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 .
Continued Examination Under 37 CFR 1.114
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 6/8/2026 has been entered.
Applicant’s amendment of claim 1, 9, 14, in the paper of 6/8/2026, is acknowledged. Applicants' arguments filed on 6/8/2026, have been fully considered and are deemed to be persuasive to overcome some of the rejections previously applied. Rejections and/or objections not reiterated from previous office actions are hereby withdrawn. Claims 1, 4, 9-16, 18. 19, 21 are still at issue and are present for examination.
Election/Restrictions
Applicant's election without traverse of the invention of Group 1, claims 1-4, 10-15 to a ATP-dependent DNA ligase, in the paper of 7/8/2025, is acknowledged. Applicant's election without traverse of species of SEQ ID NO:1, in the paper of 7/8/2025, is acknowledged.
Claims 9, 16, 18. 19, 21 are withdrawn from further consideration by the examiner, 37 CFR 1.142(b), as being drawn to a non-elected invention.
Information Disclosure Statement
The reference JP 2009-148205 listed on the information disclosure statement filed 6/8/2026 fails to comply with 37 CFR 1.98(a)(3)(i) because it does not include a concise explanation of the relevance, as it is presently understood by the individual designated in 37 CFR 1.56(c) most knowledgeable about the content of the information, of each reference listed that is not in the English language. It has been placed in the application file, but the information referred to therein has not been considered.
Claim Rejections - 35 USC § 101
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.
Claim 1, 4 and 10 are rejected under 35 U.S.C. 101 because the claimed invention is directed to non-statutory subject matter.
Claims 1-4 and 10 are directed to an isolated ATP-dependent DNA ligase, wherein the DNA ligase comprises an amino acid sequence of SEQ ID NO: 1 or comprises an amino acid sequence having at least 98 % amino acid sequence identity to SEQ ID NO:1 and wherein the DNA ligase is able to ligate a 3'-hydroxyl-deoxyribonucleic acid molecule to a 5'-end of a 5'phosphoryl-ribonucleic acid molecule in the presence of a complementary deoxyribonucleic acid molecule that spans the ligation junction; said DNA ligase being produced recombinantly in a host cell and isolated and purified therefrom wherein the host cell is a yeast cell, an insect cell, a human cell line, or an E. coli bacterial cell that is not patent-eligible pursuant to the Supreme Court decision in Association for Molecular Pathology v. Myriad Genetics, Inc., 106 USPQ2d 1972 (June 13, 2013). Uniprot Accession No. M1EZJ7, Dec 2019 which discloses SEQ ID NO:1 of the present application is an ATP dependent DNA ligase from Cronobacter phage CR9, evidence that a ATP-dependent DNA ligase or an enzymatically active fragment thereof, wherein the DNA ligase comprises the amino acid sequence of SEQ ID No. 1 is a naturally occurring ligase and is thus not patent eligible.
Applicants Response
Applicants continue to argue the rejection on much the same basis as previously submitted and discussed. Applicants continue to submit that they have amended the claims and said DNA ligase being produced recombinantly in a host cell and isolated and purified therefrom, wherein the host cell is a yeast cell, an insect cell, a human cell line, or an E. coli bacterial cell. Applicant disagrees with the Examiner's assertion that Uniprot Accession No M1EZJ7 describes a DNA sequence and an ammo acid sequence and function "Inferred from homology ". Applicants continue to submit that Uniprot Accession No M1EZJ7 provides no evidence that the putative DNA ligase described there in is in fact a "naturally occurring ligase", or that it was in fact synthesized and active in Cronobacter phage CR9.
Applicants submit that "Inferred from Homology" does not mean "exists in nature".
