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 .
Office Action: Notice
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 final rejection. 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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 4/3/2026 has been entered.
Claim Status
Claims 1, 7, 11, 15-17 have been amended (4/3/2026). Claims 19-22 are new (4/3/2026). Claims 3-4, 8-9, 12-13 have been cancelled. No new matter was added. Claims 1-2, 5-7, 10-11, 14-22 are under examination (4/3/2026).
Priority
Claims 1-2, 5-7, 10-11, 14-22 receive a priority date of 11/25/2019, the effective filing date of Japanese Provisional Patent JP2019-211933.
Rejections Withdrawn
Claim Rejections - 35 USC § 103
The rejection of claims 1-18 under 35 U.S.C. 103 as being unpatentable over O’Neil et al., (WO 2014/044,724 A1, published 3/27/2014), in view of Shao et al. (“Reconstitution of a Minimal Ribosome-Associated Ubiquitination Pathway with Purified Factors”, Molecular Cell, published 9/18/2014) and further in view of Samaha et al. (“A base pair between tRNA and 23S rRNA in the peptidyl transferase centre of the ribosome”, Nature, published 1995) is withdrawn because the newly added limitations requiring that ribosomal subunits be split using a chelating agent (and the associated chelating agent species and Mg2+ chelation limitations) were not previously searched or evaluated in the prior art combination of record. Accordingly, further search and consideration of the amended claim are required.
New Rejections
Claim Rejections - 35 USC § 103
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 (i.e., changing from AIA to pre-AIA ) 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.
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.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-2, 5-7, 10-11, 14-22 are rejected under 35 U.S.C. 103 as being unpatentable over O’Neil et al., (WO 2014/044,724 A1, published 3/27/2014), in view of Shao et al. (“Reconstitution of a Minimal Ribosome-Associated Ubiquitination Pathway with Purified Factors”, Molecular Cell, published 9/18/2014) and further in view of Samaha et al. (“A base pair between tRNA and 23S rRNA in the peptidyl transferase centre of the ribosome”, Nature, published 1995), and Miall et al. (“Structural Studies on Ribosomes”, Biochemica et Biophysica Acta, published 1969) and Todorova et al. (“Specific binding of ribosome recycling factor (RRF) with the Escherichia coli ribosomes by BIACORE”, Molecular Biology Reports, published 2002).
O’Neil teaches a method of preparing a target RNA depleted composition from an initial RNA containing composition, comprising contacting the initial RNA containing composition with one or more groups of probe molecules, an improved depletion method which effectively and specifically removes unwanted target RNA such as ribosomal RNA (rRNA) from total RNA, while ensuring recovery of mRNA and noncoding RNA from various species, including human, mouse, and rat (Abstract). Further, O’Neil teaches that the above rRNA removal techniques, in contrast to poly A enrichment-based approaches, these methods do not aim at enriching the RNA of interest, but rather aim at depleting and thus removing RNA types which are not of interest for the subsequent transcriptome analysis such as i.e., rRNA (p. 3, lines 25-35). Specifically, O’Neil teaches that the smaller the distance between the different target or split regions, the more efficient is the target RNA removal even in case of fragmented or split RNA, because the likelihood is increased that a target RNA fragment comprises at least one target region and accordingly can be efficiently captured and thus removed from the initial RNA composition via the use of more probe molecules providing more binding sites for the anti-hybrid binding agent, and thus increasing the chance to efficiently capture the target RNA also in case of fragmentation (p. 14, lines 30-40).
Further, O’Neil teaches that the previously described method of RNA removal targets RNA that can be depleted from rRNA, tRNA, snRNA, snoRNA and abundant protein mRNA, preferably, one or more types of rRNA are depleted as target RNA, respectively target RNAs (p. 22, lines 20-30).
