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 .
Priority
This application 17/797,950 filed on 08/05/2022 is a 371 national phase of PCT/SG2021/050070 filed on 02/10/2021, and claims the benefit of Singapore application SG 10202001192W filed on 02/10/2020.
The priority date of claim 1 and its dependent claims 2-6, 8, 10-14, 16, 17, 21-23, and independent claim 15 is determined to be 02/10/2020, the filing date of Singapore application SG 10202001192W.
Status of Claims
Applicant’s amendments to claims filed 10/29/2025 in response to the Non-Final Rejection mailed 08/06/2025 are acknowledged.
Claims 1, 3, 5, 6, 13, and 15 are amended.
Claims 2, 4, 8, 10, 11, 14, and 23 have been canceled.
New claims 24-29 are acknowledged.
Claims 1, 3, 5, 6, 12, 13, 15-17, 19-22, 24-29 are pending and claims 1, 3, 5, 6, 12, 13, 15-17, 21-22, and 24-29 are under examination.
Response to Remarks filed 10/29/2025
The amendments and arguments presented in the papers filed 10/29/2025 ("Remarks”) have been thoroughly considered. The issues raised in the Office action dated 08/06/2025 listed below have been reconsidered as indicated.
a) The objection to the specification regarding the use of an embedded hyperlink is withdrawn in view of the amendments to the specification.
b) The objection to claim 6 is withdrawn in view of the amendment to the claim.
c) The 35 USC 112(b) indefiniteness rejections of claim 10 has been withdrawn as being moot in view of the cancellation of claim 10.
d) The 35 USC 112(b) indefiniteness rejections of claims 13 and 33 have been withdrawn in view of the amendments to claims.
e) The rejection of claims 1-6, 8, 10-15, and 21-23 under 35 U.S.C. 101 are withdrawn in view of the amendments to claims 1 and 15 and cancellation of claims 2, 4, 8, 10, 11, 14, and 23.
f) The rejections of claim(s) 1, 3, 5, 6, 8, 12, 13, and 23 under 35 U.S.C. 102(a)(1) as being anticipated by Siegfried et al. (RNA motif discovery by SHAPE and mutational profiling (SHAPE-MaP). 2014. Nat Methods. 11(9): 1-30), are withdrawn in view of the amendments to the claims and cancellation of claims 8 and 23.
g) The rejection of claims 2, 4, 10, 11, and 14 under 35 U.S.C. 103 as being unpatentable are withdrawn as moot in view of the cancellation to the claims.
New and modified grounds of rejection necessitated by amendment are detailed below and this action is made FINAL.
Claim Objections - New
Claims 1 and 15 are objected to because of the following informalities: Claims 1 and 15 recite the limitation " a modifying agent comprising --- 2-methylnicotinic acid imidzolide-azide (NAI-N3)". “Imidzolide” appears to be a typo. It is assumed that the intended term is "2-methylnicotinic acid imidazolide-azide (NAI-N3)". Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1, 3, 5-6, 12-13, 15-17, 21-22, and 24-29 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
The term “substantially free” in claims 1 and 15 is a relative term which renders the claim indefinite. The term “substantially free” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. The specification provides a characterization of “substantially free” as “less than about 0.1%, less than about 0.01%, less than 0.001% or less than a detectable amount” or “about 0%” (p. 13). A percentage of a “detectable amount” does not clearly set forth the metes and bounds of the patent protection desired. The limitation regarding the magnesium in the medium is rendered indefinite by use of the term "substantially free".
Amended claims 1 and 15 filed on 10/29/2025 recite the limitation “fragmenting the modified RNA molecules comprising heating the modified RNA molecules in the presence of deoxynucleoside triphosphate (dNTP), wherein the fragmenting step is carried out in a medium that is substantially free of magnesium, and wherein the medium is water”. It is not clear that the fragmenting step requires a medium that is only water and nothing else. As written the claims recite limitations under the open language of comprising. The limitation requires a fragmenting step that comprises additional dNTPs and some level of magnesium, thus requiring a medium that is not only water. Therefore, the metes and bounds of claim 1 cannot be determined.
Amended claims 1 and 15 recite the limitation “sequencing the product obtained from the preceding step to generate sequencing reads. The claims recite multiple active steps preceding the step of “sequencing the product”. Therefore, the metes and bounds of claims 1 and 15 cannot be determined.
Claims 3,5,6,12,13,15-17,21,22,24-26 are similarly indefinite because they directly or indirectly depend from claim 1.
Claims 27 and 29 are similarly indefinite because they directly or indirectly depend from claim 15.