Applicants submit that the Office Action states that "the protein of SEQ ID NO:1 is a naturally occurring protein as evidenced by Uniprot M1EZJ7." (Office Action, p. 6) Applicant submits that this is a misstatement of the disclosure of Uniprot Accession No. M1EZJ7, Dec. 2019 (Uniprot M1EZJZ). Applicant submits that Uniprot M1EZJ7 was disclosed as a part of the genome sequencing of viral DNA in "Complete Genome Sequence of Cronobacter sakazakii Bacteriophage CR9" by Shin et al. (https://www.uniprot.org/citations/CI-49NMVR6VJ9NR3); Genbank YP_009015023.1). Applicant submits that this is the genetic sequence, i.e., DNA sequence, for a bacteriophage virus. Applicant submits that this publication of viral DNA genome generated 281 UniProtKB entities, i.e., protein structures which might be created from the DNA genome as information if it were translated into corresponding amino acids to form a protein.
Applicant submits that of these 281 Uniprot entries for proteins from the publication of the "Complete Genome Sequence of Cronobacter sakazakii Bacteriophage CR9," we are unable to find evidence that any of these proteins have been shown to exist in nature. Applicant submits that all entries have an annotation score of 1/5 (PE4) or 2/5 (PE3), meaning that they are "predicted" or "inferred from homology." Essentially, this means that the published DNA sequence has been computationally compared to other DNA sequences and, at best, has been found similar to DNA sequences that code for known functional proteins. Applicant submits that this this computational comparison is not determinative of the physical presence of the protein in nature or its qualities. As noted below, the physical presence of a protein in nature requires transcription and translation of the viral DNA into a protein, which the virus itself lacks the ability to perform.
Applicant submits that moreover, the computational comparison of a genome sequence into a functional protein demonstrates the inherent flaws in inferring the presence or qualities of a protein from a simple DNA sequence. Applicants submit models of the existence of a protein.
Applicant submits that it is not clear from the Office Action which of these four protein structures is inferred to exist in nature. This is because the SWISS-MODEL and other analytic tools are machine-learning aided databases that are user-friendly, promote scientific collaboration and data sharing, but would be understood by one of ordinary skill in the art not to provide evidence of existence in fact. Applicant submits that one of ordinary skill in the art would understand that UNIPROT's comparison of M1EZJ7 to known or inferred proteins and found similarities in hypothetical protein structure does not show that the claimed protein exists or ever has existed in nature.
Applicant submits that further, as protein function is dependent on tertiary structure, it is clear that the computational inference provided from the Uniprot and SWISS-MODEL are, at best, suggestions that a hypothetical protein structure may have similar function to known proteins. Applicant submits that Importantly, with each additional potential hypothetical protein, the factual certitude of any single hypothetical protein diminishes, i.e., the four hypothetical protein structures which have been inferred from homology cancel one another out. Accordingly, Applicant submits that the Uniprot M1EZJ7 publication of viral DNA code is not evidence that the functional recombinant proteins of the claimed invention exist in nature.
Applicants submit that Viruses do not make proteins from their genetic code. Applicants submit that Cronobacter phage CR9 is a certrevirus from the caudoviricietes class of viruses, which like all viruses, lack a protein-synthesizing system for synthesis of proteins from viral DNA or mRNA. See Rampersad S, Tennant P. Replication and Expression Strategies of Viruses. Viruses. 2018:55-82. doi: 10.1016/B978-0-12-811257-1.00003-6. Epub 2018 Mar 30. PMCID:
PMC7158166, previously submitted. Applicants submit that so while the strand of DNA coding disclosed in Uniprot M1EZJZ exists as potential genetic instruction within a virus, the cited references provide no evidence this gene is ever expressed within a host cell.
Applicants submit that further, there is no evidence that once Cronobacter phage CR9 enters a host cell and hijacks the metabolic processes and protein-synthesizing system of the host, that this portion of the viral DNA is actually transcribed or translated into a protein. Applicants submit that the Cronobacter sakazakii host has its own DNA ligase (Uniprot Accession No. A7MKW4, Dec. 2019, previously submitted) which may be used, or the sequence found in Uniprot M1EZJ7 may be part of the non-functional or 'junk' code which remains in the genome but is not expressed. Applicants submit that finally, even if the viral DNA from Cronobacter phage CR9 is transcribed and translated such that the corresponding amino acids are assembled into a primary polypeptide chain by the host Cronobacter sakazakii's ribosomes, the final functional structure of the resultant protein may be affected by post-translational modification in the host. See Post-translational modification, https://en.wikipedia.org/wiki/Post- translational_modification (last visited Oct. 16, 2025, previously submitted).
