DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims 1, 3, 5, 9, 11, 15, 18, 20, 26, 30, 34, 38, 42-44, 46-49, and 51 are pending. Claims 2, 4, 6-8, 10, 12-14, 16-17, 19, 21-25, 27-29, 31-33, 35-37, 39-41, 45, 50, and 52-65 are canceled. Claim 1 is amended. Claims 1, 3, 5, 9, 11, 15, 18, 20, 26, 30, 34, 38, 42-44, 46-49, and 51 are currently under examination.
Response to Amendment
The Amendment filed 11/11/25 has been entered. Claims 1, 3, 5, 9, 11, 15, 18, 20, 26, 30, 34, 38, 42-44, 46-49, and 51 are currently pending.
Response to Arguments
Applicant’s arguments, see pages 9-15, filed 11/11/25, with respect to the rejections of claims 1, 3, 5, 9, 11, 15, 18, 20, 26, 30, 34, 38, 42-44, 46-49, and 51 under 35 USC 103 have been fully considered and are persuasive. Therefore, the rejections documented in the Non-Final mailed on 6/11/25 have been withdrawn. However, upon further consideration, new grounds of rejections necessitated by claim amendments are made in this Final Office Action.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1 and 9 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Szemes et al. (2005; “Diagnostic application of padlock probes – multiplex detection of plant pathogens using universal microarrays”; Nucleic Acids Research, Volume 33, Issue 8, 1 April 2005, Page e70; https://doi.org/10.1093/nar/gni069).
This new 102(a)(1) rejection is necessitated by claim amendments filed 11/11/25.
Relevant to claim 1, Szemes et al. teaches “Padlock probes (PLPs) offer a means of combining pathogen-specific molecular recognition and universal amplification, thereby increasing sensitivity and multiplexing capabilities without limiting the range of potential target organisms. PLPs are long oligonucleotides of ~100 bases, containing target complementary regions at both their 5’ and 3’ ends (Figure 1). These regions recognize adjacent sequences on the target DNA [citation], and between these segments lie universal primer sites and a unique sequence identifier, the so-called ZipCode. Upon hybridization, the ends of the probes get into adjacent position and can be joined by enzymatic ligation. This ligation and the resulting circular molecule can only take place when both end segments recognize their target sequences correctly. Non-circularized probes are removed by exonuclease treatment, while the circularized ones may be amplified by using universal primers. Subsequently, the target-specific products are detected by a universal complementary ZipCode (cZipCode) microarray [citation]. PLPs have been shown to possess good specificity and very high-multiplexing capabilities in genotyping assays” (Introduction, paragraph 3 continued from page 1-3).
This teaching reads on claim 1 A method for analyzing a biological sample, comprising: a) contacting the biological sample with a circularizable probe, wherein: the circularizable probe comprises a 5’ hybridization region and a 3’ hybridization region that hybridize to adjacent first and second target regions, respectively, in a target nucleic acid in the biological sample… b) circularizing the circularizable probe hybridized to the target nucleic acid to form a circularized probe; and c) detecting the circularized probe or a product thereof in the biological sample.
Further relevant to claim 1, Szemes et al. teaches “The second strategy involved inserting a destabilizing mismatch in the middle of the 3’ arm-complementary sequence, and the binding of the probe was similarly stabilized by lengthening the 5’ arm (oligonucleotides A2 and A2C)” (page 6, left column, first paragraph). Szemes et al. Table 4 demonstrates that the A2 and A2C oligonucleotides have 25 nt-long 5’ arms, and 3’ arm lengths of ither 18 nt or 17 nt.
These teachings read on claim 1 a)… the 5’ hybridization region and the 3’ hybridization region are different in lengths, the 5’ hybridization region or the 3’ hybridization region comprises an interrogatory sequence complementary to a sequence of interest in the first or second target region, respectively, and the interrogatory sequence does not comprise a 5’ or 3’ terminal nucleotide.
