DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim(s) 1-20 are pending.
Priority
Acknowledgement is made that this application is a Continuation-In-Part (CIP) of PCT/CN2024/079487 filed 03/01/2024 and claims priority based on foreign application filed as CN202311152733.2 on 09/07/2023.
The present application is a continuation-in-part of a foreign-language PCT application. In view of the foreign-language disclosure, an English translation of the portions relied upon for support is required to determine whether the pending claims are entitled to the benefit of the earlier filing date. Without a translation sufficient to assess support, the examiner cannot determine the effective filing date of each limitation. See MPEP §§ 201.08, 211.05, 213.02, 213.03, 2163.01, and 2163.02.
All claims are given the filing date of the instant application, 04/29/2024.
Information Disclosure Statement
Receipt of the information disclosure statement on 09/25/2024 is acknowledged. The signed and initialed PTO-1449 form(s) has/have been mailed with this action.
Specification
Minor informalities
The disclosure is objected to because of the following informalities:
In paragraph [0034], the description of Fig. 1, does not explain what panel E or I represent. It would be remedial to add that into this description.
In paragraph [0036], the description of Fig. 3, does not explain what the arrow is pointing to in the drawing, nor does any other part of the specification. It would be remedial to add in what the arrow is pointing to.
In paragraph [0044] there is a reference to a color, “. . . and the red spectra represents.”, it would be remedial to change this to a shade since the drawings are not in color.
Appropriate correction is required.
Claim Objections
Claim 10 is objected to because of the following informalities:
It would be remedial to add “wherein” after the first, second, and third semi-colon, to improve the grammar of the claim:
“The method according to claim 1, wherein in step 1), the target sequence comprises telomere ssDNA, human c-Myc promoter ssDNA, or human immunodeficiency virus (HIV) ssDNA; wherein the telomere ssDNA has the nucleotide sequence set forth in SEQ ID NO: 1; wherein the human c-Myc promoter ssDNA has the nucleotide sequence set forth in SEQ ID NO: 2; and wherein the HIV ssDNA has the nucleotide sequence set forth in SEQ ID NO: 3.
Claim 12 is objected to because of the following informalities:
It would be remedial to add an “and/or” after the semicolon for consistency between claim 12 and claim 1.
Claim 19 is objected to because of the following informalities:
There is an additional comma after “wherein”. It would be remedial to remove this comma for consistency between all other claims; and
Appropriate correction is required.
Claim Rejections - 35 USC § 112(b) – indefiniteness
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.
Claim(s) 2 and 15 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.
Claim(s) 2 and 15 contain the trademark/trade name(s) “KpnI-HF”, “BamHI-HF”, “PstI-HF”, and “Hind III-HF”. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe “high-fidelity (HF®) restriction enzymes” which “have the same specificity as the native enymes but have been engineered for significantly reduced star activity and performance in a single buffer (rCutSmartTM).”, as taught by NEB (BamHI-HF® |New England Biolabs, 1 page, accessed 08/20/2026) and, accordingly, the identification/description is indefinite.
Claim Rejections - 35 USC § 102
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.
Claim(s) 1-20 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Wang et al (CN 117143897 A, published December 1st, 2023; cited on IDS filed 09/25/2024 as FOR #5).
Applicant cannot rely upon the certified copy of the foreign priority application to overcome this rejection because a translation of said application has not been made of record in accordance with 37 CFR 1.55. When an English language translation of a non-English language foreign application is required, the translation must be that of the certified copy (of the foreign application as filed) submitted together with a statement that the translation of the certified copy is accurate. See MPEP §§ 215 and 216.
Regarding claim 1, Wang et al discloses, “The invention provides a method for preparing stable isotope labeled ssDNA by biological fermentation, which includes the following steps:
1) Add the first restriction endonuclease site and the second restriction endonuclease site to the 5' and 3' ends of the target sequence respectively to obtain repeating units; The first restriction endonuclease and the second restriction endonuclease are two different restriction enzymes;
2) Connect the repeating units described in step 1) in series to obtain a fusion sequence;
3) Insert the fusion sequence described in step 2) into the high-copy vector to obtain a recombinant vector;
4) Introduce the recombinant vector described in step 3) into the host bacterium to obtain the recombinant bacterium;
5) After culturing the recombinant bacteria described in step 4), extract and purify the recombinant vector in the recombinant bacteria; the culture medium used for the culture uses 15NH4Cl as the only nitrogen source and/or 13C-glucose as the only carbon source. source;
6) Use the first restriction endonuclease and the second restriction endonuclease to digest the recombinant vector described in step 5) to obtain an asymmetric double-stranded DNA structure, and separate and obtain two ssDNAs of different lengths. One of the two ssDNAs is the target sequence;
7) Recover the target sequences in the two pieces of ssDNA described in step 6) to obtain stable isotope-labeled target ssDNA.”, (see page 2-3).