Regarding amended claim 15, it is not clear how the recited preamble is intended to breathe life and meaning into the claim. The preamble of the claim recites “a method of simultaneously determining a structure of an RNA molecule of a gene and an expression of the gene in a single cell”. However, the method steps in the claim only require “contacting the RNA molecule with a modifying agent ---; fragmenting the modified RNA molecule --; reverse transcribing the modified RNA molecule; and
sequencing the product ----”. Thus, it is unclear if applicant intends to cover any method of performing the active method steps, or if the method is intended to somehow require more to accomplish the goal set forth in the preamble. If it is the latter, then it appears that the claims are incomplete, as they fail to provide any active steps that clearly accomplish the goal of “simultaneously determining a structure of an RNA molecule of a gene and an expression of the gene in a single cell” as set forth by the preamble of the claim. Amending the claim to include an active process step directed towards a single cell.
Claim Rejections - 35 USC § 101 - modified
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 16, 17, 26 and 27 are/remain rejected under 35 U.S.C. 101 because the claimed invention is directed to non-statutory subject matter.
These are new and modified rejections necessitated by claim amendments filed on 10/29/2025.
35 U.S.C. § 101 requires that to be patent-eligible, an invention (1) must be directed to one of the four statutory categories, and (2) must not be wholly directed to subject matter encompassing a judicially recognized exception. M.P.E.P. § 2106. Regarding judicial exceptions, “[p]henomena of nature, though just discovered, mental processes, and abstract intellectual concepts are not patentable, as they are the basic tools of scientific and technological work.” Gottschalk v. Benson, 409 U.S. 63, 67 (1972); see also M.P.E.P. § 2106, part II.
Based upon consideration of the claims as a whole, as well as consideration of elements/steps recited in addition to the judicial exception, the present claims fail to meet the elements required for patent eligibility.
Step 1
The claimed invention is directed to the statutory category of a process.
Step 2A, Prong One
The claims are taken to be directed to an abstract idea, a judicial exception.
Claim 16 depends from claim 1 and is directed to a method comprising “characterizing a cell by: determining the structure of RNA molecules in the cell”. This limitation is an abstract mental process (see MPEP 2106.04(a)(2)(III)). As written, the characterizing by determining step encompasses the mental step of looking at the structure of RNA molecules and making a mental judgement.
Claim 17 depends from claim 1and is directed to a method comprising “classifying cells into one or more cell populations by: determining the structure of RNA molecules in each cell and classifying the cells into one or more cell populations based on similarity in the structure of their RNA molecules”. This limitation is an abstract mental process (see MPEP 2106.04(a)(2)(III)). As written, the classifying steps (by determining and classifying) encompass the mental step of looking at the structure of RNA molecules in each cell and making mental judgements.
Claim 26 depends from claim 1and is directed to a method comprising “analyzing the sequencing reads to determine the structure of the RNA molecules”. This limitation is an abstract mental process (see MPEP 2106.04(a)(2)(III)). As written, the analyzing step encompasses the mental steps of looking at and comparing sequencing reads, and making mental judgements.
Claim 27 depends from claim 15 and is directed to a method comprising “analyzing the sequencing reads to determine the structure of the RNA molecule of the gene; evaluating the amount of sequencing reads to determine the expression of the gene; and distinguishing between cell types, including cell types showing similar gene expression profiles”. These limitations are an abstract mental process (see MPEP 2106.04(a)(2)(III)). As written, the analyzing, evaluating, and distinguishing steps encompass the mental steps of looking at and comparing sequencing reads, and making mental judgements.
Step 2A, Prong Two
The judicial exception is not integrated into a practical application.
The claims do not recite any additional elements that integrate the exception into a practical application of the exception.
Claim 1, which claims 16, 17, and 26 depend from additionally recites “contacting the RNA molecules with a modifying agent -- to obtain modified RNA molecules; fragmenting the modified RNA molecules --; reverse transcribing the modified RNA molecules; and sequencing the product --”. However, these steps are not an integration of the exception into a practical application. Rather, these steps are mere data gathering required to perform the method.
Claim 15, which claim 27 depends from additionally recites “contacting the RNA molecule with a modifying agent -- to obtain a modified RNA molecule; fragmenting the modified RNA molecule --; reverse transcribing the modified RNA molecule; and sequencing the product--”. However, these steps are not an integration of the exception into a practical application. Rather, these steps are mere data gathering required to perform the method.
Step 2B
The claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception. The claim does not add a specific limitation other than what is well-understood, routine, and conventional in the field. Steps directed to “contacting RNA molecules”; “reverse transcribing “; “sequencing” and “fragmenting” "are techniques that are routine, conventional, and well-known in the art as demonstrated in the 103 rejections documented below.
