DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
2. This Application has been transferred to Examiner Haney in Art Unit 1682.
3. Applicant’s election without traverse of Group I in the reply filed on February 26, 2026 is acknowledged.
Claims 1-8, 14-15, 17-23, and 30-32 are currently pending.
Claims 30-32 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on February 26, 2026.
Claim Rejections - 35 USC § 103
4. 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.
5. Claims 1-8, 14-15, and 20-23 are rejected under 35 U.S.C. 103 as being unpatentable over Marquardt (US 2018/0274009 Pub 9/27/2018) in view of Rabbani (US 2020/0332348 Pub 10/22/2020).
Regarding Claim 1 Marquardt teaches a method of digesting a test mRNA with an RNase enzyme to produce a plurality of mRNA fragments (para 0009). Marquardt teaches that the RNase enzyme is RNase T1, a catalytic RNA (e.g., ribozyme, DNAzyme, etc.), RNase H, or Cusativin (para 0010). Marquardt teaches that the “test mRNA” is an mRNA of interest, having a known nucleic acid sequence (para 0116). Thus Marquardt teaches a method of digesting an RNA molecule having a known reference sequence into smaller RNA fragments, the method comprising: digesting the RNA molecule into the fragments with one or more sequence-specific nucleases that cleave the RNA molecule at a plurality of predetermined sequence-specific sites. It is noted that RNase T1 is a sequence specific nuclease that cuts RNA.
Regarding Claim 2 Marquardt teaches that some embodiments, RNase T1 and cusativin are used in parallel to determine the identity of a test mRNA. Use of two or more enzymes “in parallel” may refer to the use of the enzymes in the same digest, or simultaneously in separate digests of the same test mRNA(s) (para 0142). Thus Marquardt teaches a method wherein the
one or more sequence-specific nucleases comprises a plurality of nucleases. It is noted that RNase T1 and custavin are both sequence specific endonucleases that cut RNA.
Regarding claim 6 Marquardt teaches a method wherein the one or more sequence-specific nucleases comprises one or more of RNase T1, RNase A, Colicin E5, and MazF (paras 0008, 0010, 0142).
Regarding Claim 7 Marquardt teaches that a mRNA sample encoding the fluorescent protein mCherry was digested with RNase T1 (para 0178). Marquardt teaches that the cominbed length of all unique oligos was 373nt, out of a total mRNA length of 1014 nt (para 0181). Thus Marquardt teaches a method wherein the RNA molecule has a length greater than about 1,000 mers.
Regarding Claim 8 Marquardt teaches that the RNase T1 digestion of mCherry produced fragments that were between about 10 to 1,000 mers in length (see Table 3).
Regrding Claims 14 and 15 Marquardt teaches a method that comprises forming one or more oligonucleotide duplexes with the RNA molecule along specific portions of the reference sequence and digesting the RNA molecule into the fragments with RNase H. Marquardt teaches that specific nucleic acids (e.g., DNA) oligos can be designed to anneal to the test mRNA and the resulting duplexes digested with RNase H to generate a unique fragment pattern for a given test mRNA (para 0118). Marquardt teaches that the oligos can be between 10 and 50 bp long (para 0121).
Regarding Claim 20 Marquardt teaches that in some embodiments, it is desirable to map the 5′ UTR of an mRNA to identify whether the mRNA contains Cap, partial Cap, or is uncapped. Similarly, in some embodiments, it is desirable to characterize the 3′ UTR of an mRNA, for example to quantify the length of the mRNA polyA tail (para 0183). Marquardt does not specifically teach that the mRNA is cut within about 100 nucleotides of a proximal end of a 3' poly(A) tail and/or a site within about 100 nucleotides of a 5' cap, however it would have been obvious to do this for the benefit of being able to characterize the 5’cap and the 3’ poly A tail regions.
Regarding Claim 21 Marquardt teaches digesting a test mRNA with an RNase enzyme to produce a plurality of mRNA fragments and then physically separating the plurality of mRNA fragments (para 0009). Marquardt teaches that the physical separation is achieved by liquid chromatography (para 0013).
Regarding Claim 22 Marquardt teaches comparing the profile of the masses of the fragments generated to the predicted masses from the primary molecular sequence of the mRNA (para 0005). Marquardt teaches that the mass of the fragments can be determined using mass spectrometry (para 0013, 0050).
Regarding Claim 23 Marquardt teaches a method further comprising mapping the RNA fragments to the reference sequence (Example 7).
