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 .
Claims Status
Claims 1-10 & 12-15 filed on 05/15/2026 are pending. The cancellation of claim 11 in the reply filed on 05/15/2026 is acknowledged. All the amendments and arguments have been thoroughly reviewed but are deemed insufficient to place this application in condition for allowance. The following rejections are either newly applied, as necessitated by amendment, or are reiterated. They constitute the complete set being presently applied to the instant application. Response to Applicant’s argument follow. This action is FINAL.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office Action.
Any rejection not reiterated is hereby withdrawn in view of the amendments to the claims.
Claim Rejections - 35 USC § 102
Claim(s) 1, 3, 4, 6-10, & 13-15 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by McGlincy (McGlincy & Ingolia; Methods, Vol. 126, pages 112-129, June 2017), as cited on the IDS dated 05/24/2023.
Regarding claim 1, McGlincy teaches a method for ribosome profiling (determining a translatome of a cell) through steps of lysing a cell (lysing a single cell), digesting the RNA of the cell lysate (digesting RNA from the single cell) with a ribonuclease to generate a footprint, purifying the footprint RNA with a denaturing sample loading buffer that contains EDTA (inactivating the ribonuclease in the presence of a chelating agent of EDTA and releasing the RNA molecules from the ribosomes), treating the footprint RNA fragments (released RNA molecules) with T4 polynucleotide kinase (PNK) to prepare the fragments for ligation to a DNA linker (end repairing the released RNA molecules), constructing a library (constructing an RNA library from the end-repaired RNA molecules); analyzing and selecting for length distribution and concentration of the constructed library from a footprint size (ribosome footprint length) of around 28 nt (size selecting part of the prepared RNA library for fragments having a ribosome footprint length of about 20-40 nucleotides), and then sequencing the size selected library to analyze the ribosome profile (sequencing size selected RNA library and determining the translatome of the single cell) (abstract lines 6-12; pg. 118 column 2 1st full paragraph lines 1-14; pg. 118 column 2 2nd full paragraph lines 1-7; pg. 118 section 3.1.2 steps 1-6; pg. 118-119 paragraph bridging pg. 118 & 119 lines 1-11; pg. 119 column 1 1st full paragraph lines 1-7; pg. 118-119 section 3.2 steps 1-4; pg. 119 column 2 2nd full paragraph lines 1-9; pg. 119 section 3.3 steps 1-3; pg. 120 column 1 1st full paragraph lines 1-4; pg. 120 section 3.4 steps 1-2; pg. 123 section 3.9 steps 1-7; pg. 124 paragraph bridging column 1 & 2 lines 1-13; pg. 124 column 2 1st full paragraph lines 1-10; pg. 124 column 2 2nd full paragraph lines 1-9; Table 3).
Regarding claims 3 & 4, McGlincy teaches purifying the footprint RNA with a denaturing sample loading buffer that contains EDTA (inactivating the ribonuclease in the presence of a chelating agent of EDTA) (pg. 119 column 2 2nd full paragraph lines 1-9; pg. 119 section 3.3 steps 1-3; Table 3).
Regarding claims 6 & 7, McGlincy teaches treating the footprint RNA fragments (released RNA molecules) with T4 polynucleotide kinase (PNK) to prepare the fragments for ligation to a DNA linker (end repairing the released RNA molecules) in which the T4 PNK transfers phosphates to itself (a phosphate donor that is not ATP) (pg. 120 column 1 1st full paragraph lines 1-4; pg. 120 section 3.4 steps 1-2).
Regarding claim 8, McGlincy teaches the method may be used to treat HEK293 cells (translatome of two or more single cells are determined) (pg. 118 column 2 1st full paragraph lines 1-8).
Regarding claim 9, McGlincy teaches that after library construction (step v) the constructed library is treated with a DNA binding buffer to purify the products, loading the products on a gel and excising the full-length libraries corresponding to insert products (pooling the constructed RNA libraries after step v) and before step vi)) (pg. 123 section 3.9 steps 3-7).