Applicants submit that so while it is true that DNA ligases or enzymatically active fragments thereof may be isolated from a natural source (see Specification at [0210]), doing so requires processes which do not occur in nature, namely through recombinant production in a host cell.
In summary, Applicants submit that that while a strand of DNA existed in nature which was sequenced in "Complete Genome Sequence of Cronobacter sakazakii Bacteriophage CR9" which, if translated by ribosomes would correspond to an amino acid sequence of SEQ. ID. NO. 1, there is no evidence of record that the DNA sequence has ever been translated into an amino acid sequence nor is there evidence of record that if that occurred in nature, it would result in the enzymes of the present claims. Applicants submit that the applicant does not claim an isolated DNA sequence, as was claimed and invalidated in Myriad. The claimed invention here is an enzyme synthesized through recombinant processes which has qualities which no other known DNA ligase possesses. Applicants submit that while it is impossible to prove that a naturally occurring analog to the claimed enzyme does not exist, the existence and qualities of such a naturally occurring analog are fully speculative and not supported by any evidence presented in the Office Action. Applicants submit that furthermore, as explained in the Declaration of Olav Lanes, ligases with the qualities found in the present invention are not only not known in nature, the functionality is selected against in nature to prevent RNA-DNA ligation. Lanes Decl. 77 5-6.
Applicants submit that In the absence of a naturally occurring analog, the analysis of "markedly different characteristics" is moot. No evidence is provided that shows a naturally occurring analog to the claimed enzyme has been isolated from natural sources or tested for its characteristics.
Applicants amendment of the claims, Declaration filed and applicants complete argument are acknowledged and have been carefully considered, however, are not found persuasive for the reasons previously made of record and for those reasons repeated herein.
In response to applicants submission that Uniprot Accession No M1EZJ7 provides no evidence that the putative DNA ligase described there in is in fact a "naturally occurring ligase", or that it was in fact synthesized and active in Cronobacter phage CR9, applicants previous and current arguments have been carefully considered, however, not found persuasive for the reasons previously stated and repeated herein. As previously stated, Uniprot Accession No. M1EZJ7, Dec 2019 which discloses SEQ ID NO:1 of the present application is an ATP dependent DNA ligase from Cronobacter phage CR9, evidence that a ATP-dependent DNA ligase or an enzymatically active fragment thereof, wherein the DNA ligase comprises the amino acid sequence of SEQ ID No. 1 is a naturally occurring ligase and is thus not patent eligible. It is noted that Uniprot Accession No. M1EZJ7, Dec 2019 is not being used to evidence that the DNA ligase of SEQ ID NO:1 was or is in fact synthesized and active in Cronobacter phage CR9, however it is noted that such is not required to invoke that the DNA ligase of SEQ ID NO:1 is naturally occurring.
In response to applicants submission that "Inferred from Homology" does not mean "exists in nature", while this is acknowledged, inferred from homology is how the function of the protein is described by Uniprot Accession No. M1EZJ7.
Applicants statement that the Office Action’s statement that "the protein of SEQ ID NO:1 is a naturally occurring protein as evidenced by Uniprot M1EZJ7." (Office Action, p. 6) is a misstatement of the disclosure of Uniprot Accession No. M1EZJ7, Dec. 2019 (Uniprot M1EZJZ), is not found persuasive or accurate. The disclosure of Uniprot M1EZJ7 has been stated previously and is made a part of the record. As stated previously, "the protein of SEQ ID NO:1 is a naturally occurring protein as evidenced by Uniprot M1EZJ7." (Office Action, p. 6). This is not a misstatement of the disclosure of Uniprot Accession No. M1EZJ7, Dec. 2019 (Uniprot M1EZJZ). This is a statement of the Office. The office is using the disclosure of Uniprot Accession No. M1EZJ7, Dec. 2019 (Uniprot M1EZJZ) as evidence that the protein taught by Uniprot Accession No. M1EZJ7, Dec. 2019 (Uniprot M1EZJZ) is a naturally occurring protein.