Marquardt teaches a method that comprises forming one or more oligonucleotide duplexes with the RNA molecule along specific portions of the reference sequence and digesting the RNA molecule into the fragments with RNase H (para 0118). RNase H is a non-sequence-specific endonuclease that is duplex-dependent nuclease that only cuts the RNA strand in RNA/DNA hybrids.
Marquardt does not teach a method of digesting a RNA molecule into fragments with one or more sequence-specific nucleases that cleave the RNA molecule at a plurality of predetermined sequence-specific sites, wherein the one or more sequence-specific nucleases comprise one or more duplex-dependent nucleases that only act on RNA within a duplex, and wherein each of the one or more duplexes formed with the RNA molecule comprises a motif recognized by one of the one or more duplex- dependent nucleases (claim 1). Marquardt does not teach a method wherein the plurality of nucleases, comprises a plurality of duplex-dependent nucleases (clm 3). Marquardt does not teach a method wherein the one or more duplex-dependent nucleases is a restriction enzyme (claim 4). Marquardt does not teach a method wherein the one or more restriction endonucleases is from the group consisting of AvaII, AvrII, BanI, TaqI, HinfI, and HAEIII (claim 5).
However Rabbani teaches that single stranded RNA may be selectively cleaved by incorporating therein a restriction enzyme recognition site of a restriction enzyme, such as AvaII, AvrII, BanI, HaeIII, HinfI and/or TaqI, that recognizes and cleaves RNA in an RNA:DNA duplex that may be formed with said RNA molecule by hybridizing a DNA oligonucleotide, which may be synthetic, to form the hybrid restriction site and constituting a reaction mixture including the subject restriction enzyme(s) under conditions permissive for its activity (para 0092).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Marquardt by also digesting the RNA with one or more sequence specific, duplex dependent endonucleases as suggested by Rabbani. One of skill in the art wishing to digest RNA would have been motivated to additionally use a sequence specific, duplex dependent endonucleases to cut the RNA for the benefit of having greater control over where the endonuclease cuts. Further as discussed above, the prior art of Marquardt teaches methods wherein a plurality of nucleases are used to digest RNA and it would have been obvious to use a plurality of any type of nucleases, including duplex dependent nucleases, for better control of where the endonuclease cuts.
6. Claims 17-19 are rejected under 35 U.S.C. 103 as being unpatentable over Marquardt (US 2018/0274009 Pub 9/27/2018) in view of Rabbani (US 2020/0332348 Pub 10/22/2020) as applied to claim 1 and in further view of Athapattu (Nucleic Acids Research 1/28/2021 Vol 49 No 7 e41).
The teachings of Marquardt and Rabbani are presented above.
The combined references do not teach a method wherein at least one of the sequence-specific nucleases is immobilized on a solid support (clm 17). The combined references do not each a method wherein the at least one immobilized nuclease is provided in the form of an immobilized enzyme reactor (IMER) that allows flow-through digestion of the RNA molecule (clm 18). The combined references do not teach a method wherein the nuclease immobilized within the IMER is not a duplex-dependent nuclease and is used to further digest a selected fraction of the RNA fragments after digestion with a duplex-dependent nuclease (clm 19).
However Athapattu teaches that microfluidic devices, where enzymes are immobilized for biological reactions, are known as immobilized microfluidic enzymatic reactors, IMERs. Athapattu teaches an IMER containing XRN1 as the immobilized enzyme for the sequential digestion of 5′ monophosphorylated ssRNA for potential applications in single-molecule RNA exosequencing (page 2).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Marquardt and Rabbani by immobilizing a sequence specific nuclease, such as a non duplex dependent nuclease, on a solid support in the form of a IMER that allows flow through digestion of RNA as suggested by Athapattu. One of skill in the art would have been motivated to make a IMER particular since Athapattu teaches that there are several advantages of IMERs compared to solution phase bioreactors, such as enhanced enzymatic activity and stability, prevention of aggregation and auto-digestion, and reduced interference in downstream analysis (page 2). Additionally one of skill in the art would have been motivated to first cut the RNA with a duplex dependent nuclease and then further cut the remaining RNA with a non-duplex dependent nuclease for the benefit of being able to create specific breaks (using the duplex dependent nuclease) which allows for non-preferred regions to be chewed away, such that the preferred regions remain in tact, and then digestion of the prefered regions for further analysis such as mapping.
7. Any inquiry concerning this communication or earlier communications from the examiner should be directed to AMANDA HANEY whose telephone number is (571)272-8668. The examiner can normally be reached Monday-Friday, 8:15am-4:45pm EST.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Wu-Cheng Shen can be reached at 571-272-3157. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/AMANDA HANEY/ Primary Examiner, Art Unit 1682
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