Regarding claim 2, Wang et al teaches “Preferably, in step 1), the first restriction endonuclease is KpnI and the second restriction endonuclease is BamHI; or, the first restriction endonuclease is KpnI-HF and the The second restriction endonuclease is BamHI-HF; alternatively, the first restriction endonuclease is Pst I-HF and the second restriction endonuclease is HindIII-HF; alternatively, the first restriction enzyme The first restriction enzyme was Kpn I-HF and the second restriction enzyme was Hind III-HF.” (p.3, para 3).
Regarding claim 3 and 5, Wang et al discloses, “The double-stranded DNA sequence was purified by urea-denatured polyacrylamine gel electrophoresis to obtain two ssDNAs of different lengths, namely LTR-III and LTR-C.”, (see figure 8 and p.4, para 8).
Regarding claim 4, Wang et al discloses, “Preferably, the shorter one of the two ssDNAs of different lengths in step 4) is the target sequence.”, (p.3, para 4).
Regarding claim 6 and 8, Wang et al discloses, “Preferably, in step 2), every 3 to 4 repeating units are connected in series to form one large repeating unit, and the large repeating units are connected in series through a linker sequence.”, (p.3, para 5).
Regarding claim 7, Wang et al discloses, “Preferably, in step 2), the length of the fusion sequence is ≤2k.”, (p.3, para 6).
Regarding claim 9, Wang et al discloses, “Preferably, in step 3), the high-copy vector includes pUC57.”, (p.3, para 7).
Regarding claim 10, Wang et al discloses, “In the present invention, the target sequence includes telomeric ssDNA, human promoter ssDNA or HIV ssDNA; the nucleotide sequence of the telomeric ssDNA is shown in SEQ ID NO.1, specifically: 5'-tagggttagggttagggttaggg-3 ', named wtTel23; the nucleotide sequence of the human promoter c-myc ssDNA is shown in SEQ ID NO.2, specifically: 5'-ctgagggtgggtagggtgggtaa-3', named PU22C; the core of the HIV ssDNA The nucleotide sequence is shown in SEQ IDNO.3, specifically: 5'-gggaggcgtggcctgggcgggactgggg-3', named LTR-III.”, (p.5, para 14).
Wherein SEQ ID NOs: 1-3 are identical to instant SEQ ID NOs: 1-3.
Regarding claim 11, Wang et al discloses, “Preferably, when the target sequence is telomeric ssDNA, 15 repeating units are connected in series in step 2); when the target sequence is human promoter ssDNA, 20 repeating units are connected in series in step 2); when the target sequence When the sequence is HIV ssDNA, the 15 repeating units in step 2) are connected in series.”, (p. 3, para 9).
Regarding claim 12, Wang et al discloses, “Preferably, the concentration of 15NH4Cl in the culture medium in step 5) is ≥1g/L; the concentration of 13C-glucose in the culture medium is ≥4g/L.”, (p. 3, para 10).
Regarding claim 13, Wang et al discloses, “Preferably, step 7) also includes recovering another ssDNA except the target sequence.” (p. 3, para 10).
Regarding claim 14, Wang et al discloses, “The HIV ssDNA sequence LTR-III uniformly labeled with stable isotope 15N prepared by the method of the present invention can improve the sensitivity of nuclear magnetic resonance signal collection and can be used for traditional 15N filtered nuclear magnetic spectrum collection. In addition, the stable isotope 15N uniformly labeled HIV ssDNA prepared by the method of the present invention can also be used for NMR research on the structure of HIV LTR-III G4 and its interaction with ligands in the complex environment of cell extracts. Provide a new platform for drug screening targeting HIV LTR-III G4 in complex cellular environments.”, (p.8, para 2).
Regarding claim(s) 15-20, Wang et al discloses, “add irrelevant sequence connections between every four repeated target sequences in the fusion sequence, and construct the fusion sequence into the high-copy vector pUC57. An asymmetric double-stranded DNA structure was produced by double enzyme digestion of Pst I-HF and HindⅢ-HF, and two ssDNAs of different lengths were obtained by urea-denaturing polyacrylamide gel electrophoresis, which are: 5'-gggaggcgtggcctgggcgggactgggg-3 'LTR-III and 3'-acgtccctccgcaccggacccgcctgaccccctag-5'LTR-C. The plasmid was constructed by repeatedly concatenating the target DNA sequence and the linker sequence (1370 bp) through BsmBI single-enzyme digestion vector pUC57 to generate sticky end splicing, and the plasmid was named pUC57-LTR-III.”, (p. 15, para 2).