O’Neil also teaches that the previously described method of RNA removal includes a binding or chelating agent that may bind to a second antihybrid binding agent, thereby allowing to separate the hybrid/binding agent complexes from the remaining composition (p. 21, lines 35-40) via using magnetic particles as solid support for the antihybrid binding agent, i.e., having superparamagnetic, paramagnetic, ferromagnetic or ferromagnetic properties, allowing the respective magnetic particles with the bound hybrid/binding agent complexes to be easily separated by the aid of a magnetic field (p. 19, lines 20-30).
Further, O’Neil teaches that particles or targeted RNA in the previously described method can also be separated by filtration (p. 19, lines 20-30). Further, O’Neil teaches that respective purification step can be useful e.g. in order to remove short unbound probe molecules, buffer components and the like and/or to concentrate the RNA and examples for respective purification methods include but are not limited to extraction, solid-phase extraction, polysilicic acid-based purification, isolation using silica columns or magnetic silica beads, magnetic particle-based purification, phenol chloroform extraction, anion-exchange chromatography (using anion-exchange surfaces), gel-electrophoresis, precipitation, e.g. alcohol precipitation, and combinations thereof where also any other nucleic acid isolating technique known by the skilled person can be used (p. 27, Step (d)).
O’Neil teaches that the previously described method of RNA removal includes targeted RNA removal even in the case of fragmented or split RNA, because the likelihood is increased that a target RNA fragment comprises at least one target region and accordingly can be efficiently captured and thus removed from the initial RNA composition via the use of more probe molecules providing more binding sites for the anti-hybrid binding agent, and thus increasing the chance to efficiently capture the target RNA also in case of fragmentation (p. 14, lines 30-40). Specifically, O’Neil teaches that the target RNA depleted RNA composition can be used for construction of a sequencing library (p. 22, lines 5-10).
O’Neil teaches that the previously described method of RNA removal includes a kit as described subsequently and, in the claims, may be used in step b) in order to remove one or more types of unwanted target RNAs and where sequencing comprises preparing a sequencing library suitable for massive parallel sequencing and sequencing the molecules comprised in the sequencing library in parallel (p. 31, lines 30-40). Further, O’Neil teaches that when hybridized to their target region, the short probe molecules of one group are located adjacent to each other in the formed double-stranded hybrid and thus are located in close proximity and thus covers the target region followed by binding of one group to an anti-hybrid binding agent or reagent, whereby a hybrid/binding agent complex is formed and can be easily separated or removed from the remaining composition, thereby removing unwanted target RNA via hybrid capturing technology or a remover to improve the specificity and efficiency of target RNA removal (p. 4-5, lines 40-50, 1-5).
O’Neil does not teach or suggest splitting subunits utilizing specified chelating agents (i.e., EDTA, Mg2+).
Shao teaches that ribosomes stalled on aberrant mRNAs engage quality control mechanisms that degrade the partially translated nascent polypeptide, where ubiquitination of the nascent protein is mediated by the E3 ligase Listerin via a mechanism involving ribosome subunit dissociation via a method to reconstitute ribosome-associated ubiquitination with purified factors to define the minimal components and essential steps in this process Summary). Shao further teaches that the primary role of the ribosome splitting factors Hbs1, Pelota, and ABCE1 is to permit Listerin access to the nascent chain (Summary; Graphical Abstract). Shao also teaches that splitting of 80S ribosomes is mediated by the ATPase ABCE1 where recruitment of ABCE1 to empty or stalled ribosomes requires the eRF1 and eRF3 homologs Pelota and Hbs1 and in the case of stalled RNCs, splitting generates a unique 60S-nascent chain-tRNA complex that was speculated to be recognized by RQC components for protein quality control (Introduction: Paragraphs 6-8).
Samaha teaches that protein synthesis is mediated by transfer RNA, the anticodon
sequences of which recognize complementary codon sequences in messenger RNA in the structural context of the ribosome (Introduction: Paragraph 1). Further, Samaha teaches that EDTA is used as part of the chimeric reconstitution approach to monitor peptidyl transferase activity (Peptidyl transferase activity: Paragraphs 1-2; Figure 5).
