and/or other relief is required, Applicant petitions for any required relief including extensions of time and authorizes the Commissioner to charge the cost of such petitions and/or other fees due in connection with the filing of this document to Deposit Account No. 03-1952 referencing docket no. 20241-20192.00. However, the Commissioner is not authorized to charge the cost of the issue fee to the Deposit Account.

Dated: December 13, 2024 Respectfully submitted,

By / Corey Garyn /

Corey Garyn

Registration No.: 82,420

MORRISON & FOERSTER LLP

755 Page Mill Road

Palo Alto, CA 94304

+1 (212) 336-4439

NEXT OFFICE ACTION: Notice of Allowability
►	Claim(s) 99-125  as amended in the paper(s) filed  13 DEC 2024 is/are pending in this application.
►	The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA . In the event the determination of the status of the application as subject to AIA  35 U.S.C. 102 and 103 (or as subject to pre-AIA  35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
Reason(s) for Allowance
►	Claim(s) 99-125 is/are deemed to be allowable in light of the paper(s) filed 13 DEC 2024 and the persuasive argument(s) therein. The Claims have now been renumbered as Claims 1-27 in the same order as presented by the applicant.
Conclusion
C.

CITED REFERENCE SUMMARIES: Ref US20210198727A1: Claims:
A method for detecting an analyte in a sample, comprising: (i) providing a rolling circle amplification product (RCP), wherein said RCP comprises multiple repeat copies of a marker sequence indicative of said analyte; (ii) performing a hybridization chain reaction (HCR), wherein said HCR reaction is initiated by an HCR initiator which directly or indirectly hybridizes to said marker sequence or which is comprised in said marker sequence; and (iii) detecting the product of the HCR reaction, thereby detecting the analyte.
(i) providing a rolling circle amplification product (RCP), wherein said RCP comprises multiple repeat copies of a marker sequence indicative of said analyte;
(ii) performing a hybridization chain reaction (HCR), wherein said HCR reaction is initiated by an HCR initiator which directly or indirectly hybridizes to said marker sequence or which is comprised in said marker sequence; and
(iii) detecting the product of the HCR reaction, thereby detecting the analyte.
The method of claim 1 , wherein the analyte is a nucleic acid molecule, a lipid or a protein.
The method of claim 2 , wherein the nucleic acid molecule is DNA or RNA.
The method of claim 1 , wherein the sample comprises cells.
The method of claim 4 , wherein the cells are immobilized, fixed or in suspension.
The method of claim 1 , wherein the analyte is detected in situ in a tissue sample.
The method of claim 1 , wherein the analyte is detected in single cells.
The method of claim 1 , wherein a padlock probe comprising a complement of the marker sequence is hybridized to the analyte, and is circularized to form a rolling circle amplification (RCA) template, which is subjected to RCA to form the RCP.
The method of claim 1 , wherein the method is used to detect multiple analytes in a sample, and at least one RCP is provided for each analyte.
The method of claim 1 , wherein HCR is performed using HCR monomers in the form of hairpins, wherein the HCR monomers comprise first HCR monomers and second HCR monomers, wherein the first HCR monomers comprise a toehold region which is complementary to the HCR initiator and to the interacting region in the second HCR monomers, and the toehold region in the second HCR monomers is complementary to the interacting region in the first HCR monomers.
The method of claim 1 , wherein the HCR initiator is provided separately to the RCP and comprises a marker-binding domain capable of hybridizing to the marker sequence in the RCP, and wherein the HCR initiator is hybridized to the RCP before the HCR reaction is performed.
The method of claim 1 , wherein at least a fraction of at least one of the HCR monomers in an HCR reaction is labelled with a detectable label.
The method of claim 9 , wherein for each RCP, multiple sequential HCR reactions are performed, and the respective HCR products are detected together to provide for detection of the analyte.
The method of claim 15 , wherein sequential HCR reactions are performed using differentially labelled HCR monomer sets, such that each RCP is combinatorially labelled to detect the analyte.
The method of claim 1 , for detecting multiple analytes in sample, wherein a RCP is provided for each analyte, and said method is performed sequentially in cycles and a different set of RCPs is detected in each cycle, to detect a different set of analytes.
The method of claim 17 , wherein in a first cycle, a first set of RCPs is detected using a first set of HCR initiators each specific for a different member of the first set of RCPs and the HCR products are removed after the detection, and in a second or subsequent cycle, a second or subsequent set of RCPs is detected using a second or subsequent set of HCR initiators each specific for a different member of the first or subsequent set of RCPs, and the HCR products are removed after the detection.
The method of claim 18 , wherein in each cycle the same sets of HCR monomers are used.
The method of claim 1 , wherein the marker sequence is between 5 and 50 nucleotides in length.
- 23 . (canceled)
The method of claim 1 , wherein the rolling circle amplification for generating the RCP is performed for no more than 3 hours.
The method of claim 1 , wherein the rolling circle amplification for generating the RCP is performed for no more than 2 hours.
Ref US20230374573A1: Claims:
A method for analyzing a biological sample, comprising: (a) contacting the biological sample with a circular or circularizable probe, wherein the circular or circularizable probe comprises a hybridization region that hybridizes to a target RNA sequence in a target RNA in the biological sample to form a duplex, (b) contacting the biological sample with a primer oligonucleotide, a ribonuclease H (RNase H), and a polymerase, wherein the primer oligonucleotide hybridizes to the circular or circularizable probe, and wherein the RNase H cleaves the target RNA in the duplex at one or more sites in the target RNA sequence; (c) performing rolling circle amplification (RCA) using the circular probe or a circularized probe formed by circularizing the circularizable probe as template; and (d) detecting one or more products of the RCA, thereby detecting the target RNA or a sequence thereof in the biological sample.
(a) contacting the biological sample with a circular or circularizable probe,
wherein the circular or circularizable probe comprises a hybridization region that hybridizes to a target RNA sequence in a target RNA in the biological sample to form a duplex,
(b) contacting the biological sample with a primer oligonucleotide, a ribonuclease H (RNase H), and a polymerase, wherein the primer oligonucleotide hybridizes to the circular or circularizable probe, and wherein the RNase H cleaves the target RNA in the duplex at one or more sites in the target RNA sequence;
(c) performing rolling circle amplification (RCA) using the circular probe or a circularized probe formed by circularizing the circularizable probe as template; and
(d) detecting one or more products of the RCA, thereby detecting the target RNA or a sequence thereof in the biological sample.
The method of claim 1 , wherein the RCA is performed using the circularized probe as a template, and wherein the method comprises ligating the circularizable probe to form the circularized probe.
The method of claim 2 , wherein the ligating is performed using the target RNA as a template.
- 6 . (canceled)
The method of claim 1 , wherein the ligating is performed prior to contacting the biological sample with the RNase H.
- 10 . (canceled)
The method of claim 1 , wherein the circular or circularizable probe comprises deoxyribonucleotide residues.
- 13 . (canceled)
The method of claim 1 , wherein the circular or circularizable probe comprises two, three, four, five, six, or more consecutive ribonucleotide residues.
The method of claim 1 , wherein the biological sample is contacted with the primer oligonucleotide, the RNase H, and the polymerase simultaneously.
The method of claim 1 , wherein the biological sample is contacted with the RNase H prior to being contacted with the primer oligonucleotide and the polymerase.
The method of claim 1 , wherein the biological sample is contacted with the RNase H prior to being contacted with the polymerase.
The method of claim 1 , wherein the polymerase is a Phi29 DNA polymerase or a Bst DNA polymerase.
The method of claim 1 , wherein the RNase is RNase H1 or an RNase H2.
- 24 . (canceled)
The method of claim 1 , wherein the contacting with the RNase H comprises: a first step performed in a first buffer that is free or substantially free of a cofactor for the RNase H, and a second step performed in a second buffer comprising the cofactor.
a first step performed in a first buffer that is free or substantially free of a cofactor for the RNase H, and
a second step performed in a second buffer comprising the cofactor.
The method of claim 25 , further comprising: incubating the biological sample with the RNase H in the first buffer, thereby allowing the RNase H to bind the target RNA, the circular or circularizable probe, and/or the duplex; removing excess RNase H from the biological sample; and incubating the biological sample in the second buffer, thereby allowing the RNase H to cleave at one or more sites in the target RNA sequence.
incubating the biological sample with the RNase H in the first buffer, thereby allowing the RNase H to bind the target RNA, the circular or circularizable probe, and/or the duplex;
removing excess RNase H from the biological sample; and
incubating the biological sample in the second buffer, thereby allowing the RNase H to cleave at one or more sites in the target RNA sequence.
The method of any one of claim 1 , wherein the one or more products of the RCA comprise an RCA product generated by using the primer oligonucleotide as a primer.
- 32 . (canceled)
The method of claim 1 , wherein the biological sample is contacted with the RNase H at a concentration of between about 1Ã10 â3 U/Î¼L and about 2Ã10 â2 U/Î¼L.
- 40 . (canceled)
The method of claim 1 , wherein the detecting step comprises contacting the one or more RCA products with a plurality of detectable probes and/or a plurality of intermediate probes in sequential cycles.
- 64 . (canceled)
A method for analyzing a biological sample, comprising: (a) contacting the biological sample with a circularizable probe comprising a hybridization region that hybridizes to a target RNA sequence in a target RNA in the biological sample to form a duplex, (b) ligating the circularizable probe to form a circularized probe; (c) contacting the biological sample with (i) a primer oligonucleotide that hybridizes to the circularized probe, (ii) a ribonuclease H (RNase H), and (iii) a Phi29 DNA polymerase, wherein the contacting with the RNase H comprises: (i) a first step that is performed in a first buffer that is free or substantially free of a cofactor for the RNase H, and (ii) a second step that is performed in a second buffer comprising the cofactor, (d) performing rolling circle amplification (RCA) using the circularized probe as template; and (e) detecting one or more products of the RCA, thereby detecting the target RNA or a sequence thereof in the biological sample.