Regarding claim 10, McGlincy teaches preparation of a library of end-repaired footprint RNA fragments (library preparation step v)) comprising ligation of linker to the 5’ end and McGlincy teaches dual-ligation strategies that employ sequential ligation of 3’ & 5’ adapters to the footprint during library preparation (ligating and first adapter and a second adapter to the end-repaired RNA molecules), reverse transcribing the linker-ligated sample (reverse-transcribing the adapter-ligated RNA molecules to obtain cDNA), and amplifying the sample with reverse and forward primers (amplifying the cDNA with a first and second primer) wherein barcodes can be added during linker ligation and at the PCR stage using indexed reverse library PCR primers (preferably at least one adapter comprises a barcode and preferably at least one of the first and second primer comprises a barcode) (pg. 116 column 2 5th full paragraph lines 1-6; pg. 117 column 2 1st full paragraph lines 1-9; pg. 120 section 3.4 steps 2 & 3; pg. 121 section 3.6 steps 1-7; pg. 123 section 3.9 steps 1-8; pg. 128 column 1 2nd full paragraph lines 1-8; Table 16).
Regarding claim 13, McGlincy teaches end repairing the released RNA molecules comprises ligation of a linker (adapter) performed in a buffer that comprises PEG-800 wherein a highest ligation efficiency may be achieved by increasing the concentration of PEG-800 to 25% w/v (ligation of first and/or second adapter is performed in a buffer comprising PEG in a concentration of preferably about 15%-25%) (pg. 120 column 1 1st full paragraph lines 6-11).
Regarding claim 14, McGlincy teaches performing a gel and excising full-length libraires compared to no-insert products of library amplification products (a complexity reduction step comprising resulting in the enrichment of full-length libraries (a specific target sequence) within the complex starting material) (pg. 123 section 3.9 step 7).
Regarding claim 15, McGlincy teaches the method may be used to treat HEK293 cells (the cell is preferably a human cell) (pg. 118 column 2 1st full paragraph lines 1-8).
Response to Arguments
The response traverses the rejection. The response asserts that claim 1 has been amended and specified that the method concerns a method for determining a translatome of a single cell wherein the RNA from a single cell has been digested and that McGlincy fails to teach each and every element of amended claim 1. Specifically, the response asserts that McGlincy fails to teach a method for determining a translatome of a single cell but concerns bulk methods comprising for mammalian cells lysate containing 30 μg total RNA and for at least these reasons McGlincy does not teach each and every element of amended claim 1. This argument has been thoroughly reviewed but was not found persuasive. First, claim 1 as currently amended, is a comprising (open) claim and does not exclude additional steps or elements. Therefore, the claim as currently amended does not exclude determining the translatome of multiple single cells, and further, claim 8 as currently amended, which depends from claim 1 is focused on this embodiment of “wherein the translatome of two or more single cells are determined”. Further, McGlincy teaches a method for ribosome profiling (determining a translatome of a cell) through steps of lysing a cell (lysing a single cell) and digesting the RNA of the cell lysate (digesting RNA from the single cell) with a ribonuclease to generate a footprint and therefore teaches determining a translatome of a single cell wherein the RNA from a single cell has been digested (abstract lines 6-12; pg. 118 column 2 1st full paragraph lines 1-14; pg. 118 column 2 2nd full paragraph lines 1-7; pg. 118 section 3.1.2 steps 1-6; pg. 118-119 paragraph bridging pg. 118 & 119 lines 1-11; pg. 119 column 1 1st full paragraph lines 1-7). Therefore, McGlincy teaches each and every element of claim 1 as currently amended.
For these reasons, and the reasons already made of record and modified to address the claims as currently amended, the rejections are maintained and applied to the newly amended claims.
Claim Rejections - 35 USC § 103
Claim(s) 2 & 5 is/are rejected under 35 U.S.C. 103 as being unpatentable over McGlincy (McGlincy & Ingolia; Methods, Vol. 126, pages 112-129, June 2017), as cited on the IDS dated 05/24/2023, in view of Reid (Reid, Shenolikar, & Nicchitta; Methods, Vol. 91, pages 69-74, July 2015), as cited on the IDS dated 05/24/2023.
The teachings of McGlincy with respect to claim 1 is discussed above and incorporated herein.