Accordingly, claims 1-20 are anticipated by Wang et al.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claim(s) 1, 3-8 and 13, are rejected under 35 U.S.C. 103 as being unpatentable over Ramanathan et al (A Novel Approach for Uniform 13C and 15N Labeling of DNA for NMR studies, Biochemical and Biophysical Research Communications, Vol 290, pages 928-932, published January 25th, 2002) in view of Nelissen et al (Preparation of selective and segmentally labeled single-stranded DNA for NMR by self-primed PCR and asymmetrical endonuclease double digestion, Nucleic Acids Res, vol 37, issue 17, pages 1-10 published June 24th, 2009; on IDS filed 09/25/2024 as NPL #5).
Claim 1 recites: “A method for preparing stable isotope-labeled single-stranded DNA
(ssDNA) by biosynthesis with Escherichia coli (E. coli), comprising the steps of:
adding a site of a first restriction endonuclease and a site of a second restriction endonuclease to 5' and 3' ends of a target sequence, respectively, to obtain a repeating unit; wherein the first restriction endonuclease and the second restriction endonuclease are different;
ligating the repeating unit obtained in step 1) in tandem to obtain a fusion sequence;
inserting the fusion sequence in step 2) into a high-copy vector to obtain a recombinant vector;
introducing the recombinant vector in step 3) into host strain to obtain a recombinant E. coli strain;
extracting and purifying the recombinant vector from the recombinant E. coli strain after culture of the recombinant E. coli strain in step 4); where the culture is conducted in a medium with 15NH4Cl as a sole source of nitrogen and/or 13C-glucose as a sole source of carbon;
digesting the recombinant vector obtained in step 5) using the first restriction endonuclease and the second restriction endonuclease to obtain an asymmetric double-stranded DNA (dsDNA) structure, and isolating two ssDNAs of unequal lengths, with one of the two ssDNAs as a target sequence; and
recovering the target sequence in the two ssDNAs in step 6) to obtain the stable isotope-labeled target ssDNA.”
Regarding claim 1, Ramanathan et al teaches a method “for large-scale synthesis of 13C- and 15N-labeled DNA for NMR studies. In this methodology, endonuclease-sensitive repeat amplification (ESRA), a modified PCR strategy, has been used to amplify tandem repeats of the target DNA sequence. The design of the template is such that restriction enzyme (RE) sites separate repeats of the target sequence. The ESRA product is then cloned into a suitable vector. The Escherichia coli cells harboring the plasmid are grown in minimal medium containing [13C] glucose and 15NH4Cl as the sole source of carbon and nitrogen, respectively. The target sequence is released by RE digestion of the plasmid, followed by purification using PAGE (Abstract).
Regarding claim 7, Ramanathan et al teaches insert length of 1.6kb or less in figure 2. Specifically, Ramanathan et al teaches two repeat sequences concatenated to make a 1.6kb or less fragment (see figure 2 and p. 928 to 929 under materials).
Ramanathan et al teaches, “This results in a highly cost effective method for the synthesis of DNA oligonucleotides for NMR studies as described below.”, (p. 928, col 2, para 3).
Ramanathan et al teaches, “We believe that this method will boost structural analysis of biologically important target DNA sequences that form recognition motifs in several protein–DNA interactions.”, (p.931, col 2, para 2).
Ramanathan et al does not explicitly teach (a) different restriction enzymes on the 5’ and 3’ ends of the target repeat sequence (found in step 1)); (b) production of an asymmetric dsDNA sequence (found in step 6) via the digestion with different RE) or (c) wherein the shorter strand of the asymmetric dsDNA duplex is the target sequence (also found in step 6)).
Nelissen et al teaches PCR-generated tandem repeats of a target sequence, that are separated by two different restriction enzymes to produce single-stranded DNA, which contain 15N and 13C nucleotides (Figure 1 and table 1).
Regarding claim 1 and 13, Nelissen et al teaches a method of target sequence production wherein “Synthesis of ssDNA, instead of only dsDNA, is now possible by introducing asymmetrical digestion” (p. 2, col 1, para 5). To do so, “the long DNA is digested with a blunt-end-generating endonuclease followed by digestion with a sticky-end-generating endonuclease. The asymmetry in the digested DNA molecules allows for separation of the four individual strands on denaturing PAGE” (Figure 1 legend). Nelissen et al uses PvuII as a blunt end restriction enzyme site on the 5’ of the target sequence and XhoI as a sticky end on the 3’ end of the target sequence (see figure 1).
