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 .
DETAILED ACTION
Status of Claims
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after allowance or after an Office action under Ex Parte Quayle, 25 USPQ 74, 453 O.G. 213 (Comm'r Pat. 1935). Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, prosecution in this application has been reopened pursuant to 37 CFR 1.114. Applicant's submission filed on 08/05/2026 has been entered.
Claims 1, 3-4, 12, 14-16 and 41-51 are pending and the subject of this NON-FINAL Office Action.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claim(s) 1, 3-4, 12, 14-16, 41-43 and 45-48 and 50-51 is/are rejected under 35 U.S.C. 103 as being unpatentable over PHAM (WO20190226659, cited on IDS 08/05/2026; all cites to US20190360043, the national stage entry of WO20190226659), in view of SONG (US 20200370114).
The prior art as a whole demonstrates that it would have been obvious to a skilled artisan at the time of filing to apply familiar bisulfite-free TET-assisted pyridine borane sequencing (TAPS) to the method of PHAM as suggested by PHAM, to detect methylation in DNA without damaging DNA with a reasonable expectation of success.
As to claims 1 and 50, PHAM teaches a method of sequencing a nucleic acid molecule, wherein the nucleic acid molecule comprises, from 5' to 3', a first strand, a first primer binding sequence, a second strand comprising a modified cytosine nucleobase, and a second primer binding sequence, wherein the second strand is complementary to the first strand, the method comprising (Figs. 3-5): (a) annealing a blocking primer to the first primer binding sequence of the nucleic acid molecule and extending the blocking primer with a polymerase to form a blocking strand hybridized to the first strand that blocks the first strand from annealing to the second strand (206/208 in Figs. 3-5); (b) converting the modified cytosine nucleobase of the second strand to a uracil nucleobase, or uracil nucleobase analog (para. 0085); (c) removing the blocking strand (Step 5 w/ exo in Figs. 3-5); and (d) sequencing the second strand to generate a sequencing read (Figs. 3-5).
As to claims 3-4, the modified cytosine is 5-mC (para. 0085).
As to claim 12, the polymerase is strand displacing (e.g. para. 0035).
As to claim 14, the sequencing is SBS (paras. 0086, 0098ff).
As to claims 15-16, the sequencing uses reversible terminators (paras. 0098ff).
As to claims 42 and 46-48, a probe with streptavidin/biotin is attached to a solid support is hybridized to second strand, and used to pull down or enrich (Figs. 3-5).
As to claims 43 and 45, any nucleotide can be the “terminating” or last nucleotide because this is undefined.
PHAM does not explicitly teach contacting the modified cytosine nucleobase with a ten-eleven translocation (TET) enzyme to generate a 5-carboxylcytosine (5caC) nucleobase; and contacting the 5caC nucleobase with a borane-containing reducing agent to generate a uracil nucleobase or a uracil nucleobase analog; or amplification can be performed before removing blocking strand.
However, PHAM teaches to use familiar methylation detection sequencing methods (para. 0085), of which TAPS was a well-known option at the time of effective filing here, offering reduced DNA damage and other familiar benefits compared to other options like bisulfite. Specifically, SONG teaches TAPS:
5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the two major epigenetic marks found in the mammalian genome. 5hmC is generated from 5mC by the ten-eleven translocation (TET) family dioxygenases. Tet can further oxidize 5hmC to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), which exists in much lower abundance in the mammalian genome compared to 5mC and 5hmC (10-fold to 100-fold lower than that of 5hmC). Together, 5mC and 5hmC play crucial roles in a broad range of biological processes from gene regulation to normal development. Aberrant DNA methylation and hydroxymethylation have been associated with various diseases and are well-accepted hallmarks of cancer. Therefore, the determination of 5mC and 5hmC in DNA sequence is not only important for basic research, but also is valuable for clinical applications, including diagnosis and therapy.