Furthermore, the courts have recognized the following laboratory techniques as well-understood, routine, conventional activities in the life science arts when they are claimed in a merely generic manner or as insignificant extra-solution activity:
i. Amplifying and sequencing nucleic acid sequences, University of Utah Research Foundation v. Ambry Genetics, 774 F.3d 755, 764, 113 USPQ2d 1241, 1247 (Fed. Cir. 2014); and
For these reasons, the claims are rejected under section 101 as being directed to non-statutory subject matter.
Response to Arguments against Claim Rejection - 35 U.S. C § 101
The response asserts that the amendments to independent claims 1 and 15 render moot this rejection. Claims 1 and 15 have further been amended to recite, inter alia, a novel and non-obvious combination of transformative steps comprising contacting RNA molecules with a modifying agent, fragmenting the modified RNA molecules, reverse transcribing the modified RNA molecules and sequencing the product obtained from the preceding steps. The combination of steps recited in instant claims 1 and 15 is not a "well understood, routine, and conventional method in the field". The response further asserts that claims 1 and 15 have been amended to delete the alleged abstract
mental process step (p. 9-10).
Applicant's arguments regarding independent claims 1 and 15 have been fully considered and are persuasive. Amendments deleting the abstract mental processes cited in the 35 U.S.C. 101 rejection in the office action dated 08/06/2025 are sufficient to overcome the rejections of independent claims 1 and 15 and their dependent claims 3, 5, 6, 12, 13, 21, and 22.
The response additionally asserts claims 16 and 17 depend on claim 1 and incorporate all of its elements and should therefore have their 35 U.S.C rejections withdrawn (p. 10).
Applicant's arguments have been fully considered but are not persuasive.
Claims 16 and 17 were not amended by applicant and remain directed to non-statutory subject matter for the reasons of record and the reasons set forth above. The rejection has been modified as needed in order to address applicant’s amendments to the claims.
New rejection to claims 26 and 27 have been added as necessitated by amendments, for the reasons provided above.
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.
Claims 1,3,5-6,12-13, 15, 22, 24-26 and 28-29 are rejected under 35 U.S.C. 103 as being unpatentable over Siegfried et al. (RNA motif discovery by SHAPE and mutational profiling (SHAPE-MaP). 2014. Nat Methods. 11(9):959-965) in view of Spitale et al. (Structural imprints in vivo decode RNA regulatory mechanisms. 2015. Nature. 519: 1-19, on the IDS dated 08/05/2022) and Bibillo et al. (WO2018089860).
These are new rejections necessitated by claim amendments filed on 10/29/2025.
Regarding claim 1, Siegfried teaches determining the structure of RNAs, the method comprising: treating RNA with a SHAPE reagent that modifies the RNA (p. 1, col 2); fragmenting the RNA molecules after SHAPE modification by incubating at 94°C (i.e. heating) (p. 9, col 1); reverse-transcribing the modified RNA (p. 1, col 2); and sequencing the product to generate sequencing reads (p. 8, col 1).
Siegfried does not teach (i) “contacting the RNA molecules of a single cell”; (ii) “a modifying agent comprising 2- methylnicotinic acid imidazolide (NAI) or 2-methylnicotinic acid imidazolide-azide (NAI-N3)”, (iii) fragmenting the modified RNA “in the presence of deoxynucleoside triphosphate (dNTP), wherein the fragmenting step is carried out in a medium that is substantially free of magnesium, and wherein the medium is water”; or (iv) “wherein the fragmenting step is carried out in the same vessel as the reverse transcribing step”.
Regarding (ii), Siegfried teaches the use of SHAPE modifying reagents (including 1M6, 1M7, and NMIA) (p.2, col 1) but Siegfried does not teach the specific modifying agent comprises 2- methylnicotinic acid imidazolide (NAI) or 2-methylnicotinic acid imidazolide-azide (NAI-N3).
Spitale teaches the use of NAI-N3, a derivative of NAI, as a SHAPE reagent and states that NAI-N3 generated identical profiles to previously designed SHAP reagents (p.1, col 1). Spitale further states that the use of NAI-N3 gives a more complete representation of RNA structure (p. 2, col. 1).
Regarding (iii), Spitale teaches a fragmenting step free of magnesium (p. 5 col 2 to p. 6 col 1).
Regarding (i), (iii) and (iv), neither Siegfried nor Spitale teach the RNA molecules are from a single cell or the fragmenting step is carried out in the presence of dNTP and in the same vessel as the reverse transcribing step.