Miall teaches that the effect of removing Mg2+ from 30- and 50-S ribosomal subunits and ribosomal RNA by the direct addition of EDTA at room temperature has been followed using the analytical ultracentrifuge where 50-S subunits unfolded by a cooperative process, without the dissociation of the protein and RNA moieties, via two discrete, partially unfolded species, to give a 16-S particle and ribosome-like particles were reformed when the unfolded species were dialysed into magnesium acetate (Abstract). Further, Miall teaches that the unfolded ribosomes showed a limited polyelectrolyte behaviour on altering the Na+ concentration or removing Mg2+ and a tendency to specifically refold as the Na+ concentration was increased and by contrast the sedimentation coefficient of 23-S RNA showed progressive changes as Mg2+ was removed with EDTA. No discrete intermediate species were formed, where the native 50-S ribosome and the unfolded species had the same total amount of secondary structure and the denaturation behaviour of the 16-S species was the same as 23-S RNA in EDTA (Abstract).
Todorova teaches that the direct assays on Biacore with immobilised RRF and purified L11 from E. coli in the flow trough have shown unspecific binding between the both proteins, where the interaction of RRF with GTPase domain of E. coli ribosomes, a functionally active complex of L11 with 23S r RNA and L10.(L7/L12)4 was studied by Biacore and in the experiments of binding of RRF with 30S, 50S and 70S ribosomes from E. coli were used the antibiotics thiostrepton, tetracycline and neomycin and factors, influencing the 70S dissociation Mg2+,NH4Cl, EDTA (Abstract). Further, Todorova teaches that the effect of [Mg2+] on the binding of RRF with the ribosome has been evaluated (Figure 1 A,B,C) and maximal binding has been detected at 5 mM [Mg2+] for 70S ribosomal particles comparing with 50S and
30S subunits (Results and Discussion: Paragraph 2). Specifically, Todorova teaches that the effect of [Mg2+] is evidence that the interaction between the ribosome (30S, 50S, 70S) and RRF is charge-dependent and the process of binding is also dependent from the concentrations of EDTA, a chelating agent with a maximal effect at about 10 mM in the flow, where the presence of EDTA influences most strongly the binding of RRF with 50S ribosomal subunit, but also interacted with 70S ribosomes as well as 50S and 30S subunits (Figure 4; Results and Discussion: Paragraph 4).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to modify the RNA depletion and sequencing preparation method of O’Neil in view of Shao and further in view of Samaha, Miall and Todorova. O’Neil teaches preparing a target-RNA-depleted composition from total RNA, including removal of ribosomal RNA and use of the resulting material for sequencing library construction, thereby providing the primary framework for producing a non-ribosomal RNA-containing sample suitable for downstream transcriptomic analysis. Shao teaches the biological mechanism and conditions under which ribosome subunits are dissociated and removed from stalled ribosome-nascent chain complexes, demonstrating that ribosome splitting and removal are predictable and controllable processes in RNA-associated systems. Samaha further teaches the structural and functional interaction of tRNA, rRNA and mRNA within the ribosome, providing foundational knowledge of ribosomal architecture and confirming that manipulation or dissociation of ribosomal components to access RNA substrates was well understood in the art. Miall further teaches that removal of Mg2+ by direct addition of EDTA affects 30S and 50S ribosomal subunits and ribosomal RNA, demonstrating that chelation-mediated destabilization of ribosomal structures was known in the art. Todorova likewise teaches that ribosome interactions are dependent upon Mg2+ concentration and that EDTA, a chelating agent, strongly affects interactions involving 30S, 50S, and 70S ribosomal particles.