Regarding claim 2, McGlincy teaches digesting the RNA of the cell lysate with a ribonuclease of RNase 1 to generate a footprint and that micrococcal nuclease (MNase) has been utilized in ribosomal profiling (pg. 127 column 2 2nd full paragraph lines 1-14).
McGlincy does not teach that the ribonuclease in step ii) is MNase.
Reid teaches a method of ribosomal profiling that uses MNase to generate ribosome footprints in cellular extracts (abstract lines 5-7; pg. 70 column 1 2nd full paragraph lines 1-8; pg. 70 section 2.2 steps 1-3). Reid also teaches that this method is simplified enabling the removal of expensive purification ribosomes such as RNase I with the addition of simpler and more cost effective ribonuclease of MNase and should ease the barrier to employing ribosomal profiling (abstract lines 5-11; pg. 69 column 2 1st full paragraph lines 1-9).
McGlincy and Reid are considered to be analogous to the claimed invention because they are all in the same field of ribosomal profiling (determining a translatome) of a cell. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method digesting the RNA of the cell lysate with RNase I in McGlincy to incorporate the use of the MNase as taught in Reid because Reid teaches that doing so would provide a simpler and more cost effective method for conducting ribosomal profiling of a cell.
Regarding claim 5, McGlincy does not teach that step iii) further comprises a chaotropic agent.
Reid teaches adding guanidinium thiocyanate to the completed digestion reaction (step iii) further comprising the presence of a chaotropic agent which is preferably guanidium thiocyanate) (pg. 70 section 2.3 step (8)). Reid also teaches that this method is simplified enabling the removal of expensive purification ribosomes such as RNase I with the addition of simpler and more cost effective ribonuclease of MNase and should ease the barrier to employing ribosomal profiling (abstract lines 5-11; pg. 69 column 2 1st full paragraph lines 1-9).
McGlincy and Reid are considered to be analogous to the claimed invention because they are all in the same field of ribosomal profiling (determining a translatome) of a cell. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method purifying the footprint RNA with a denaturing sample loading buffer that contains EDTA in McGlincy to incorporate the addition of a chaotropic agent in the buffer as taught in Reid because Reid teaches that doing so would provide a simpler and more cost effective method for conducting ribosomal profiling of a cell.
Claim(s) 12 is/are rejected under 35 U.S.C. 103 as being unpatentable over McGlincy (McGlincy & Ingolia; Methods, Vol. 126, pages 112-129, June 2017), as cited on the IDS dated 05/24/2023, in view of Heyer (Heyer et al.; Nucleic Acids Research, Vol. 43, pages 1-14, December 2014).
The teachings of McGlincy with respect to claims 1 & 10 is discussed above and incorporated herein.
Regarding claim 12, McGlincy does not teach ligating linker (adapter) at a temperature below about 10°C.
Heyer teaches obtaining RNA fragments from ribonucleoprotein particle footprinting (determining a translatome) in which the ligation of adapters to the RNA are optimized through ligation at different temperatures and time periods including ligation of adapters to RNA at 4 °C for a time period of 0 to 24 hours (ligation of first and/or second adapter is performed at a temperature below about 10°C, preferably about 4°C, preferably for a time period of at least about 0.5, 1, 2, 4, 6, 8, 10, 12, 14, or 16 hours) (abstract lines 7-11; pg. 4-5 paragraph bridging pg. 4 & 5 lines 1-21; Figure 2F). Heyer also teaches that the use of T4 RNA ligases for library preparation employ a wide range of reaction times and temperatures but colder temperature should stabilize secondary structures of the RNA (pg. 4-5 paragraph bridging pg. 4-5 lines 1-8).
McGlincy and Heyer are considered to be analogous to the claimed invention because they are all in the same field of preparing libraries for ribosomal profiling (determining a translatome) of a cell. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of end repairing the released RNA molecules comprising ligation of a linker (adapter) in McGlincy to incorporate performing ligation of adapters at a temperature of below about 10°C as taught in Heyer because Heyer teaches that doing so would provide a method to stabilize the RNA when ligating an adapter for preparation of a library for analysis of ribonucleoprotein footprinting.