Applicants characterization of Uniprot M1EZJ7 as a part of the genome sequencing of viral DNA in "Complete Genome Sequence of Cronobacter sakazakii Bacteriophage CR9" by Shin et al. (https://www.uniprot.org/citations/CI-49NMVR6VJ9NR3); Genbank YP_009015023.1), is acknowledged, however, regardless of applicants characterization, it remains that Uniprot Accession No. M1EZJ7 evidences that the DNA ligase of SEQ ID NO:1 is a naturally occurring protein, regardless of how and when it may or may not be expressed.
Applicants submission that additional evidence must be found to show that the protein of Uniprot Accession No. M1EZJ7 is expressed is not found persuasive.
Applicants submission of various three dimensional models (i.e. SWISS-MODEL) of the existence of a protein are not found persuasive in refuting the evidence of the existence of a naturally occurring DNA ligase comprising the amino acid sequence of SEQ ID NO:1.
In response to applicants submission that viruses do not make proteins from their genetic code, this is acknowledged. Applicants are reminded that the current rejection is based upon the evidence presented that the DNA ligase comprising the amino acid sequence of SEQ ID NO: 1 is a naturally occurring protein, not whether it is made by a virus from its genetic code.
In response to applicants submission that while the strand of DNA coding disclosed in Uniprot M1EZJZ exists as potential genetic instruction within a virus, the cited references provide no evidence this gene is ever expressed within a host cell is acknowledged, however, not found persuasive on the basis that whether or not or even when it is expressed is not what is being evidenced. What is being evidenced is that the DNA ligase comprising the amino acid sequence of SEQ ID NO:1 is a naturally occurring protein.
In summary applicants submission that applicants have amended the claims and said DNA ligase being produced recombinantly in a host cell and isolated and purified therefrom, this is not found persuasive as regardless of applicant’s amendment to the claims such that it now comprises a “product by process” type of limitation, it remains that the protein of SEQ ID NO:1 is the protein of SEQ ID NO:1 regardless of how it is produced and/or isolated. As evidenced by Uniprot Accession No M1EZJ7 the protein of SEQ ID NO:1 is a naturally occurring protein. In response to applicants submission that Uniprot Accession No M1EZJ7 provides no evidence that the putative DNA ligase described there in is in fact a "naturally occurring ligase", or that it was in fact synthesized and active in Cronobacter phage CR9, this is not found persuasive on the basis that Uniprot Accession No M1EZJ7 does provide evidence that the protein of SEQ ID NO:1 is a naturally occurring protein. The required conditions wherein said protein would or would not be expressed though unknown does not provide evidence that the protein of SEQ ID NO:1 is not a naturally occurring protein.
Claim Rejections - 35 USC § 112
The rejection of claim(s) 1-4 and 10-15 under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, based upon a lack of written description is withdrawn based upon applicants amendment of the claims in the paper of 10/24/2025.
The rejection of claim(s) 1-4 and 10-15 under 35 U.S.C. 112, first paragraph, based upon a scope of enablement is withdrawn based upon applicants amendment of the claims in the paper of 10/24/2025.
The rejection of claim(s) 1, 4 and 10-15 under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement because the recitation “said DNA ligase being produced recombinantly in a host cell and isolated and purified therefrom, wherein the host cell is not a Cronobacter sp.” is not supported by applicants specification at the time of filing and is considered new matter is withdrawn based upon the deletion of this recitation in the paper of 6/8/2026.
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim(s) 1 and 4 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Uniprot Accession No. M1EZJ7, May 2013.
This rejection was stated in the previous office action as it applied to previous claims 1-4 and 10. In response applicants have amended the claims and traverse the rejection as it applies to the newly amended claims. For applicants convenience the original rejection is repeated herein.