One of ordinary skill in the art would have been motivated to incorporate the ribosome-splitting principles taught by Shao, as informed by the structural understanding provided by Samaha, into the RNA-depletion workflow of O’Neil in order to improve removal of ribosome-associated RNA species. Such motivation would have been further supported by Miall and Todorova, which demonstrate that EDTA-mediated Mg2+ chelation was a known and predictable technique for disrupting or destabilizing ribosomal subunit interactions. Applying a known chelating agent such as EDTA to facilitate ribosomal dissociation in O’Neil’s depletion workflow would merely involve the use of known ribosome-handling techniques according to their established functions to achieve the predictable result of the improved separation of ribosomal components from target RNA to thereby enhance the efficiency and specificity of generating non-ribosomal RNA samples for sequencing. Further, such modification merely applies known ribosome dissociation and RNA-handling techniques to the known RNA-depletion method of O’Neil to achieve the predictable result of improved separation of ribosomal components from target RNA, which constitutes the use of prior art elements according to their established functions.
Moreover, a person of ordinary skill in the art would have had a reasonable expectation of success in making this combination because each reference operates in the same technical field of ribosome associated RNA processing and sequencing preparation, and the mechanisms of ribosome splitting, RNA removal, and sequencing library preparation were well characterized and routinely combined in molecular biology workflows. Specifically, Shao teaches ribosome disassociation, while Miall and Todorova independently demonstrate that EDTA-mediated Mg2+ removal predictably alters ribosomal subunit structure and interactions. Because the cited references collectively establish that ribosomal dissociation through chelation-based manipulation of Mg2+ was well understood and routinely employed, incorporating such teachings into the RNA depletion workflow of O’Neil would have represented no more that routine optimization of known techniques yielding predictable results. Further, integrating Shao’s ribosome disassociation teachings and Samaha’s structural insights into O’Neil’s depletion and sequencing framework would therefore have represented no more than routine optimization of known techniques yielding predictable results.
Applicant’s Response: The Applicant argues that the cited references fail to teach or suggest splitting ribosomal subunits and mRNAs using a chelating agent and subsequently removing the split ribosomal subunits by ultrafiltration or size exclusion chromatography, as recited in amended independent claims 1, 7 and 11. Applicant further contends that the references do not teach the specifically recited chelating agents, Mg2+ chelating agents, or the claimed separation techniques, and therefore do not render the amended claims obvious.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully and fully considered and are found to be partially persuasive, as discussed below. In response to the Applicant’s arguments, as written above, the rejection has been modified to additionally rely upon Miall and Todorova.
As evidenced by the modified rejection, Todorova teaches that interaction of ribosomal subunits is dependent upon Mg2+ concentration and further teaches that EDTA, a chelating agent, strongly affects interactions involving 50S, 30S, and 70S ribosomal subunits, thereby evidencing ribosomal dissociation through Mg2+ chelation. Miall similarly teaches removal of Mg2+ directly via EDTA, resulting in unfolding and dissociation of 50S ribosomal subunits. Accordingly, the combined references teach or suggest the claimed use of chelating agents, including Mg2+ chelating agents, for splitting ribosomal subunits.
Applicant’s arguments regarding ultrafiltration and size exclusion chromatography are likewise unpersuasive. O’Neil teaches purification and separation of the target RNA-depleted composition using numerous known isolation and purification techniques, including chromatography and further teaches other nucleic acid isolation technique known in the art may be employed. Therefore, selection of ultrafiltration or size exclusion chromatography as a known separation technique for removing dissociated ribosomal components would have been obvious matter of routine optimization yielding predictable results.
Therefore, the Applicant’s arguments were considered partially persuasive, because they identified deficiencies in the previously applied combination in light of the new amendments; however, those deficiencies were remedied by the additional teachings of Miall and Todorova as set forth in the modified rejection above. To overcome the rejection, Applicant may amend the claims to recite structural or process limitations directed to a particular ribosome-spitting mechanism, chelation condition or separation methodology.
Conclusion
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ELIZABETH ROSE LAFAVE whose telephone number is (703)756-4747. The examiner can normally be reached Compressed Bi-Week: M-F 7:30-4:30.
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/ELIZABETH ROSE LAFAVE/ Examiner, Art Unit 1684
/HEATHER CALAMITA/ Supervisory Patent Examiner, Art Unit 1684
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