Bibillo teaches methods for analyzing nucleic acid samples by preparing libraries that can be used for single cell nucleic acid (e.g., RNA) sequencing (para 32). Bibillo teaches the RNA can come from a single cell (para 6); RNA fragmentation, including using higher temperatures in the presence of metals such as Mn (para 328). Bibillo further teaches using reverse transcriptase to produce a cDNA library for sequencing (para 32).
Bibillo teaches the presence of dNTPs in a reaction mixture along with nucleic acid fragments for reverse transcription (paras 85, 149-156), but does not explicitly teach a fragmenting step in the presence of dNTP.
However, Bibillo teaches that in some embodiments, all the steps of the method of the present disclosure is performed in a single tube (single vessel) (para 149). Bibillo teaches that a 1-pot or 1-step method provides the benefit of no losses (Fig. 2) while performing the method.
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Siegfried and Spitale with Bibillo to arrive at the instantly claimed invention. The modification would have entailed (a) substituting the SHAPE agent NAI-N3 of Spitale in place of the SHAPE agents taught by Siegfried and substituting the magnesium-free fragmentation step of Spitale. One would have been motivated by the ability of NAI-N3 to produce a more complete representation of RNA structures as stated by Spitale. The substitution of the magnesium-free fragmentation step of Spitale for the fragmentation step of Siegfried. One would have been motivated to make the substitution by the expectation that the fragmenting step of Spitale would work with the modified RNA generated by the modifying agent of Spitale. The modification would have further entailed using the method of Bibillo to adapt the method of Siegfried and Spitale for use on RNA from single cells and performing reactions in a single vessel. One of ordinary skill in the art would have been motivated to adopt the method of Bibillo in order to increase the resolution of Siegfried and Spitale to analyze the structure of single cell RNAs and simplify the active steps of the method and minimize sample loss by using a single vessel. Furthermore, by performing the fragmenting and reverse transcription in a single vessel as in Bibillo, the Mn of the reverse transcription buffer in Siegfried could serve as the divalent cation during fragmentation (a substitution for magnesium recognized by Bibillo). Differences in the order and location of fragmenting of RNA and reverse transcription are routine optimizations known in the art. Siegfried and Spitale present two known optimizations and Bibillo teaches numerous embodiments capable of generating a cDNA library from RNA for sequencing. It would have been obvious to try different steps of fragmenting and reverse transcription to optimize the method. There would have been a reasonable expectation of success given the underlying materials and methods are widely known, successfully demonstrated, and commonly used as evidenced by the prior art.
Regarding claim 3, Siegfried teaches a reverse transcription buffer that includes manganese (p. 8, col 1 and p. 9, col. 1).
Regarding claim 5, Siegfried teaches use of the Moloney murine leukemia virus (MMLV) reverse transcriptase (p. 8, col 2).
Regarding claim 6, Siegfried teaches performing reverse transcription for a long incubation, specifically 3 hours (p.9, col 1), but does not teach the specific limitation of the reverse transcribing step is carried out for at least about 8 hours.
However, Siegfried teaches screening reverse transcriptase enzymes for multiple parameters, including reaction time, in order to minimize reverse transcription stops and maximize full-length cDNA products (p. 8, col. 1).
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Siegfried to arrive at the instantly claimed invention. The modification would have entailed extending the reverse transcription reaction time from 3 hours to at least about 8 hours. Determining an appropriate reaction time is deemed merely a matter of judicious selection and routine optimization which is well within the purview of the skilled artisan, as is taught by Siegfried. The courts have found "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). See MPEP 2144.05.01. One of ordinary skill in the art would have been motivated to optimize for a reaction time that minimized reverse transcription stops and maximized full-length cDNA products. There would have been a reasonable expectation of success given the underlying materials and methods are widely known, successfully demonstrated, and commonly used as evidenced by the prior art.
Regarding claim 12, Siegfried teaches amplifying reverse-transcribed products before sequencing (p. 9, col 2).
Regarding claim 13, Siegfried teaches amplifying reverse-transcribed products and purifying the amplicons (p. 9, col 2).
Regarding claim 15, Siegfried teaches determining the structure of RNAs, the method comprising: treating RNA with a SHAPE reagent that modifies the RNA (p. 1, col 2); fragmenting the RNA molecules after SHAPE modification by incubating at 94°C (i.e. heating) (p. 9, col 1); reverse-transcribing the modified RNA (p. 1, col 2); and sequencing the product to generate sequencing reads (p. 8, col 1).