5fC and 5caC are the two final oxidized derivatives of 5mC and can be converted to unmodified cytosine by Thymine DNA glycosylase (TDG) in base excision repair pathway. Therefore, 5fC and 5caC are two important key intermediates in the active demethylation process, which plays important role in embryonic development. 5fC and 5caC are found in these contexts and may serve as indicator of nearly complete 5mC demethylation. 5fC and 5caC may also play additional functions such as bind specific proteins and affect the rate and specificity of RNA polymerase II.
5mC is also a post-transcriptional RNA modification that has been identified in both stable and highly abundant tRNAs and rRNAs, and in mRNAs. In addition, 5mC has been detected in snRNA (small nuclear RNA), miRNA (microRNA), lncRNA (long noncoding RNA) and eRNA (enhancer RNA). However, there appears to be differences in the occurrence of 5mC in specific RNA types in different organisms. For example, 5mC appears not to be present in tRNA and mRNA from bacteria, while it has been found in tRNA and mRNA in eukaryotes and archaea.
5hmC has also been detected in RNA. For example, mRNA from Drosophila and mouse has been found to contain 5hmC. The same family of enzymes that oxidize 5mC in DNA was reported to catalyze the formation of 5hmC in mammalian total RNA. In flies, a transcriptome wide study using methylation RNA immunoprecipitation sequencing (MeRIP-seq) with 5hmC antibodies, detected the presence of 5hmC in many mRNA coding sequences, with particularly high levels in the brain. It was also reported that active translation is associated with high 5hmC levels in RNA, and flies lacking the TET enzyme responsible for 5hmC deposition in RNA have impaired brain development.
The current gold standard and most widely used method for DNA methylation and hydroxymethylation analysis is bisulfite sequencing (BS), and its derived methods such as Tet-assisted bisulfite sequencing (TAB-Seq) and oxidative bisulfite sequencing (oxBS). All of these methods employ bisulfite treatment to convert unmethylated cytosine to uracil while leaving 5mC and/or 5hmC intact. Through PCR amplification of the bisulfite-treated DNA, which reads uracil as thymine, the modification information of each cytosine can be inferred at a single base resolution (where the transition of C to T provides the location of the unmethylated cytosine). There are, however, at least two main drawbacks to bisulfite sequencing. First, bisulfite treatment is a harsh chemical reaction, which degrades more than 90% of the DNA due to depurination under the required acidic and thermal conditions. This degradation severely limits its application to low-input samples, such as clinical samples including circulating cell-free DNA and single-cell sequencing. Second, bisulfite sequencing relies on the complete conversion of unmodified cytosine to thymine. Unmodified cytosine accounts for approximately 95% of the total cytosine in the human genome. Converting all these positions to thymine severely reduces sequence complexity, leading to poor sequencing quality, low mapping rates, uneven genome coverage and increased sequencing cost. Bisulfite sequencing methods are also susceptible to false detection of 5mC and 5hmC due to incomplete conversion of unmodified cytosine to thymine.
Bisulfite sequencing has also been used to detect cytosine methylation in RNA. Unlike other methods for detecting 5mC in RNA such as methylated-RNA-immunoprecipitation, RNA-bisulfite-sequencing (RNA-BS-seq) has the advantage of being able to determine of the extent of methylation of a specific C position in RNA. RNA-BS-seq, however, suffers from the same drawbacks described above for bisulfite sequencing of DNA. In particular, the reaction conditions can cause substantial degradation of RNA.
There is a need for a method for DNA methylation and hydroxymethylation analysis that is a mild reaction that can detect the modified cytosine (5mC and 5hmC) at base-resolution quantitatively without affecting the unmodified cytosine. Likewise, there is a need for a method for RNA methylation and hydroxymethylation analysis that employs mild reaction conditions and can detect the modified cytosine quantitatively at base resolution without affecting the unmodified cytosine.