These teachings are relevant to claim 9 as well, since the “mismatch” is within the 3’ hybridization region, which is shorter than the 25 nt-long 5’ hybridization region. Thus, these teachings read on claim 9 the interrogatory sequence is in the shorter one of the 5' hybridization region and the 3' hybridization region.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 3, 5, 11, 18, 26, 30, 34, 38, 42, and 51 are rejected under 35 U.S.C. 103 as being unpatentable over Szemes et al. (2005; “Diagnostic application of padlock probes – multiplex detection of plant pathogens using universal microarrays”; Nucleic Acids Research, Volume 33, Issue 8, 1 April 2005, Page e70; https://doi.org/10.1093/nar/gni069) as applied to claims 1 and 9 above, and further in view of Krzywkowski et al. (2018; NPL citation U in PTO-892 filed 6/11/25; "Chapter 14: Padlock Probes to Detect Single Nucleotide Polymorphisms". Imre Gaspar (ed.), RNA Detection: Methods and Protocols, Methods in Molecular Biology, vol. 1649, pages 209-229. DOI 10.1007/978-1-4939-7213-5_14).
The teachings of Szemes et al. are applied to instantly rejected claims 3, 5, 11, 18, 26, 30, 34, 38, 42, and 51 as they were previously applied to claims 1 and 9 as anticipating a method for analyzing a biological sample.
Szemes et al. is silent to circularizable probe design limitations relevant to claims 3, 5, 11, 18, 26, 30, 34, 38, 42, and 51. However, these limitations were known in the prior art and taught by Krzywkowski et al.
Relevant to claim 3, Krzywkowski et al. Abstract teaches “We hereby present the most recent protocol for multiplexed, in situ detection of mRNAs and single nucleotide polymorphisms using padlock probes and rolling circle amplification.”
This teaching reads on claim 3 the target nucleic acid is an mRNA.
Relevant to claim 5, Krzywkowski et al. teaches “This chapter introduces an up-to-date protocol and outlines the list of notes and hints to conduct successful multiplexed mRNA SNP detection in situ, using padlock probes (PLP) and rolling circle amplification (RCA). PLP is a continuous, single stranded DNA oligonucleotide with two terminal, target-specific arms (3’ arm and 5’ arm) connected by a DNA linker” (page 210, paragraph 2).
Further relevant to claim 5, Krzywkowski et al. teaches “To maximize the conversion rate, target specific RT [reverse transcription] primer contains several chemically modified DNA bases where 2’-O, 4’-methylene bridge locks the ribose pucker in C3’ endo conformation [citation]. Such ‘locked’ nucleic acids DNA analogues (LNAs) display much higher RNA/DNA hybridization affinity than conventional nucleic acids” (page 210, paragraph 3).
These teachings read on claim 5 the circularizable probe comprises a DNA backbone and one or more ribonucleotide residues at and/or near a ligatable 3' end of the circularizable probe.
Relevant to claim 11, Krzywkowski et al. Figure 2 teaches that the 5' hybridization region is shorter than the 3' hybridization region.
Relevant to claim 18, Krzywkowski et al. Figure 2 and associated caption teach that the interrogatory sequence is an interrogatory nucleotide, and the sequence of interest is a single nucleotide of interest selected from the group consisting of a single-nucleotide polymorphism (SNP).
Relevant to claim 26, Krzywkowski et al. Note 1 on pages 222-223 teaches that “1. Tth DNA ligase exhibits significantly greater thermal stability and ligation specificity over traditional DNA ligases [citation] and it works very well for SNP genotyping in our hands… Alternative DNA ligases can be used for conventional mRNA detection and genotyping [citation] or mitochondrial DNA genotyping [citation], however the SNP detection specificity should be evaluated experimentally for every target with appropriate controls (see Subheading 3.1)”.
This teaching reads on claim 26 the circularizing step comprises enzymatic ligation using a ligase having an RNA-templated DNA ligase activity and/or an RNA-templated RNA ligase activity.
Relevant to claim 30, Krzywkowski et al. 3.3.3 mRNA Degradation, Padlock Probe Hybridization and Ligation section teaches that, following ligation of the padlock probe, the protocol includes “4. Wash the chamber twice with 1x PBS-T.” Krzywkowski et al. 3.4 Anticipated Results section teaches that “1. Every discrete, fluorescent detected signal originates from a single, successful detection of the SNP (Fig. 3).”
Thus, following washing of non-bound probes and ensuring that only correctly bound probes are present, these teachings read on claim 30 further comprising prior to the circularizing step, a step of removing molecules of the circularizable probe that are bound to the target nucleic acid but comprise one or more mismatches in the interrogatory sequence from the biological sample, and/or allowing the molecules (or portions thereof) comprising one or more mismatches to dissociate from the target nucleic acid while the molecules comprising no mismatch in the interrogatory sequence remain bound to the target nucleic acid.
Relevant to claim 34, Krzywkowski et al. Abstract teaches that “Lastly, circularized probes are replicated in situ, using rolling circle amplification mechanism to facilitate detection.”