Also, the “method is based on the difference in migration distance on denaturing PAGE of the asymmetric DNA fragments that are generated thanks to the sticky-end restriction endonuclease digestion.” (p. 7-8, para 5).
Further, “The major modification we introduce lies in the enzymatic digestion of the PCR products, which allows for separation of the PCR products into ssDNAs. Instead of using a single blunt-end digestion, we generate asymmetrical dsDNA in two successive digestions. The double digested products migrate distinguishably on denaturing PAGE. The desired ssDNA fragment can be used for NMR structural analysis directly after purification from gel.”, (p.4, col 1, para 2 to col 2, para 1).
Lastly, Nelissen et al teaches, “A host of labeling patterns is possible, thereby making NMR structural studies on (larger) ssDNA more accessible. With this labeling, NMR spectral crowding is reduced, specific resonances can be eliminated or selected and line widths reduced by deuteration.”, (p.7, col 2, para 3).
Regarding claim(s) 3 and 5, Nelissen et al teaches, “The digested DNA was purified over a ResourceQ column, desalted in an YM-3 centricon (Millipore) and subsequently, electrophorized on a preparative 20% denaturing polyacrylamide gel (PAGE) containing 8M urea. The band of 36-nt three-way junction ssDNA was electroeluted from the gel in an Elutrap device (Schleicher & Schuell) and subsequently washed with 20mM sodium phosphate buffer (pH 7.5) containing 1M NaCl+50mM EDTA and Milli-Q water.”, (p.3, col 1 para 3 to col 2 para 1; and figures 3-4).
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Regarding claim 4, As depicted in the bottom segment of Figure 1 (see below) of Nelissen et al, the shorter segment of the asymmetrical dsDNA contains the target sequence while the longer segment contains the complementary segment.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Ramanathan et al, i.e., biosynthetically producing tandem-repeat uniformly isotope labeled dsDNA and separating the repeats via PAGE after digestion with the teachings of Nelissen et al, i.e., using different restriction enzymes 5’ (e.g., PvuII) and 3’ (e.g., XhoI) of each target sequence in a tandem repeat and separating the repeats via a PAGE after digestion, to yield the predictable results of biosynthetically producing tandem-repeat uniformly isotope labeled ssDNA. One of skill would have been motivated to do so because (1) Ramanathan et al teaches that their method is highly cost effective for producing uniformly labeled DNA (for NMR) and will boost structural analysis of biologically important target DNA sequences that form recognition motifs in several protein–DNA interactions, and (2) Nelissen et al teaches that the major modification they introduce lies in the enzymatic digestion of the PCR products, which allows for separation of the PCR products into ssDNAs, and that a host of labeling patterns is possible, thereby making NMR structural studies on (larger) ssDNA more accessible. One of skill could have modified the teachings of Ramanathan et al with Nelissen et al to arrive at the claimed invention with a high likelihood of success.
Regarding claim(s) 6 and 8, Nelissen et al teaches a target sequence flanked by different restriction enzyme sites and a 3’ spacer, wherein the amount of fragments is denoted as n (see figure 1). Further, “Finally, our segmental labeling method can be extended to more than two segments without significant loss in ligation yield, e.g. by designing two nicks on a hybrid of three segments with a DNA splint or by using two DNA splints and three segments in one ligation reaction.”, (p.9, col 1, para 1). Further, “The amplification of the DNA primers by means of self-primed PCR followed by double digestion requires attention to their design. The sequences should not become trapped in duplexes or intramolecular structures blocking PCR. When DINAMelt predicts such alternative stable structures, the spacer fragment can be shortened, lengthened or altered. The role of the spacer is essentially only to facilitate efficient restriction endonuclease digestion in the second digestion step and is of no further interest.”, (p.7, col 2, para 4).
Therefore, it would have been obvious to try to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the above teachings of Ramanathan et al in view of Nelissen et al, i.e., biosynthetically producing tandem-repeat uniformly isotope labeled ssDNA with Nelissen et al, i.e., a target sequence flanked by different restriction enzyme sites and a 3’ spacer, wherein the amount of fragments is denoted as n, to yield the predictable results of at 3 target sequences (reading on repeating units) to produce a large repeating unit and then ligating the large repeat units in tandem by a spacer sequence. One of skill would be motivated to do so because Nelissen et al teaches that their method can be extended to three segments in one ligation reaction, which would entail a starting product of three fragments, followed by a spacer that becomes the primer and template. One of skill could look to the teachings of Ramanathan et al and Nelissen et al and arrived at the claimed invention with a high likelihood of success.
Accordingly, claim(s) 1, 3-8, and 13 are unpatentable over Ramanathan et al in view of Nelissen et al.