Uniprot Accession No. M1EZJ7, Dec 2019 discloses SEQ ID NO:1 of the present application. It is an ATP dependent DNA ligase from Cronobacter phage CR9. While Uniprot Accession No; M1EZJ7 is silent about the activity of ligating 3’-OH DNA to 5’-phosphate RNA in the presence of a cDNA molecule that spans the ligation junction, this is considered an inherent property of the taught ATP dependent DNA ligase based upon its identical amino acid sequence. Uniprot Accession No. M1EZJ7, Dec 2019 discloses SEQ ID NO:1 of the present application. While Uniprot Accession No. M1EZJ7 may not teach that the DNA ligase is produced recombinantly in a host cell (yeast, insect human cell of E. coli bacterial cell) and subsequently isolated and purified, this does not change the claimed or taught DNA ligase comprising an amino acid sequence of SEQ ID NO:1.
Applicants Response:
Applicants continue to submit that Uniprot M1EZJ7 discloses a strand of DNA in a virus which has no ability to synthesize protein on its own Uniprot M1EZJ7 does not disclose the existence or properties of a protein. Applicants submit that the inherent features of prior art proteins are not relevant here because, as described above, no prior art proteins have been isolated or analyzed. Only the present inventors have reduced the claimed DNA ligases to practice. Applicants submit that the claims have been amended to specify that the protein is produced recombinantly in a host cell and isolated and purified therefrom, wherein the host cell is a yeast cell, an insect cell, a human cell line, or an E. coli bacterial cell. Applicants submit that there is no evidence of record that even if the protein of Uniprot M1EZJ7 were expressed in nature, it would necessarily be the same as a protein produced recombinantly in a host cell and isolated and purified therefrom wherein the host cell is a yeast cell, an insect cell, a human cell line, or an E. coli bacterial cell.
Applicants amendment of the claims and applicants complete argument is acknowledged and has been carefully considered, however, is not found persuasive for the reasons previously made of record and for those reasons repeated herein.
In response to applicants submission that Uniprot M1EZJ7 discloses a strand of DNA in a virus which has no ability to synthesize protein on its own and that Uniprot M1EZJ7 does not disclose the existence or properties of a protein, this is not found persuasive on the basis that as stated previously and repeated above, Uniprot Accession No. M1EZJ7, Dec 2019 discloses the protein of SEQ ID NO:1 of the present application. It is an ATP dependent DNA ligase from Cronobacter phage CR9 as identified by Uniprot M1EZJ7. In response to applicants submission that only the present inventors have reduced the claimed DNA ligases to practice, this is not found persuasive as previously stated, Uniprot Accession No. M1EZJ7, Dec 2019 discloses the protein of SEQ ID NO:1 of the present application. It is an ATP dependent DNA ligase from Cronobacter phage CR9 as identified by Uniprot M1EZJ7.
In response to applicants submission that the claims have been amended to specify that the protein is produced recombinantly in a host cell and isolated and purified therefrom, wherein the host cell is a yeast cell, an insect cell, a human cell line, or an E. coli bacterial cell, this is not found persuasive because as stated above, while Uniprot Accession No. M1EZJ7 may not teach that the DNA ligase is produced recombinantly in a host cell (yeast, insect human cell of E. coli bacterial cell) and subsequently isolated and purified, this does not change the claimed or taught DNA ligase comprising an amino acid sequence of SEQ ID NO:1.
In response to applicants submission that is no evidence of record that even if the protein of Uniprot M1EZJ7 were expressed in nature, it would necessarily be the same as a protein produced recombinantly in a host cell and isolated and purified therefrom wherein the host cell is a yeast cell, an insect cell, a human cell line, or an E. coli bacterial cell, this is not found persuasive on the basis that as stated previously and above, Uniprot Accession No. M1EZJ7 may not teach that the DNA ligase is produced recombinantly in a host cell (yeast, insect human cell of E. coli bacterial cell) and subsequently isolated and purified, this does not change the claimed or taught DNA ligase comprising an amino acid sequence of SEQ ID NO:1.
Thus claim(s) 1 and 4 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Uniprot Accession No. M1EZJ7, Dec 2019.
Claim Rejections - 35 USC § 103
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.