SUMMARY OF THE INVENTION
The present invention provides methods for identifying the location of one or more of 5-methylcytosine, 5-hydroxymethylcytosine, 5-carboxylcytosine and/or 5-formylcytosine in a nucleic acid. The methods described herein provide for DNA or RNA methylation and hydroxymethylation analysis involving mild reactions that detect the modified cytosine quantitatively with base-resolution without affecting the unmodified cytosine. Provided herein is a new method for identifying 5mC and 5hmC by combining TET oxidation and reduction by borane derivatives (e.g., pyridine borane and 2-picoline borane (pic-BH3)), referred to herein as TAPS (TET Assisted Pyridine borane Sequencing) (Table 1). TAPS detects modifications directly with high sensitivity and specificity, without affecting unmodified cytosines, and can be adopted to detect other cytosine modifications. It is non-destructive, preserving RNA and DNA up to 10 kbs long. Compared with bisulfite sequencing, TAPS results in higher mapping rates, more even coverage and lower sequencing costs, enabling higher quality, more comprehensive and cheaper methylome analyses. Variations of this method that do not employ the oxidation step are used to identify 5fC and/or 5caC as described herein.
In one aspect, the present invention provides a method for identifying 5-methylcytosine (5mC) in a target nucleic acid comprising the steps of:
a. providing a nucleic acid sample comprising the target nucleic acid;
b. modifying the nucleic acid comprising the steps of:
i. adding a blocking group to the 5-hydroxymethylcytosine (5hmC) in the nucleic acid sample;
ii. converting the 5mC in the nucleic acid sample to 5-carboxylcytosine (5caC) and/or 5-formylcytosine (5fC); and
iii. converting the 5caC and/or 5fC to dihydrouracil (DHU) to provide a modified nucleic acid sample comprising a modified target nucleic acid; and
c. detecting the sequence of the modified target nucleic acid; wherein a cytosine (C) to thymine (T) transition in the sequence of the modified target nucleic acid compared to the target nucleic acid provides the location of a 5mC in the target nucleic acid.
(paras. 0003-17). An amplification step can be included (paras. 0026-42, 0068; Fig. 5). This technique is shown in Figure 5:
PNG
media_image1.png
542
462
media_image1.png
Greyscale
A skilled artisan would have been familiar with TAPS as an option for methylation sequencing detection without harsh bisulfite treatment, that has base-level resolution, without affecting unmodified cytosines; resulting in higher mapping rates, more even coverage and lower sequencing costs, thus enabling higher quality, more comprehensive and cheaper methylome analyses compared to bisulfite sequencing.
Claim(s) 49 is/are rejected under 35 U.S.C. 103 as being unpatentable over PHAM (WO2019226659, cited on IDS 08/05/2026; all cites to US20190360043, the national stage entry of WO2019226659), in view of SONG (US 20200370114), in further view of TRAVERS (US 20160237485).
The prior art as a whole demonstrates that it would have been obvious to a skilled artisan using SMRTBell sequencing at the time of filing to apply familiar LNA, PNA, etc. to increase primer affinity with the SMRTbell loop with a reasonable expectation of success.
Neither PHAM nor SONG specifically teach the primer that binds to the SMRTbell loop has PNA, LNA, etc.
However, this was familiar in the SMRTbell art to allow primers that are engineered to provide substantially higher affinity for the template, without the concurrent concern that such higher affinity to the template will yield a higher rate of random or non-specific priming. This is explained in TRAVERS (para. 0069). Thus, a skilled artisan would have found it obvious to include an LNA, PNA, etc. in the primer of PHAM used for the same SMRTbell sequencing to increase primer affinity and specificity.
Claim Objection- Allowable Subject Matter
Claim 44 is objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Aaron Priest whose telephone number is (571)270-1095. The examiner can normally be reached 8am-6pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Gary Benzion can be reached at (571) 272-0782. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/AARON A PRIEST/ Primary Examiner, Art Unit 1681
Prosecution Insights