Siegfried does not teach (i) “a modifying agent comprising 2- methylnicotinic acid imidazolide (NAI) or 2-methylnicotinic acid imidazolide-azide (NAI-N3)”, (ii) fragmenting the modified RNA “in the presence of deoxynucleoside triphosphate (dNTP), wherein the fragmenting step is carried out in a medium that is substantially free of magnesium, and wherein the medium is water”; or (iii) “wherein the fragmenting step is carried out in the same vessel as the reverse transcribing step”.
Regarding (i), Siegfried teaches the use of SHAPE modifying reagents (including 1M6, 1M7, and NMIA) (p.2, col 1) but Siegfried does not teach the specific modifying agent comprises 2- methylnicotinic acid imidazolide (NAI) or 2-methylnicotinic acid imidazolide-azide (NAI-N3).
Spitale teaches the use of NAI-N3, a derivative of NAI, as a SHAPE reagent and states that NAI-N3 generated identical profiles to previously designed SHAP reagents (p.1, col 1). Spitale further states that the use of NAI-N3 gives a more complete representation of RNA structure (p. 2, col. 1).
Regarding (ii), Spitale teaches a fragmenting step free of magnesium (p. 5 col 2 to p. 6 col 1).
Regarding (ii) and (iii), neither Siegfried nor Spitale teach the RNA molecules are from a single cell or the fragmenting step is carried out in the presence of dNTP and in the same vessel as the reverse transcribing step.
Bibillo teaches methods for analyzing nucleic acid samples by preparing libraries that can be used for single cell nucleic acid (e.g., RNA) sequencing (para 32). Bibillo teaches the RNA can come from a single cell (para 6); RNA fragmentation, including using higher temperatures in the presence of metals such as Mn (para 328). Bibillo further teaches using reverse transcriptase to produce a cDNA library for sequencing (para 32).
Bibillo teaches the presence of dNTPs in a reaction mixture along with nucleic acid fragments for reverse transcription (paras 85, 149-156), but does not explicitly teach a fragmenting step in the presence of dNTP.
However, Bibillo teaches that in some embodiments, all the steps of the method of the present disclosure is performed in a single tube (single vessel) (para 149). Bibillo teaches that a 1-pot or 1-step method provides the benefit of no losses (Fig. 2) while performing the method.
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Siegfried and Spitale with Bibillo to arrive at the instantly claimed invention. The modification would have entailed (a) substituting the SHAPE agent NAI-N3 of Spitale in place of the SHAPE agents taught by Siegfried and substituting the magnesium-free fragmentation step of Spitale. One would have been motivated by the ability of NAI-N3 to produce a more complete representation of RNA structures as stated by Spitale. The substitution of the magnesium-free fragmentation step of Spitale for the fragmentation step of Siegfried. One would have been motivated to make the substitution by the expectation that the fragmenting step of Spitale would work with the modified RNA generated by the modifying agent of Spitale. The modification would have further entailed using the method of Bibillo to adapt the method of Siegfried and Spitale for use on RNA from single cells and performing reactions in a single vessel. One of ordinary skill in the art would have been motivated to adopt the method of Bibillo in order to increase the resolution of Siegfried and Spitale to analyze the structure of single cell RNAs and simplify the active steps of the method and minimize sample loss by using a single vessel. Furthermore, by performing the fragmenting and reverse transcription in a single vessel as in Bibillo, the Mn of the reverse transcription buffer in Siegfried could serve as the divalent cation during fragmentation (a substitution for magnesium recognized by Bibillo). Differences in the order and location of fragmenting of RNA and reverse transcription are routine optimizations known in the art. Siegfried and Spitale present two known optimizations and Bibillo teaches numerous embodiments capable of generating a cDNA library from RNA for sequencing. It would have been obvious to try different steps of fragmenting and reverse transcription to optimize the method. There would have been a reasonable expectation of success given the underlying materials and methods are widely known, successfully demonstrated, and commonly used as evidenced by the prior art.
Regarding claim 22, Siegfried teaches purifying amplicons, but does not teach purifying the amplicons for at least two times.
However, it would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Siegfried to arrive at the instantly claimed invention. The modification would have entailed adding an additional purification step to the method of Siegfried. Adjusting the number of purification steps is deemed merely a matter of judicious selection and routine optimization which is well within the purview of the skilled artisan. The courts have found "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). See MPEP 2144.05.01. One of ordinary skill in the art would have been motivated to optimize for a number of purification steps that resulted in minimal loss of product balanced with cleaner samples for analysis. There would have been a reasonable expectation of success given the underlying materials and methods are widely known, successfully demonstrated, and commonly used as evidenced by the prior art.
Regarding claim 24, Siegfried teaches carrying out reverse transcription at 42°C (p. 9, col. 1).
Regarding claim 25, Siegfried teaches using Superscript II, a genetically engineered MMLV reverse transcriptase for reverse transcription (p. 9, col. 1).