Claim(s) 1, 4 and 10-15 is/are rejected under 35 U.S.C. 103 as being unpatentable over Uniprot Accession No. M1EZJ7, Dec 201 and Sekiguchi et al. (Biochemistry, Vol 36, pgs 9073-9079, July 1997).
This rejection was stated in the previous office action as it applied to previous claims 1, 4 and 10-15. In response applicants have amended the claims, submit a Declaration under 37 CFR 1.132 by one of the inventors Olav Lanes and traverse the rejection as it applies to the newly amended claims. For applicants convenience the original rejection is repeated herein.
Sekiguchi et al. (Biochemistry, Vol 36, pgs 9073-9079, July 1997) disclose that they are studying the structure and function of the eukaryotic ligases using the vaccinia virus enzyme as a model. Sekiguchi et al. teach studies and assays for characterizing ligation of RNA-containing duplexes by Vaccinia DNA ligase. In their studies, Sekiguchi et al. disclose that they analyzed the ability of vaccinia DNA ligase to seal nicked substrates containing one or more RNA strands, including 3’-OH-terminated RNA to 5’-phosphate-terminated DNA. Sekiguchi et al. disclose that vaccinia DNA ligase is much less effective at joining 3’-Oh-terminated DNA to 5’-phosphate-terminated RNA and is extremely weak at phosphodiester formation between two RNA strands (pg 9074). In order to perform the above assays, Sekiguchi et al. teach compositions comprising the various components necessary to perform the assays, including the vaccinia DNA ligase, a ligation buffer comprising ATP, Mn2+ and Mg2+, RNA 5’-end adapters, a 3’-OH DNA molecule, a 5-phosphate RNA molecule and a complementary cDNA.
Uniprot Accession No. M1EZJ7, Dec 2019 discloses SEQ ID NO:1 of the present application. It is an ATP dependent DNA ligase from Cronobacter phage CR9. While Uniprot Accession No; M1EZJ7 is silent about the activity of ligating 3’-OH DNA to 5’-phosphate RNA in the presence of a cDNA molecule that spans the ligation junction, this is considered an inherent property of the taught ATP dependent DNA ligase based upon its identical amino acid sequence.
One of skill in the art before the time of invention would have been motivated to create the compositions taught by Sekiguchi et al. to perform the assays taught by Sekiguchi et al. using the DNA ligase from Cronobacter phage CR9, as taught by Uniprot Accession No. M1EZJ7, instead of the vaccinia DNA ligase as a means of characterizing the DNA ligase from Cronobacter phage CR9. These compositions include the DNA ligase from Cronobacter phage CR9 comprising the amin acid sequence of SEQ ID NO:1 and a ligation buffer comprising ATP, Mn2+, Mg2+, RNA 5’-end adapters, a 3’-OH DNA molecule, a 5-phosphate RNA molecule and a complementary cDNA. One would have been further motivated to include all the other reagents necessary to perform the assays taught by Sekiguchi et al. together with the obvious composition thus comprising a kit. The expectation of success is high based upon the high level of expertise in the art of recombinant DNA technology as exemplified by the teaching of Sekiguchi et al.
Applicants Response:
Applicants submit that while the tools for protein prediction and analysis have grown exponentially in power in the last decade, the four different potential protein structures predicted by SWISS-MODEL above reiterate the unpredictable nature of protein synthesis and function. Applicants submit that even if a person of ordinary skill in the art were motivated to recombinantly synthesize and test every potentially functional ATP-dependent DNA ligase on Uniprot, of which there are over 34,000 identified options, (https://www.uniprot.org/uniprotkb?query=%28family%3A%22ATP-dependent+DNA+ligase+family%22%29), there is no expectation of success and no expectation of achieving the surprising and beneficial properties of the claimed protein.
Applicants submit that the ligases according to the present claims have "the ability to ligate with high efficiency a single-stranded break in a double stranded nucleic acid complex comprising a first DNA molecule comprising a 5' phosphoryl end-group to be ligated to the 3' end of a RNA molecule comprising a 3'hydroxyl end-group in the presence of a complementary second deoxyribonucleic acid molecule that spans the ligation junction.". Applicants submit that this functionality has not previously been described.