(a) contacting the biological sample with a circularizable probe comprising a hybridization region that hybridizes to a target RNA sequence in a target RNA in the biological sample to form a duplex,
(b) ligating the circularizable probe to form a circularized probe;
(c) contacting the biological sample with (i) a primer oligonucleotide that hybridizes to the circularized probe, (ii) a ribonuclease H (RNase H), and (iii) a Phi29 DNA polymerase,
wherein the contacting with the RNase H comprises: (i) a first step that is performed in a first buffer that is free or substantially free of a cofactor for the RNase H, and (ii) a second step that is performed in a second buffer comprising the cofactor,
(i) a first step that is performed in a first buffer that is free or substantially free of a cofactor for the RNase H, and
(ii) a second step that is performed in a second buffer comprising the cofactor,
(d) performing rolling circle amplification (RCA) using the circularized probe as template; and
(e) detecting one or more products of the RCA, thereby detecting the target RNA or a sequence thereof in the biological sample.
- 69 . (canceled)
The method of claim 25 , wherein the cofactor for the RNase H is Mg 2+ .
The method of claim 1 , wherein the biological sample is a cell or tissue sample.
The method of claim 71 , wherein the one or more products of the RCA are detected at location(s) in the biological sample immobilized on a solid support.
- 80 . (canceled)
Ref US20210198727: Claims:
1. A method for detecting an analyte in a sample, comprising:
(i) providing a rolling circle amplification product (RCP), wherein said RCP comprises multiple repeat copies of a marker sequence indicative of said analyte; (ii) performing a hybridization chain reaction (HCR), wherein said HCR reaction is initiated by an HCR initiator which directly or indirectly hybridizes to said marker sequence or which is comprised in said marker sequence; and (iii) detecting the product of the HCR reaction, thereby detecting the analyte.
2. The method of claim 1, wherein the analyte is a nucleic acid molecule, a lipid or a protein.
3. The method of claim 2, wherein the nucleic acid molecule is DNA or RNA.
4. The method of claim 1, wherein the sample comprises cells.
5. The method of claim 4, wherein the cells are immobilized, fixed or in suspension.
6. The method of claim 1, wherein the analyte is detected in situ in a tissue sample.
7. The method of claim 1, wherein the analyte is detected in single cells.
8. The method of claim 1, wherein a padlock probe comprising a complement of the marker sequence is hybridized to the analyte, and is circularized to form a rolling circle amplification (RCA) template, which is subjected to RCA to form the RCP.
9. The method of claim 1, wherein the method is used to detect multiple analytes in a sample, and at least one RCP is provided for each analyte.
10. (canceled)
11. The method of claim 1, wherein HCR is performed using HCR monomers in the form of hairpins, wherein the HCR monomers comprise first HCR monomers and second HCR monomers, wherein the first HCR monomers comprise a toehold region which is complementary to the HCR initiator and to the interacting region in the second HCR monomers, and the toehold region in the second HCR monomers is complementary to the interacting region in the first HCR monomers.
12. The method of claim 1, wherein the HCR initiator is provided separately to the RCP and comprises a marker-binding domain capable of hybridizing to the marker sequence in the RCP, and wherein the HCR initiator is hybridized to the RCP before the HCR reaction is performed.
13. The method of claim 1, wherein at least a fraction of at least one of the HCR monomers in an HCR reaction is labelled with a detectable label.
14. (canceled)
15. The method of claim 9, wherein for each RCP, multiple sequential HCR reactions are performed, and the respective HCR products are detected together to provide for detection of the analyte.
16. The method of claim 15, wherein sequential HCR reactions are performed using differentially labelled HCR monomer sets, such that each RCP is combinatorially labelled to detect the analyte.
17. The method of claim 1, for detecting multiple analytes in sample, wherein a RCP is provided for each analyte, and said method is performed sequentially in cycles and a different set of RCPs is detected in each cycle, to detect a different set of analytes.
18. The method of claim 17, wherein in a first cycle, a first set of RCPs is detected using a first set of HCR initiators each specific for a different member of the first set of RCPs and the HCR products are removed after the detection, and in a second or subsequent cycle, a second or subsequent set of RCPs is detected using a second or subsequent set of HCR initiators each specific for a different member of the first or subsequent set of RCPs, and the HCR products are removed after the detection.
19. The method of claim 18, wherein in each cycle the same sets of HCR monomers are used.
20. The method of claim 1, wherein the marker sequence is between 5 and 50 nucleotides in length.
21-23. (canceled)
24. The method of claim 1, wherein the rolling circle amplification for generating the RCP is performed for no more than 3 hours.
25. The method of claim 1, wherein the rolling circle amplification for generating the RCP is performed for no more than 2 hours.