Claim 2 is rejected under 35 U.S.C. 103 as being unpatentable over Ramanathan et al (supra) in view of Nelissen et al (supra) as applied to claim(s) 1, 3-8, and 13 above, and further in view of Nagahora (US 2019/0309283 A1, published October 10, 2019).
Ramanathan et al in view of Nelissen et al teach biosynthetically producing tandem-repeat uniformly isotope labeled ssDNA, wherein the 5’ end of the target sequence and the 3’ end have different restriction enzyme sequences, namely PvuII and XhoI.
Regarding claim 2, Nelissen et al teaches, “Furthermore, potentially any restriction site can be chosen to flank the DNA fragment of interest. Amongst the wide variety of available enzymes, it is likely that a combination can be found that generates digested ends exactly matching the 5’-end- and 3’-end of the DNA fragment of interest. Our method is based on the difference in migration distance on denaturing PAGE of the asymmetric DNA fragments that are generated thanks to the sticky-end restriction endonuclease digestion.”, (p.7, col 2, para 5).
Despite Nelissen et al teaching the use of any RE for generating a ssDNA fragment of interest, Ramathan and Nelissen et al do not teach KpnI and BamHI flanking the sequence of interest.
Nagahora teaches a “method for preparing a long-chain single-stranded DNA that has an accurate sequence having neither internal mutation nor terminal deletion, is homogeneous and is not contaminated with double-stranded DNAs. A target long-chain single-stranded DNA is prepared by: cloning the target DNA using a vector having nicking endonuclease recognition sites or a nicking endonuclease recognition site and a sequence-specific double-strand cleaving endonuclease recognition site; cleaving the vector by using appropriate enzyme(s); electrophoresing the same; and then cutting out a gel that contains the target single-stranded DNA to thereby prepare the target long-chain single-stranded DNA.”, (abstract).
Regarding claim 2, Nagahora teaches using a 5’ blunt end restriction enzyme site, and a 3’ sticky end restriction enzyme site in figure 2c. Nagahora teaches blunt end enzymes such as PstI, KpnI, etc., and sticky ends such as XhoI, HindIII, BamHI, etc.
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Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the two different restriction enzymes of Ramanathan et al in view of Nelissen et al, i.e., 5’ PvuII and 3’ XhoI with 5’KpnI and 3’ BamHI, as taught by Nagahora, to yield the predictable results of a target sequence with a 5’ blunt end cut site, i.e., KnpI and a 3’ sticky end cut site, i.e., BamHI. PvuII, XhoI, KpnI, and BamHI sites and their functions were known in the art before the effective filing date of the claimed invention. One of skill in the art could readily interchange 5’ blunt end cut sites as well as the 3’ end sticky end sites and arrive at the claimed invention because both Nelissen et al and Nagahora teach that any restriction enzyme can be chosen to flank the DNA fragment of interest, and both Nelissen et al and Nagahora teach the production of ssDNA through asymmetrical cut-sites.
Accordingly, claim 2 is unpatentable over Ramanathan et al in view of Nelissen et al in further view of Nagahora.
Claim 9 is rejected under 35 U.S.C. 103 as being unpatentable over Ramanathan et al (supra) in view of Nelissen et al (supra) as applied to claim(s) 1, 3-8, and 13 above, and further in view of Yong et al (CN 112877325 A, published June 1st, 2021; as evidenced by the machine translation).
Ramanathan et al in view of Nelissen et al teach biosynthetically producing tandem-repeat uniformly isotope labeled ssDNA, wherein the 5’ end of the target sequence and the 3’ end have different restriction enzyme sequences, namely PvuII and XhoI.
Ramanathan et al teaches using the recombinant plasmid, pBSKS (p. 929, col 1, para 3).
Ramanathan et al and Nelissen et al do not teach wherein plasmid is puc57.
Regarding claim 9, Yong et al teaches, “The invention claims a preparation method of ssDNA with high purity. The method comprises the following steps: the first step: synthesizing the needed target gene to the puc57 carrier. . .”, (abstract).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the recombinant vector of Ramanathan et al in view of Nelissen et al, i.e., pBSKS, with puc57, as taught by Yong et al, to yield the predictable results of using pUC57 as the recombinant vector for transforming into E.coli. pBSKS and pUC57 structures and functions were known in the art before the effective filing date of the claimed invention. One of skill could readily substitute the plasmids and the results of the substitution would have been predictable and yielded the claimed invention with a high likelihood of success.
Accordingly claim 9 is unpatentable over Ramanathan et al in view of Nelissen et al in further view of Yong et al.