Applicants submit that the ability to ligate a nucleic acid molecule to the 5' end of a RNA molecule is desirable in many molecular biology techniques such as classic cloning and cloning using Gibson approach (Gibson, D.G. et al., Enzymatic assembly of DNA molecules up to several hundred kilobases, Nature Methods, 2009,vol.6, page 343-345), adapter ligation during library preparations (e.g. illumine, Head, S.R. et al., Library construction for next-generation sequencing: overviews and challenges, Biotechniques, 2014, vol. 56, no. 2, p. 1-31), DNA synthesis, sequencing by ligation (e.g. SOLiD) (Voelkerding, K.V. et al. Next-Generation Sequencing: from basic research to diagnostics, Clinical Chemistry, 2009, vo155, no.4, p.641-658), Ligase Chain reaction (J; Czajka, J; Luo, J; Barany, F; Batt, CA (Feb 1994). "Ligase chain reaction (LCR)--overview and applications". PCR Methods and Applications. 3 (4 ): S51-64), SNP detection (Etter, P.D. et al., SNP discovery and genotyping for evolutionary genetics using RAD sequencing, Methods Mol. Biol., 2011, vol.772, p.157-178) and 5'-end labeling of RNA. See Description at p. 20, 1. 33 - p. 21, 1. 5. This unique functionality fulfills a long-unmet need for highly efficient ligases with unique substrate specific ligation properties. Description at p. 2, 11. 25-34; Lanes Decl. 77 3-4.
Applicants submit that in addition to this unprecedented functionality, the DNA ligases of the present claims have improved efficiency over known ligases: "The DNA ligase of the present invention has an improved ligation efficiency compared to T4 DNA ligase, T3DnL,SplintR or DNA ligase from Vaccinia virus in its ability to ligate a DNA molecule to the 5'end of a RNA molecule in the presence of a complementary DNA template that spans the ligation junction. Applicants submit that wherein the improved ligation efficiency is at least 2-to 100-fold compared to the ligation efficiency of T4 DNA ligase, T3DnL,SplintR or DNA ligase from Vaccinia virus. Such that the increased ligation efficiency of the DNA ligase of the present invention compared to T4 DNA ligase, T3DnL,SplintR or DNA ligase from Vaccinia virus is as at least greater than 2-fold, at least greater than 5-fold at least greater than 10-fold, at least greater than 12-fold, at least greater than 15-fold at least greater than 20-fold or at least greater than 100-fold."
Applicants submit that these novel and unexpected features of the claimed ligases are an improvement over known ligases which enables new functionality.
Applicants submit that these substantial improvements in functionality over prior art ligases would not be predicted or expected by those of skill in the unpredictable field of biochemistry and protein synthesis. Lanes Decl. 77 7-9, 14. Applicants submit that there is no teaching, suggestion, or motivation in the prior art that by cloning, recombinant expression, and isolation of SEQ ID No. 1 would yield a new family of ATP-dependent DNA ligases which are able to ligate with high efficiency a single stranded DNA molecule to the 5' end of a RNA molecule in the presence of a complementary single stranded DNA template that spans the ligation junction. See Description at p. 2, 11. 29-34; Lanes Decl. 77 10-11. Applicants submit that In view of the clear differences between claimed invention and prior art, the level of unpredictability in the art, and the secondary considerations of long-felt but unsolved needs and unexpected results, the balance of factors in factual analysis under Graham v. John Deere Co., 383
Applicants amendment of the claims, Declaration under 37 CFR 1.132 by the inventor Olav Lanes and traversal is acknowledged and has been carefully considered, however, is found non persuasive for the reasons previously made of record and for those reasons repeated herein.