This teaching reads on claim 34 the product is generated using rolling circle amplification (RCA) of the circularized probe at a location in the biological sample
Relevant to claims 38 and 42, Krzywkowski et al. Figure 1 and associated caption teaches that “RCA generates a single-stranded DNA concatamer, which collapses into a typically half micrometer-sized DNA ball and contains hundreds of tandem repeated sequences that are complementary to the original padlock probe. Finally, fluorescently labeled decorator probes hybridize with their complementary motifs on the RCA product.”
This teaching reads on claim 38 the biological sample is imaged to detect a signal associated with a fluorescently labeled probe that directly or indirectly binds to the rolling circle amplification product of the circularized probe at the location in the biological sample; and claim 42 the rolling circle amplification product comprises one or more barcode sequences or complements thereof corresponding to the target nucleic acid and/or the sequence of interest.
Relevant to claim 51, Krzywkowski et al. Abstract teaches that “We provide a method for automated characterization and quantitation of target mRNA in single cells or chosen tissue area.”
This teaching reads on claim 51 the biological sample is a tissue sample.
Although Szemes et al. does not include the circularizable probe design limitations of Krzywkowski et al., it would have been prima facie obvious to the skilled artisan. Szemes et al. and Krzywkowski et al. are analogous disclosures to the instant circularizable probe-mediated nucleic acid detection.
The skilled artisan would have been motivated to combine the analogous disclosures and include the Krzywkowski et al. design limitations within the methodology anticipated by Szemes et al.
Krzywkowski et al. teaches flexibility in the circularizable probe design, emphasized by the teaching of “Once the full PLP sequence is designed we strongly advice evaluation of probe secondary structure and off-target binding. We typically use mfold [citation and link] or OligoAnalyzer for secondary structure predictions. Use prediction parameters adjusted for ligation reaction conditions (see Note 5). Every change in PLP sequence (different decorator motif or linker sequence) should be followed by a secondary structure prediction (see Note 9). To prevent formation of false-positive signals PLP target sequence (cDNA) should be blasted [link] against refseq mRNA database” (page 214, paragraph 1).
Krzywkowski et al. further emphasizes this flexibility feature within their teaching of “Robustness and specificity of the protocol, combined with straightforward RCP localization allow user for easy SNP and mRNA mapping with subcellular resolution” (page 211-212) within their “multiplexed, in situ detection of mRNAs and single nucleotide polymorphisms using padlock probes and rolling circle amplification” (Abstract).
Thus, the skilled artisan would have been motivated to take advantage of the Krzywkowski et al. probe design flexibility and modify the Szemes et al. methodology to achieve “multiplexed, in situ detection of mRNAs and single nucleotide polymorphisms using padlock probes and rolling circle amplification.” The skilled artisan would have a reasonable expectation of success based on the disclosures of Szemes et al., and further in view of Krzywkowski et al., as discussed in the preceding paragraphs.
Claims 15, 20, 46, and 47-49 are rejected under 35 U.S.C. 103 as being unpatentable over Szemes et al. (2005; “Diagnostic application of padlock probes – multiplex detection of plant pathogens using universal microarrays”; Nucleic Acids Research, Volume 33, Issue 8, 1 April 2005, Page e70; https://doi.org/10.1093/nar/gni069) in view of Krzywkowski et al. (2018; NPL citation U in PTO-892 filed 6/11/25; "Chapter 14: Padlock Probes to Detect Single Nucleotide Polymorphisms". Imre Gaspar (ed.), RNA Detection: Methods and Protocols, Methods in Molecular Biology, vol. 1649, pages 209-229. DOI 10.1007/978-1-4939-7213-5_14), as applied to claims 3, 5, 11, 18, 26, 30, 34, 38, 42, and 51 above, and further in view of Brunstein (2018; NPL citation V in PTO-892 filed 6/11/25; "Padlock probes". Medical Laboratory Observer. March 22, 2018. https://www.mlo-online.com/home/article/13009460/padlock-probes).
The teachings of Szemes et al. in view of Krzywkowski et al. are applied to instantly rejected claims 15, 20, 46, and 47-49 as they were previously applied to claims 3, 5, 11, 18, 26, 30, 34, 38, 42, and 51 as obviating a method for analyzing a biological sample.
(i) Krzywkowski et al. teaches limitations relevant to claims 47-49.