Regarding claim 26, Siegfried teaches using sequencing reads to predict RNA structure (p. 9, col 2).
Regarding claims 28 and 29, neither Siegfried nor Spitale teach the fragmenting step is carried out in the presence of oligo dT.
Bibillo teaches the use of an oligo dT primer (Figs. 3 and 12A).
Bibillo does not teach the oligo DT is present during the fragmenting step. However, Bibillo teaches all the steps of the method of the present disclosure is performed in a single tube (single vessel) (para 149) and as 1-pot, 1-step workflows (Fig. 2). Bibillo further teaches that the use of a 1-pot, 1-step method minimizes loss (Fig. 2).
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Siegfried and Spitale with Bibillo to arrive at the instantly claimed invention. The modification would have entailed using the 1-pot method of Bibillo with the RNA structure identifying method of Siegfried and Spitale. One would have been motivated to use the 1-pot approach of Bibillo in order to simplify and shorten the method of Siegfried and Spitale in addition to minimizing loss. Further, differences in the order and location of fragmenting of RNA and reverse transcription are routine optimizations known in the art. It would have been obvious to try different steps of fragmenting and reverse transcription to optimize the method. There would have been a reasonable expectation of success given the underlying materials and methods are widely known, successfully demonstrated, and commonly used as evidenced by the prior art.
Claims 16, 17, 21, and 27 are rejected under 35 U.S.C. 103 as being unpatentable over Siegfried et al. (RNA motif discovery by SHAPE and mutational profiling (SHAPE-MaP). 2014. Nat Methods. 11(9):959-965) in view of Spitale et al. (Structural imprints in vivo decode RNA regulatory mechanisms. 2015. Nature. 519: 1-19, on the IDS dated 08/05/2022) and Bibillo et al. (WO2018089860) as applied to claims 1,3,5-6,12-13, 15, 22, 24-26 and 28-29 above, and further in view of Lafzi et al. (Tutorial: guidelines for the experimental design of single-cell RNA sequencing studies. 2018. Nat Protoc 13: 2742–2757).
Lafzi is directed to guidelines for the experimental design of single-cell RNA sequencing studies to improve understanding of complexity in tissues, organs, and organisms (p. 2742, col 1). Lafzi teaches single-cell RNA sequencing (i.e. analyzing the RNA of a single cell).
Regarding claim 16, Siegfried teaches using sequencing reads to determine the structure of RNA molecules (p. 9, col 2).
Siegfried does not teach characterizing a cell by determining the structure of RNA molecules in the cell.
Lafzi teaches demultiplexing sequencing reads using cell-specific barcodes (p. 2751, col 1 and p. 2743, Fig. 1 “Data processing”) and assigning an identity to a group of cells on the basis of expression (p. 2744, Table 1).
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Spitale and Lafzi to arrive at the instantly claimed invention. The modification would have entailed using reads from a library prepared in the method of Siegfried with the cell-specific barcodes of Lafzi to produce reads that could determine the structure of RNA molecules (per Siegfried p. 9, col, 2) with the barcodes and data processing of Lafzi to demultiplex reads from single cells. One would have motivated to do so for the increased resolution of single-cell RNA sequencing as taught by Lafzi, and insight into cell types and subpopulations as taught by Lafzi (p. 2742, col 1). Both Siegfried and Lafzi used routine methods of library preparation and RNA sequencing. There would have been a reasonable expectation of success given the underlying materials and methods are widely known, successfully demonstrated, and commonly used as evidenced by the prior art.
Regarding claims 17 and 21, Siegfried teaches using sequencing reads to determine the structure of RNA molecules (p. 9, col 2).
Siegfried does not teach classifying cells into one or more cell populations by determining the structure of RNA molecules in each cell and classifying the cells into one or more cell populations based on similarity in the structure of their RNA molecules.
Lafzi teaches demultiplexing sequencing reads using cell barcodes (p. 2751, col 1 and p. 2743, Fig. 1 “Data processing”) and assigning an identity to a group of cells on the basis of expression (p. 2744, Table 1). Lafzi further teaches cluster annotation, assigning an identity (classifying cells into one or more populations) to a group of cells on the basis of the expression of genes (p. 2744, Table 1).
