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 action is in response to the papers filed August 21, 2026. Applicant’s remarks and amendments have been fully and carefully considered but are not found to be sufficient to put the application in condition for allowance. Any new grounds of rejection presented in this Office Action are necessitated by Applicant's amendments. Any rejections or objections not reiterated herein have been withdrawn. This action is made FINAL.
Claims 1, 4-8, 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Claims 30-32 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.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
3. 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.
4. Claims 1, 4, 5, 8, 14, and 15 are rejected under 35 U.S.C. 103 as being unpatentable over Wang (US 2008/0124713 Pub 5/29/2008) in view of Rattan (US 2023/0174973 Filed 12/1/2022 with priority back to 63/285,402 Filed 12/2/2021).
Regarding Claim 1 Wang teaches a method that includes contacting a sample of RNA with a set of oligodeoxynucleotides to provide a DNA/RNA duplex and then contacting the DNA/RNA duplex with an enzyme having a DNA:RNA nuclease activity to provide a digested RNA sample (abstract, para 0004, and 0042). Wang teaches that the enzyme having the DNA:RNA nuclease activity may be any enzyme known to be capable of specifically cleaving at DNA/RNA duplexes (para 0051). Wang teaches that a typical example of an enzyme having the DNA:RNA nuclease activity is RNase H (para 0052). It is noted that it is a property of RNase H that it only cleaves RNA in a RNA/DNA duplex. However RNase H is NOT considered to be sequence specific. Thus Wang teaches a method comprising: forming one or more oligonucleotide duplexes with the RNA molecule; digesting the RNA molecule into fragments with a nucleases that cleaves the RNA molecule, wherein the nuclease only acts on RNA within an oligonucleotide duplex formed with the RNA molecule.
Regarding Claim 8 Wang teaches that the digested RNA sample includes a set of RNA fragments, wherein each of RNA fragments is less than about 500 bases long (para 0046).
Regarding Claim 14 Wang teaches that the oligodeoxynucleotides are 6-14 bp long (para 0045).
Regarding Claim 15 Wang teaches that the two or more duplexes are form with oligodeoxynucleotides (abstract, para 0004, 0042).
Wang does not teach a method comprising digesting the RNA molecule into fragments with “sequence specific” nucleases that cleave the RNA molecule at a plurality of predetermined sequence specific sites, wherein the sequence specific nucleases comprise two or more duplex-dependent nucleases that only act on RNA within an oligonucleotide duplex formed with the RNA molecule, and wherein each of the oligonucleotide duplexes formed with the RNA molecule comprises a motif recognized by at least one of the two or more duplex-dependent nucleases (clm 1). Wang does not teach a method wherein the two or more duplex-dependent nucleases are restriction endonucleases (clm 4). Wang does not teach a method wherein the two or more restriction endonucleases are HinfI and HAEIII (clm 5).
However Rattan teaches cleaving a DNA/RNA complex with at least one restriction enzyme (para 0005). Rattan teaches cleaving the DNA/RNA complex with the at least one restriction enzyme selected from the group consisting of AvaII, AvrII, BanI, BstNI, MvaI, HaeIII, HinfI, NciI, PFLMI, Sau3AI, and TaqI (para 0014). Further Rattan teaches that in some embodiments, the at least one restriction enzyme comprises two or more restriction enzymes that recognize different recognition sequences (para 0015). It is noted that it is a property of HaeIII and HinfI that they only cleave RNA in a RNA/DNA duplex (see Table 1 of the instant specification).
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 Wang by digesting the RNA with two or more sequence specific duplex dependent nucleases that only act on RNA within a RNA/DNA duplex as suggested by Rattan. The claim would have been obvious because the substitution of one duplex dependent nuclease that only cleaves RNA in a RNA/DNA duplex (RNase H) for another (HinfI/HAEIII) would have yielded predictable results to one of ordinary skill in the art at the time of the invention. Further one of skill in the art wishing to digest RNA would have been motivated to 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 Rattan teaches methods wherein two or more sequence specific duplex dependent nucleases are used to digest RNA in a RNA/DNA duplex and it would have been obvious to use two or more of these nucleases, for the benefit of creating a larger number of RNA fragments.
5. Claims 6-7 and 20-23 are rejected under 35 U.S.C. 103 as being unpatentable over Wang (US 2008/0124713 Pub 5/29/2008) in view of Rattan (US 2023/0174973 Filed 12/1/2022 with priority back to 63/285,402 Filed 12/2/2021) as applied to claim 1 above and in further view of Marquardt (US 2018/0274009 Pub 9/27/2018).