Relevant to claims 47-49, Krzywkowski et al. Figure 1 and caption teach detectably distinct first and second circularizable probes targeting mouse ACTB SNP and human ACTB SNP. Krzywkowski et al. 3.3.6 Image Acquisition and Analysis section teaches " 2. Make sure to use filters appropriate for nuclear staining and fluorophores used in the experiment (see Note 31)". Krzywkowski et al. Note 31. teaches "31. We use a Zeiss Axioplan II Epifluorescence microscope equipped with a 100-W mercury lamp and a Hamamatsu, C4742-95 CCD camera. The following filter setup provides good wavelength separation and minimal crosstalk between different channels. 38HE (Zeiss) for imaging GFP/FITC/FAM dyes; SP102v2 (Chroma) for imaging Cy3 (minimal crass talk with 38HE filter); SP103v2 (Chroma) for imaging Cy3.5/TexasRed; SP104v2 (Chroma) for imaging Cy5; 49007 (Chroma) for imaging Cy7/Alexa 7.5 dyes."
Thus, Krzywkowski et al. teachings read on claim 47 ii) a second circularizable probe comprising the 5' hybridization region and the 3' hybridization region, except that the second circularizable probe comprises a second interrogatory nucleotide complementary to a second variant of the single nucleotide of interest; claim 48 the first circularizable probe comprises a first barcode sequence corresponding to the first variant and the second circularizable probe comprises a second barcode sequence corresponding to the second variant, and a signal detected in the detecting step at a given location in the biological sample is associated with the first or second barcode sequence or complement thereof; and claim 49 detecting the first or second variant of the single nucleotide of interest in situ at multiple locations in the biological sample.
(ii) Szemes et al. in view of Krzywkowski et al. is silent to certain specifics regarding hybridization region design and interrogatory nucleotide positioning (claims 15, 20, 46-47). However, these limitations were known in the prior art and taught by Brunstein.
Brunstein teaches an overview of padlock probes and applications.
Brunstein teaches that “The most common application at present is probably in the detection of single nucleotide polymorphisms (SNPs). By placing the SNP of interest directly under either side of the R1-R2 junction, a mismatch to padlock probe will block ligation, or, conversely, a match will allow ligation and selective signal generation only when a perfect match occurs” (page 6, paragraph 4). The R1-R2 junctions refer to the hybridization regions depicted within Brunstein Figure 1.
Thus, Brunstein teaches that the interrogatory sequence is capable of placement in either hybridization region. Furthermore, the Krzywkowski et al. teaching of manual probe design for optimal and efficient SNP genotyping (page 213, paragraph 1) would allow for flexibility in the interrogatory sequence position and hybridization regions. Krzywkowski et al. further teaches that “Once the full PLP sequence is designed we strongly [advise] evaluation of probe secondary structure and off-target binding” (page 214, paragraph 1). These teachings indicate that the circularizable probe design is flexible in order to properly and efficiently allow for SNP genotyping while minimizing deleterious effects (off-target binding and secondary structures).
Taken together, these teachings and the teachings relevant to above rejections read on:
claim 15 the 5' hybridization region comprises the interrogatory sequence and is shorter than the 3' hybridization region by about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides;
claim 20 the interrogatory nucleotide is the fourth, fifth, or sixth nucleotide of the 5' hybridization region, with the 5' terminal nucleotide of the circularizable probe being the first nucleotide of the 5' hybridization region;
claim 46 a)… the 5' hybridization region is shorter than the 3' hybridization region and comprises an interrogatory nucleotide complementary to a single nucleotide of interest in the first target region of a first molecule of the target RNA, wherein a second molecule of the target RNA comprises a mismatch with the interrogatory nucleotide, and the interrogatory nucleotide is any one of the second to the tenth nucleotides from the 5' end of the circularizable probe;
and claim 47 a)… the shorter one of the 5' hybridization region and the 3' hybridization region comprises a first interrogatory nucleotide complementary to a first variant of a single nucleotide of interest in the first or second target region, respectively, and the first interrogatory nucleotide is any one of the second to the tenth nucleotides from the 5' end of the first circularizable probe.
Thus, the skilled artisan would find it prima facie obvious to include the Brunstein interrogatory sequence positioning within the methodology rendered obvious by Szemes et al. in view of Krzywkowski et al. It is noted that Szemes et al., Krzywkowski et al., and Brunstein are all analogous disclosures to the instant method for analyzing a biological sample. The skilled artisan would be motivated to include the Brunstein interrogatory sequence positioning within the flexible methodologies taught by Krzywkowski et al.-modified Szemes et al. in order to enable increased hybridization flexibility, as Krzywkowski et al. provided the foundation for flexible SNP genotyping while minimizing deleterious effects. The skilled artisan would have a reasonable expectation of success based on the disclosures of Szemes et al. in view of Krzywkowski et al., and further in view of Brunstein, as discussed in the preceding paragraphs.
