Office Action 2: Notice of Allowance (2025-02-05)

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\. (Currently Amended) 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, wherein the oligonucleotide hybridization region is adjacent to the 3' end of a target sequence in the target RNA or is overlapping with the 3' end of a target sequence in the target RNA;
b) cleaving the target RNA with an RNase H in the oligonucleotide hybridization region to generate a cleaved target RNA, wherein the cleaved target RNA comprises at least a portion of the target sequence;
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 \[\[a\]\]the target sequence i ;
'
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\. (Previously presented) 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\. (Previously presented) 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\. (Previously presented) The method of claim 99, wherein the oligonucleotide hybridization region and the target sequence overlap by between 1 and 20 nucleotides.

103\. (Previously presented) 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\. (Previously presented) 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\. (Previously presented) The method of claim 99, wherein the RNase H comprises an RNase Hi and/or an RNase H2.

106\. (Previously presented) 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\. (Previously presented) The method of claim 99, wherein the method comprises imaging the biological sample to detect the RCP.

108\. (Previously presented) 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\. (Previously presented) 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\. (Previously presented) 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\. (Currently Amended) 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 metalof 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\. (Currently Amended) The method of claim 111, wherein the non-catalytic metal of the polymerase is barium, strontium, iron, cobalt, nickel, tin, zinc, or europium.

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

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

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

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

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

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

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

125\. (Previously presented) 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:
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.

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

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Palo Alto, CA 94304

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