The teachings of Wang in view of Rattan are presented above.
The combined references do not teach a method wherein the two or more sequence- specific nucleases further comprise two or more of Colicin E5 and MazF (clm 6).
However Marquardt teaches digesting a test mRNA with one or more endonucleases to produce a plurality of mRNA fragments. Marquart teaches that the one more endonucleases can be MazF and colicin E5 (para 0008).
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 Wang and Rattan by further digesting the RNA with colicin E5 and MazF as suggested by Marquardt. One of skill in the art would have been motivated to use these two nucleases for the benefit of being able to digest sequence specific regions on the single stranded portions of the target RNA hybridized to the oligonucleotides. One of skill in the art would have been motivated use of two or more of both types of nucleases (those that are duplex-dependent and those that are not), for the benefit of creating a larger number of RNA fragments.
The combined references do not teach a method wherein the RNA molecule has a length greater than about 1,000 mers (clm 7).
However Marquardt teaches that a mRNA sample encoding the fluorescent protein mCherry was digested with RNase T1 (para 0178). Marquardt teaches that the combined 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.
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 Wang and Rattan by using a RNA molecule that has a length greater than about 1,000 mers as suggested by Marquardt. One of skill in the art would have been motivated to use a RNA molecule of this length since most mRNA sequences are greater than 1000 bp long. Further there was a reasonable expectation of success that the methods could be adapted to RNA molecules of any length.
The combined references do not teach a method wherein the RNA molecule is an mRNA molecule and the plurality of predetermined sequence-specific sites comprises a site 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 (clm 20).
However 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).
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 Wang and Rattan by using a mRNA molecule with sequence specific sites near the poly(A) tail or 5’ cap as suggested by Marquardt. One of skill in the art would have been motivated to do this for the benefit of being able to identify whether the mRNA contains Cap, partial Cap, or is uncapped or to quantify the length of the mRNA polyA tail. While none of the references 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, this would have been obvious to do for the benefit of being able to characterize the 5’cap and the 3’ poly A tail regions.
The combined references do not teach a method further comprising separating one or more of the RNA fragments based on length using liquid chromatography (clm 21).
However Marquardt teaches digesting a test mRNA with a nuclease 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).
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 Wang and Rattan by further separating the fragments based on length using liquid chromatography as suggested by Marquardt. As demonstrated by Marquardt liquid chromatography was a routine method of separated fragments by length. One of skill in the art would have been motivated to separate the fragments by length for further downstream applications such as enrichment of a desired size or removing unwanted fragments.
The combined references do not teach a method further comprising measuring the mass of one or more of the RNA fragments using mass spectrometry (clm 22).
However Marquardt teaches comparing the profile of the masses of the fragments generated to 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).
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 Wang and Rattan by further measuring the mass of the RNA fragments using mass spectrometry as suggested by Marquardt. One of skill in the art would have been motivated to measure the mass of the fragments by mass spectrometry since Marquardt teaches that by analyzing the mass pattern it is possible to identify the test mRNA (para 0009).
The combined references do not teach a method further comprising mapping the RNA fragments to the reference sequence (clm 23).
However Marquardt teaches a method further comprising mapping the RNA fragments to the reference sequence (Example 7).
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 Wang and Rattan by further mapping the fragments to a references as suggested by Marquardt. One of skill in the art would have been motivated to map the fragments for the benefit of being able to identify which RNA molecule produced a fragment in a sample or to compare the cleavage patterns of different nucleases.
6. Claims 17-19 are rejected under 35 U.S.C. 103 as being unpatentable over Wang (US 2008/0124713 Pub 5/29/2008) in view of Rattan (US 2023/0174973 Filed 12/1/2022 with priority back to 63/285,402 Filed 12/2/2021) as applied to claim 1 above and in further view of Athapattu (Nucleic Acids Research 1/28/2021 Vol 49 No 7 e41).
The teachings of Wang and Rattan 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 Wang and Rattan 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 intact, and then digestion of the preferred regions for further analysis such as mapping.
Response To Arguments
7. In the response the Applicants traversed the rejections under 35 USC 103. In the response, the Applicants traversed the rejections over the combination of (i) Marquardt and Rabbani and (ii) Marquardt, Rabbani, and Athapattu. The arguments are moot because the claims as amended are rejected over a new combination of references.
8. 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 extension fee 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 date of this final action.
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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/AMANDA HANEY/ Primary Examiner, Art Unit 1682
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