Claims 43-44 are rejected under 35 U.S.C. 103 as being unpatentable over Szemes et al. (2005; “Diagnostic application of padlock probes – multiplex detection of plant pathogens using universal microarrays”; Nucleic Acids Research, Volume 33, Issue 8, 1 April 2005, Page e70; https://doi.org/10.1093/nar/gni069) in view of Krzywkowski et al. (2018; NPL citation U in PTO-892 filed 6/11/25; "Chapter 14: Padlock Probes to Detect Single Nucleotide Polymorphisms". Imre Gaspar (ed.), RNA Detection: Methods and Protocols, Methods in Molecular Biology, vol. 1649, pages 209-229. DOI 10.1007/978-1-4939-7213-5_14), as applied to claims 3, 5, 11, 18, 26, 30, 34, 38, 42, and 51 above, and further in view of Gyllborg et al. (2020; NPL citation W in PTO-892 filed 6/11/25; "Hybridization-based In Situ Sequencing (HybISS): spatial transcriptomic detection in human and mouse brain tissue". bioRxiv preprint doi: https://doi.org/10.1101/2020.02.03.931618; this version posted February 3, 2020)
The teachings of Szemes et al. in view of Krzywkowski et al. are applied to instantly rejected claims 43-44 as they were previously applied to claims 3, 5, 11, 18, 26, 30, 34, 38, 42, and 51 as obviating a method for analyzing a biological sample.
Szemes et al. in view of Krzywkowski et al. is silent to specifics regarding dehybridization of probes. However, these limitations were known in the prior art and taught by Gyllborg et al.
Gyllborg et al. teaches “Hybridization-based In Situ Sequencing (HybISS): spatial transcriptomic detection in human and mouse brain tissue” (Title).
Relevant to claims 43-44, Gyllborg et al. Figure 1 and caption teach “Every cycle consists of hybridizing bridge-probes to RCPs [rolling circle products] and reading them out with fluorophore conjugated readout detection probes. For sequential cycles, bridge-probes are then stripped off to allow for rehybridizing next round of bridge-probes.” The Gyllborg et al. readout detection probes are equivalent to the instant detectably-labeled probes. The Gyllborg et al. bridge-probes are equivalent to the instant intermediate probes.
These teachings read on claim 43 the detecting comprises: contacting the biological sample with one or more detectably-labeled probes that directly or indirectly bind to the one or more barcode sequences in the rolling circle amplification product, and dehybridizing the one or more detectably-labeled probes from the rolling circle amplification product; and claim 44 the detecting comprises: contacting the biological sample with one or more intermediate probes that hybridize to the one or more barcode sequences in the rolling circle amplification product, wherein the one or more intermediate probes are detectable using one or more detectably-labeled probes, and dehybridizing the one or more intermediate probes and/or the one or more detectably- labeled probes from the rolling circle amplification product.
Although Szemes et al. in view of Krzywkowski et al. does not include the Gyllborg et al. dehybridization of probes, it would have been prima facie obvious to the skilled artisan to include the Gyllborg et al. positioning within the methodology rendered obvious by Krzywkowski et al.-modified Szemes et al.
It is noted that Szemes et al., Krzywkowski et al., and Gyllborg et al. are analogous disclosures to the instant method for analyzing a biological sample. The skilled artisan would be motivated to include the Gyllborg et al. probe dehybridizations within the methodology rendered obvious by Krzywkowski et al.-modified Szemes et al. because Gyllborg et al. teaches that their “hybridization-based in situ sequencing (HybISS)… allows for increased flexibility and multiplexing, increased signal-to-noise, all without compromising throughput efficiency of imaging large fields of view” (Abstract). The skilled artisan would be motivated to include the dehybridizations in order to achieve flexible, sensitive, and efficient imaging, as taught by Gyllborg et al. The skilled artisan would have a reasonable expectation of success based on the disclosures of Szemes et al. in view of Krzywkowski et al., and further in view of Gyllborg et al., as discussed in the preceding paragraphs.
Conclusion
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.
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/SARAH JANE KENNEDY/Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682