Claim(s) 10-11, 14, and 16-19 are rejected under 35 U.S.C. 103 as being unpatentable over Ramanathan et al (supra) in view of Nelissen et al (supra) as applied to claim(s) 1, 3-8, and 13 above, and further in view of Butovskaya et al (Major G-Quadruplex For of HIV-1 LTR Reveals a (3+1) Folding Topology Containing a Stem-Loop, Journal American Chemical Society, vol 140, issue 42, pages 13654-13662, published October 9th, 2018).
Ramanathan et al in view of Nelissen et al teach biosynthetically producing tandem-repeat uniformly isotope labeled ssDNA.
Regarding claim 19, Ramanathan et al teaches insert length of 1.6kb or less in figure 2. Specifically, Ramanathan et al teaches two repeat sequences concatenated to make a 1.6kb or less fragment (see figure 2 and p. 928 to 929 under materials).
Regarding claim 16, Nelissen et al teaches, “The digested DNA was purified over a ResourceQ column, desalted in an YM-3 centricon (Millipore) and subsequently, electrophorized on a preparative 20% denaturing polyacrylamide gel (PAGE) containing 8M urea. The band of 36-nt three-way junction ssDNA was electroeluted from the gel in an Elutrap device (Schleicher & Schuell) and subsequently washed with 20mM sodium phosphate buffer (pH 7.5) containing 1M NaCl+50mM EDTA and Milli-Q water.”, (p.3, col 1 para 3 to col 2 para 1; and figures 3-4).
Regarding claim 17, As depicted in the bottom segment of Figure 1 (see photo in the above rejection) of Nelissen et al, the shorter segment of the asymmetrical dsDNA contains the target sequence while the longer segment contains the complementary segment.
Ramanathan et al and Nelissen et al do not teach wherein the target sequence comprises telomere ssDNA, human c-Myc promoter ssDNA, or human immunodeficiency virus (HIV) ssDNA.
Butovskaya et al teaches, “Among all the G-quadruplexes formed in the LTR sequence, LTR-III was shown to be the major G-quadruplex conformation in vitro. Here we report the NMR structure of LTR-III in K+ solution, revealing the formation of a unique quadruplex−duplex hybrid consisting of a three-layer (3 + 1) G-quadruplex scaffold, a 12-nt diagonal loop containing a conserved duplex-stem, a 3-nt lateral loop, a 1-nt propeller loop, and a V-shaped loop. Our structure showed several distinct features including a quadruplex−duplex junction, representing an attractive motif for drug targeting.”, (abstract).
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Regarding claim 10 and 14, Butovskaya et al teaches, instant SEQ ID NO:3 in Figure 1 and Figure 2A, see below Figure 1 below.
Further, Butovskaya et al teaches, “Therefore, selective targeting of the major LTR-III G-quadruplex component may be a promising strategy for viral transcription inhibition. Such a singular structure of LTR-III G-quadruplex opens the possibility of improving selectivity by targeting the quadruplex-duplex junction.”, (p. 13660, col 1, para 1-2).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Ramanathan et al and Nelissen et al i.e., biosynthetically producing tandem-repeat uniformly isotope labeled ssDNA, with the teachings of Butovskaya et al, i.e., performing NMR on the LTR-III fragment of the HIV-1 LTR, to yield the predictable results of a target sequence (or obtaining a target sequence) of HIV-1 LTR-III that is uniformly labeled via biosynthetic production. One of skill in the art would be motivated to do so because Butovskaya et al teaches using the LTR-III sequence for studying G4 structures by NMR and Ramanathan et al in view of Nelissen et al teaches that their method is highly cost effective for producing uniformly labeled DNA (for NMR) and will boost structural analysis of biologically important target DNA sequences that form recognition motifs in several protein–DNA interactions. One could look to the teachings of Ramanathan et al and Nelissen et al and combine such teachings with Butovskaya et al to arrive at the claimed invention with a high likelihood of success.
Regarding claim 11, Nelissen et al teaches a target sequence flanked by different restriction enzyme sites and a 3’ spacer, wherein the amount of fragments is denoted as n (see figure 1). Further, “Finally, our segmental labeling method can be extended to more than two segments without significant loss in ligation yield, e.g. by designing two nicks on a hybrid of three segments with a DNA splint or by using two DNA splints and three segments in one ligation reaction.”, (p.9, col 1, para 1).