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Spitale and Lafzi to arrive at the instantly claimed invention. The modification would have entailed using reads from a library prepared in the method of Siegfried with the cell-specific barcodes of Lafzi to produce reads that could determine the structure of RNA molecules (per Siegfried p. 9, col, 2) with the barcodes and data processing of Lafzi to demultiplex reads from single cells. The modification would further entail using the data processing of Lafzi to classify cells on the basis of expression in the cells. One would have motivated to do so for the increased resolution of single-cell RNA sequencing as taught by Lafzi, and insight into cell types and subpopulations as taught by Lafzi (p. 2742, col 1). Both Siegfried and Lafzi used routine methods of library preparation and RNA sequencing. There would have been a reasonable expectation of success given the underlying materials and methods are widely known, successfully demonstrated, and commonly used as evidenced by the prior art.
Regarding claim 27, Siegfried teaches aligning reads to reference sequences (p. 10, col 1) and teaches differences in read depth and number of reads for sequenced RNA molecules (p. 11, col 2). Siegfried further teaches quantifying the relative abundance of variant sequences.
Siegfried does not teach evaluating the amount of sequencing reads to determine the expression of the gene.
Spitale teaches calculating the expression level of all transcripts in the mouse transcriptome in terms of reads per kilobase per million mapped reads (RPKM) (p. 8, col. 1).
Neither Siegfried nor Spitale teach distinguishing between cell types, including cell types showing similar gene expression profiles.
Lafzi teaches clustering (grouping) cells in such a way that cells in the same group (cluster) are more similar to each other than to cells of another group (p. 2744, Table 1) and clustering with marker identification (p. 2743, Fig. 1D), which read on distinguishing between cell types, including cell types showing similar gene expression profiles. Lafzi teaches that single cell RNA sequencing can be used to resolve sample heterogeneity (p. 2753, col. 1)
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Siegfried and Spitale with Lafzi to arrive at the instantly claimed invention. The modification would have involved adding the calculation of expression level by Spitale with the sequencing steps of Spitale. Spitale teaches methods that generate read data necessary for calculating expression levels. Calculating expression levels from aligned sequencing reads and evaluating RNA reads to determine gene levels was routine and known in the art before the effective filing date of the claimed invention. One would have been motivated to do so for the purpose of accurately interpreting the RNA structure sequence data. As Siegfried states, the level of reads affects the precision of the RNA structure data (p. 11, col 2). The modification would further have entailed using the clustering and marker identification of Lafzi to distinguish between cell types. One with ordinary skill in the art would also have been motivated to use the available data from sequencing reads to maximize insight into cell to cell differences in gene expression or RNA structure, for example by providing insight into sample heterogeneity. There would have been a reasonable expectation of success given the underlying materials and methods are widely known, successfully demonstrated, and commonly used as evidenced by the prior art.
Response to Arguments against Claim Rejection - 35 U.S. C § 102
The response asserts that the amendments to independent claims render moot
this rejection. And further cancellation of claims 8 and 23 render moot the rejection. The response further states that claim 1 has been amended to incorporate the elements of claims 2, 10 and 14, all of which were recognized as novel over Siegfried (p. 10)
Applicant’s arguments, filed 10/29/2025, with respect to the rejection(s) of claim(s) 1, 3, 5, 6, 8, 12, 13, and 23 under 35 U.S.C. 102(a)(1) have been fully considered and are persuasive. Therefore, the rejection has been withdrawn. However, upon further consideration, a new ground(s) of rejection under 35 U.S.C. 103 are presented above.
Response to Arguments against Claim Rejection - 35 U.S. C § 103
The response asserts that the instant claims are not obvious over the cited references because a skilled person considering the teachings of Siegfried, Spitale and Lafzi would not have had a reason to modify their teachings to arrive at a method comprising an RNA fragmentation step by heating the RNA modified molecules in the presence of dNTP in water, wherein the medium is substantially free of magnesium (p. 11)
Regarding Siegfried, the response asserts Siegfried does not teach or suggest an RNA fragmentation step by heating the RNA modified molecules in the presence of dNTP in water, wherein the medium is substantially free of magnesium or a method of analyzing RNA molecules in a single cell, wherein the fragmenting step is carried out in the same vessel as the reverse transcribing step (p. 11).
Regarding Spitale, the response asserts Spitale et al also does not teach or suggest an RNA fragmentation step by heating the RNA modified molecules in the presence of dNTP in water, wherein the medium is substantially free of magnesium. In addition, Spitale et al does not teach or suggest a method of analyzing RNA molecules in a single cell, wherein the fragmenting step is carried out in the same vessel as the reverse transcribing step (p. 11).
Regarding Lafzi, the response asserts Lafzi does not teach or suggest an RNA fragmentation step by heating the RNA modified molecules in the presence of dNTP in water, wherein the medium is substantially free of magnesium (p. 12).
Applicant's arguments have been fully considered but are not persuasive.