Response to Arguments
The response traverses the rejection. The response asserts that claim 1 has been amended and specifies that the method concerns a method for determining a translatome of a single cell and the cited prior art does not teach or render obvious determining the translatome of a single cell. The response also asserts, that as set forth above, McGlincy fails to teach how to determine the translatome of a single cell and that the method of McGlincy requires for mammalian cells with lysate containing 30 μg total RNA and a single cell contains around 10-30 picograms RNA and McGlincy represents approximately one to three million cells. Further, the response asserts that McGlincy includes an intermediate purification and size selection steps that result in substantial material loss, making the method incompatible with single-cell analysis. This argument has been thoroughly reviewed but was not found persuasive. First, claim 1 as currently amended, is a comprising (open) claim and does not exclude additional steps or elements. Therefore, the claim as currently amended does not exclude determining the translatome of multiple single cells, and further, claim 8 as currently amended, which depends from claim 1 is focused on this embodiment of “wherein the translatome of two or more single cells are determined”. Further, McGlincy teaches a method for ribosome profiling (determining a translatome of a cell) through steps of lysing a cell (lysing a single cell) and digesting the RNA of the cell lysate (digesting RNA from the single cell) with a ribonuclease to generate a footprint and therefore teaches determining a translatome of a single cell wherein the RNA from a single cell has been digested (abstract lines 6-12; pg. 118 column 2 1st full paragraph lines 1-14; pg. 118 column 2 2nd full paragraph lines 1-7; pg. 118 section 3.1.2 steps 1-6; pg. 118-119 paragraph bridging pg. 118 & 119 lines 1-11; pg. 119 column 1 1st full paragraph lines 1-7). Second, applicants arguments cannot take place of evidence in the record.
The response also asserts that Reid cannot remedy the deficiencies of McGlincy and the method of Reid requires 10 μg RNA and Reid performs size selection before library construction and that. For example, when size selection takes place before library preparation, a significant amount of RNA is lost and losing information in the form of small RNA fragments makes it impossible to prepare and complete an accurate library from a single cell. Further, the response asserts that Reid also includes additional material-loss steps including phenol-chloroform extraction with guanidinium thiocyanate, multiple precipitations, and gel purification, and these preclude single-cell applications and the skilled person developing single-cell methods would not look at Reid’s methods. This argument has been thoroughly reviewed but was not found persuasive. First, 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). Further, Reid is not relied upon for teachings of total RNA. Second, applicants arguments cannot take place of evidence in the record. Further, as discussed above, claim 1 as currently amended, is a comprising (open) claim and does not exclude additional steps or elements and claim 5 as currently amended, which depends from amended claim 1, recites wherein step iii) further comprises the presence of a chaotropic agent which is preferably guanidium thiocyanate, therefore focuses on an embodiment with the preferable use of guanidium thiocyanate.
The response also asserts that Heyer also fails to remedy the deficiencies of McGlincy as Heyer concerns library preparation after the RNA has already been isolated, size selected, and purified and thus, Heyer does not provide any hint on how to modify the method of McGlincy so that it can be used for determining the translatome of a single cell. This argument has been thoroughly reviewed but was not found persuasive. First, 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). Further, Heyer is not relied upon for teachings of determining the translatome of a single cell and instead is relied upon temperature for ligating a linker (adapter).
The response also asserts that for at least these reasons, McGlincy in view of Reid and/or Heyer do not teach or render obvious the method of independent amended claim 1 and therefore, claim 1, and the claims dependent thereon are allowable over the cited art. This argument has been thoroughly reviewed but was not found persuasive for the reasons set forth above.
For these reasons, and the reasons already made of record and modified to address the claims as currently amended, the rejections are maintained and applied to the newly amended claims.
Conclusion
Claims 1-10 & 12-15 are rejected.
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to BAILEY C BUCHANAN whose telephone number is (703)756-1315. The examiner can normally be reached Monday-Friday 8:00am-5:00pm ET.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Winston Shen can be reached on (571) 272-3157. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/BAILEY BUCHANAN/Examiner, Art Unit 1682
/JEHANNE S SITTON/Primary Examiner, Art Unit 1682
Prosecution Insights