Therefore, it would have been obvious to try to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the above teachings of Ramanathan et al in view of Nelissen et al in further view of Butovskaya et al, i.e., biosynthetically producing tandem-repeat uniformly isotope labeled HIV-1 LTRIII ssDNA with Nelissen et al, i.e., a target sequence flanked by different restriction enzyme sites and a 3’ spacer, wherein the amount of fragments is denoted as n, to yield the predictable results of at 14 target sequences (reading on repeating units) ligated in tandem to obtain a fusion sequence. One of skill would be motivated try any range of sequences from 1 to n that is sufficient to amount to enough single stranded DNA for NMR experiments as taught by Nelissen et al in the abstract and by the annotation of n in figure 1 between the repeating target sequences and because Ramanathan et al teaches that their method is highly cost effective for producing uniformly labeled DNA (for NMR) and will boost structural analysis of biologically important target DNA sequences that form recognition motifs in several protein–DNA interactions. One of skill could look to the teachings of Ramanathan et al in view of Nelissen et al in further view of Butovskaya et al and arrived at the claimed invention with a high likelihood of success.
Regarding claim 18, Nelissen et al teaches a target sequence flanked by different restriction enzyme sites and a 3’ spacer, wherein the amount of fragments is denoted as n (see figure 1). Further, “Finally, our segmental labeling method can be extended to more than two segments without significant loss in ligation yield, e.g. by designing two nicks on a hybrid of three segments with a DNA splint or by using two DNA splints and three segments in one ligation reaction.”, (p.9, col 1, para 1). Further, “The amplification of the DNA primers by means of self-primed PCR followed by double digestion requires attention to their design. The sequences should not become trapped in duplexes or intramolecular structures blocking PCR. When DINAMelt predicts such alternative stable structures, the spacer fragment can be shortened, lengthened or altered. The role of the spacer is essentially only to facilitate efficient restriction endonuclease digestion in the second digestion step and is of no further interest.”, (p.7, col 2, para 4).
Therefore, it would have been obvious to try to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the above teachings of Ramanathan et al in view of Nelissen et al in further view of Butovskaya et al, i.e., biosynthetically producing tandem-repeat uniformly isotope labeled ssDNA of the HIV-1 LTR III with Nelissen et al, i.e., a target sequence flanked by different restriction enzyme sites and a 3’ spacer, wherein the amount of fragments is denoted as n, to yield the predictable results of at 3 target sequences (reading on repeating units) to produce a large repeating unit and then ligating the large repeat units in tandem by a spacer sequence. One of skill would be motivated to do so because Nelissen et al teaches that their method can be extended to three segments in one ligation reaction, which would entail a starting product of three fragments, followed by a spacer that becomes the primer and template. One of skill could look to the teachings of Ramanathan et al in view of Nelissen et al in further view of Butovskaya et al and arrived at the claimed invention with a high likelihood of success.
Accordingly, claim(s) 10-11, 14, and 16-19 are unpatentable over Ramanathan et al in view of Nelissen et al in further view of Butovskaya et al.
Claim(s) 12 is rejected under 35 U.S.C. 103 as being unpatentable over Ramanathan et al (supra) in view of Nelissen et al (supra) as applied to claim(s) 1, 3-8, and 13 above, and further in view of Werner et al (Chapter 12: Uniform 13C/15N-Labeling of DNA by Tandem Repeat Amplification, Methods of Enzymology, Part of Volume 338: Nuclear Magnetic Resonance of Biological Macromolecules Part A; pages 282-304, published 2002).
Despite Ramanathan et al teaching uniform isotope labeling of oligonucleotides, Ramanathan et al does not teach explicit grams per liter.
Werner et al teaches uniform 13C/15N-Labeling of DNA by tandem repeat amplification in E.coli, (see title and p.285, para 1).
Regarding claim 12, Werner et al teaches, “For 13C/15N labeling, add 2.0 g/liter [15N]ammonium chloride and 5.0 g/liter [13C]glucose.”, (p.286, para 1).
Therefore, it would have been obvious to one of ordinary skill in the art to apply the known technique of Werner et al, i.e., adding 2.0 g/liter [15N]ammonium chloride and 5.0 g/liter [13C]glucose to the teachings of Ramanathan et al in view of Nelissen et al to yield the predicable results of medium that has a concentration of at least 1 g/liter of [15N]ammonium chloride and/or at least 4 g/liter of [13C]glucose. The prior art, i.e., Werner et al contained the known technique of uniformly labeling 13C/15N DNA, which is applicable to the combined teachings of Ramanathan et al in view of Nelissen et al. One of skill in the art would have recognized that applying the known technique would have arrived at the claimed invention with a high likelihood of success, especially because all three teachings involved the use of DNA in NMR spectroscopy.
Accordingly, claim 12 is unpatentable over Ramanathan et al in view of Nelissen et al in further view of Werner et al.
Claim 15 is rejected under 35 U.S.C. 103 as being unpatentable over Ramanathan et al (supra) in view of Nelissen et al (supra) in view of Butovskaya et al (supra) as applied to claim(s) 10-11, 14, and 16-19 above, and further in view of Nagahora (supra).