Regarding the arguments against Siegfried, Spitale, and Lafzi, in response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
The response asserts that the present inventors have successfully developed a novel and inventive method which they have named 'deciphering identity of single cells operated through structure' or DISCOS, which as the name suggests, is capable of determining the structure of RNA at a single-cell resolution. The response further asserts that the method allows the structure of RNA molecules from a single cell to be determined with a high degree of sensitivity, reproducibility and accuracy and that the method captures both RNA expression levels and RNA structure information simultaneously, enabling one to combine structural and gene expression differences between single cells to better classify cellular populations. In support the response points to page 31 and Figures 3 and 4 of the specification as well as page 32 and Figure 5 (p. 12).
The response further cites experimental data (including Table 3 and Fig. 2B) in the present application to assert that the applicants have discovered that dNTP when used in fragmentation buffers prior to reverse transcription advantageously aids denaturing and/or annealing producing RNA fragments of desirable and/or uniform/similar sizes (p. 12-13).
Applicant's arguments have been fully considered but are not persuasive.
The response presents statements regarding the method and novelty of the invention (p. 12-13), citing experimental data of the present application. In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
In response to applicant's argument that the applicant's method is capable of determining the structure of RNA at a single-cell resolution and that the method captures both RNA expression levels and RNA structure information simultaneously, a recitation of the intended use or capability of the claimed invention must result in a structural difference between the claimed invention and the prior art in order to patentably distinguish the claimed invention from the prior art. If the prior art structure is capable of performing the intended use, then it meets the claim.
The response asserts none of the cited art teach the presently claimed method
of determining the structure of RNA molecules in a single cell (p. 12).
Applicant's arguments have been fully considered but are not persuasive.
Regarding claim 1, in response to applicant's argument that new rejections are presented above in view of the amended claim.
Regarding claim 15, applicant’s arguments rely on language solely recited in preamble recitations in claim 15. When reading the preamble in the context of the entire claim, the recitation “in a single cell” is not limiting because the body of the claim describes a complete invention and the language recited solely in the preamble does not provide any distinct definition of any of the claimed invention’s limitations. Thus, the preamble of the claim(s) is not considered a limitation and is of no significance to claim construction. See Pitney Bowes, Inc. v. Hewlett-Packard Co., 182 F.3d 1298, 1305, 51 USPQ2d 1161, 1165 (Fed. Cir. 1999). See MPEP § 2111.02.
The response asserts that Spitale only discloses that the 10 x RNA Fragmentation Reagent (Ambion) contains Zn2+ but does not specifically disclose that the reagent is free of Mg2+. Without this detail, a person skilled in the art following the teachings of Spitale et al would not have expected the 10 x RNA Fragmentation Reagent (Ambion) to work without Mg2+ as the agent only mentions working in the presence of Zn2 (p. 13).
Applicant's arguments have been fully considered but are not persuasive.
As written, the amended claims recites the limitation “substantially free of magnesium” and not “free of Mg2+”. Claims do not exclude the presence of any magnesium. Furthermore, regarding the argument that “a person skilled in the art following the teachings of Spitale et al would not have expected the 10 x RNA Fragmentation Reagent (Ambion) to work without Mg2+”, as noted by the applicant, the reagent taught by Spitale uses Zn2+ and not Mg2+.
The response asserts the inventors of the present application have developed a simpler and quicker method of determining the structure of RNA molecules in a single cell wherein the fragmenting step and reverse transcribing step can be carried out in a single vessel. As the medium of the fragmenting step is substantially free of magnesium, it is not necessary to include an additional step of removing Mg+ that normally affects the reverse transcription reaction. The method of the present application does not require a step of separating the fragmented RNA or a step of removing one or more components from the medium between the fragmenting and the reverse transcribing step (p. 13-14).
Applicant's arguments have been fully considered but are not persuasive.
Rejections addressing the amended claims are presented in the 103 rejection above. Furthermore, the claims as written do not exclude the additional steps cited in the assertion.
The response asserts that the present application provides an advantageous method to study RNA structure by including a fragmenting step that is advantageous for subsequent processing of the modified RNA molecules, and where fragmentation and reverse transcription can be performed in the same vessel. A person skilled in the art would not have expected studying the structure of RNA molecules in a single cell to work with a single vessel method as there is no expectation that a fragmentation buffer substantially free of magnesium would work (p. 14).
Applicant's arguments have been fully considered but are not persuasive.
New grounds of rejection under 35 U.S.C. 103 over Siegfried in view of Spitale and Bibillo are presented above. In addition, as cited above, Bibillo teaches the RNA fragmentation may occur in the presence of metals such as Mg or Mn (para 328), and does not require the presence of magnesium.
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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/JESSICA GRAY/Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682