In response to applicants submission and opinion of the unpredictable nature of protein synthesis and function, such a broad statement is unclear in the context of the current rejection. It is unclear as to the specifics of applicants statement of the unpredictable nature of protein synthesis and function. As stated previously and repeated above, Uniprot Accession No. M1EZJ7, Dec 2019 discloses SEQ ID NO:1 of the present application. They teach that it is an ATP dependent DNA ligase from Cronobacter phage CR9. While Uniprot Accession No; M1EZJ7 is silent about the activity of ligating 3’-OH DNA to 5’-phosphate RNA in the presence of a cDNA molecule that spans the ligation junction, this is considered an inherent property of the taught ATP dependent DNA ligase based upon its identical amino acid sequence.
Further as stated previously and repeated above one of skill in the art before the time of invention would have been motivated to create the compositions taught by Sekiguchi et al. to perform the assays taught by Sekiguchi et al. using the DNA ligase from Cronobacter phage CR9, as taught by Uniprot Accession No. M1EZJ7, instead of the vaccinia DNA ligase as a means of characterizing the DNA ligase from Cronobacter phage CR9. These compositions include the DNA ligase from Cronobacter phage CR9 comprising the amino acid sequence of SEQ ID NO:1 and a ligation buffer comprising ATP, Mn2+, Mg2+, RNA 5’-end adapters, a 3’-OH DNA molecule, a 5-phosphate RNA molecule and a complementary cDNA. One would have been further motivated to include all the other reagents necessary to perform the assays taught by Sekiguchi et al. together with the obvious composition thus comprising a kit. The expectation of success is high based upon the high level of expertise in the art of recombinant DNA technology and protein expression as exemplified by the teaching of Sekiguchi et al.
The expectation of success is high as stated previously. The expectation of success is not based upon applicants submitted expectation of achieving surprising and beneficial properties of the claimed protein according to applicants..
In response to applicants submission that the ligases according to the present claims have "the ability to ligate with high efficiency a single-stranded break in a double stranded nucleic acid complex comprising a first DNA molecule comprising a 5' phosphoryl end-group to be ligated to the 3' end of a RNA molecule comprising a 3'hydroxyl end-group in the presence of a complementary second deoxyribonucleic acid molecule that spans the ligation junction.", while this is appreciated, this is not the basis of the motivation to express the protein of Uniprot Accession No. M1EZJ for the reasons stated previously and above.
In response to applicants submission that the ability to ligate a nucleic acid molecule to the 5' end of a RNA molecule is desirable in many molecular biology techniques such as classic cloning and cloning using Gibson approach, adapter ligation during library preparations, DNA synthesis, sequencing by ligation, Ligase Chain reaction, SNP detection and 5'-end labeling of RNA, while this is appreciated, as stated above this is not the basis of the motivation to express the protein of Uniprot Accession No. M1EZJ for the reasons stated previously and above.
In response to applicants submission that in addition to this unprecedented functionality, the DNA ligases of the present claims have improved efficiency over known ligases, while this is appreciated, as stated above this is not the basis of the motivation to express the protein of Uniprot Accession No. M1EZJ for the reasons stated previously and above.
In response to applicants submission that these substantial improvements in functionality over prior art ligases would not be predicted or expected by those of skill in the unpredictable field of biochemistry and protein synthesis, while this is appreciated, as stated above this is not the basis of the motivation to express the protein of Uniprot Accession No. M1EZJ for the reasons stated previously and above.
In response to applicants submission that there is no teaching, suggestion, or motivation in the prior art that by cloning, recombinant expression, and isolation of SEQ ID No. 1 would yield a new family of ATP-dependent DNA ligases which are able to ligate with high efficiency a single stranded DNA molecule to the 5' end of a RNA molecule in the presence of a complementary single stranded DNA template that spans the ligation junction, while this is appreciated, as stated above this is not the basis of the motivation to express the protein of Uniprot Accession No. M1EZJ for the reasons stated previously and above.
Thus, claim(s) 1, 4 and 10-15 is/are rejected under 35 U.S.C. 103 as being unpatentable over Uniprot Accession No. M1EZJ7, Dec 201 and Sekiguchi et al. (Biochemistry, Vol 36, pgs 9073-9079, July 1997).
Remarks
No claim is allowed.
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rgh
7/15/2026
/RICHARD G HUTSON/Primary Examiner, Art Unit 1652
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