Ramanathan et al in view of Nelissen et al in further view of Butovskaya et al teach biosynthetically producing tandem-repeat uniformly isotope labeled ssDNA of the HIV LTR III, wherein the 5’ end of the target sequence and the 3’ end have different restriction enzyme sequences, namely PvuII and XhoI.
Regarding claim 15, Nelissen et al teaches, “Furthermore, potentially any restriction site can be chosen to flank the DNA fragment of interest. Amongst the wide variety of available enzymes, it is likely that a combination can be found that generates digested ends exactly matching the 5’-end- and 3’-end of the DNA fragment of interest. Our method is based on the difference in migration distance on denaturing PAGE of the asymmetric DNA fragments that are generated thanks to the sticky-end restriction endonuclease digestion.”, (p.7, col 2, para 5).
Despite Nelissen et al teaching the use of any RE for generating a ssDNA fragment of interest, Ramathan, Nelissen et al, and Butovskaya et al do not teach KpnI and BamHI flanking the sequence of interest.
Nagahora teaches a “method for preparing a long-chain single-stranded DNA that has an accurate sequence having neither internal mutation nor terminal deletion, is homogeneous and is not contaminated with double-stranded DNAs. A target long-chain single-stranded DNA is prepared by: cloning the target DNA using a vector having nicking endonuclease recognition sites or a nicking endonuclease recognition site and a sequence-specific double-strand cleaving endonuclease recognition site; cleaving the vector by using appropriate enzyme(s); electrophoresing the same; and then cutting out a gel that contains the target single-stranded DNA to thereby prepare the target long-chain single-stranded DNA.”, (abstract).
Regarding claim 15, Nagahora teaches using a 5’ blunt end restriction enzyme site, and a 3’ sticky end restriction enzyme site in figure 2c. Nagahora teaches blunt end enzymes such as PstI, KpnI, etc., and sticky ends such as XhoI, HindIII, BamHI, etc.
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Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the two different restriction enzymes of Ramanathan et al in view of Nelissen et al in further view of Butovskaya et al, i.e., 5’ PvuII and 3’ XhoI with 5’KpnI and 3’ BamHI, as taught by Nagahora, to yield the predictable results of a target sequence with a 5’ blunt end cut site, i.e., KnpI and a 3’ sticky end cut site, i.e., BamHI. PvuII, XhoI, KpnI, and BamHI sites and their functions were known in the art before the effective filing date of the claimed invention. One of skill in the art could readily interchange 5’ blunt end cut sites as well as the 3’ end sticky end sites and arrive at the claimed invention because both Nelissen et al and Nagahora teach that any restriction enzyme can be chosen to flank the DNA fragment of interest, and both Nelissen et al and Nagahora teach the production of ssDNA through asymmetrical cut-sites.
Accordingly, claim 15 is unpatentable over Ramanathan et al in view of Nelissen et al in view of Butovskaya et al in further view of Nagahora.
Claim 20 is rejected under 35 U.S.C. 103 as being unpatentable over Ramanathan et al (supra) in view of Nelissen et al (supra) in view of Butovskaya et al (supra) as applied to claim(s) 10-11, 14, and 16-19 above, and further in view of Yong et al (supra).
Ramanathan et al in view of Nelissen et al in further view of Butovskaya et al teach biosynthetically producing tandem-repeat uniformly isotope labeled ssDNA of the HIV LTR III, wherein the 5’ end of the target sequence and the 3’ end have different restriction enzyme sequences, namely PvuII and XhoI.
Ramanathan et al teaches using the recombinant plasmid, pBSKS (p. 929, col 1, para 3).
Ramanathan et al and Nelissen et al do not teach wherein plasmid is puc57.
Regarding claim 20, Yong et al teaches, “The invention claims a preparation method of ssDNA with high purity. the method comprises the following steps: the first step: synthesizing the needed target gene to the puc57 carrier. . .”, (abstract).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the recombinant vector of Ramanathan et al in view of Nelissen et al in further view of Butovskaya et al, i.e., pBSKS, with puc57, as taught by Yong et al, to yield the predictable results of using pUC57 as the recombinant vector for transforming into e.coli. pBSKS and pUC57 structures and functions were known in the art before the effective filing date of the claimed invention. One of skill could readily substitute the plasmids and the results of the substitution would have been predictable and yielded the claimed invention with a high likelihood of success.
Accordingly, claim 20 is unpatentable over Ramanathan et al in view of Nelissen et al in view of Butovskaya et al in further view of Yong et al.
Conclusion
No claims allowed.
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/L.M.T./Examiner, Art Unit 1637
/Jennifer Dunston/Supervisory Patent Examiner, Art Unit 1637
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