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 .
Election/Restrictions
Applicant’s election without traverse of Group I, drawn to a splint single stranded DNA (ssDNA), a ssDNA-ligating linear ssDNA monomeric complex and a kit comprising the ssDNA, claims 1-11, and 20-21 and the species of ssDNA wherein the linear splint sequence comprises at least 10 contiguous nucleotides, claims 2 and 7, in the reply filed on July 14, 2026 is acknowledged.
In view of the prior art, the restriction requirement has been withdrawn and the species of linear splint sequence comprising at least 12 contiguous nucleic acids (claims 3 and 8) have been rejoined. Claims 12-19 corresponding to Group 2, drawn to a method of producing a single stranded DNA circle have also been rejoined.
Claims 1-21 are examined on the merits.
Priority
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. The instant application claims domestic benefit from U.S. provisional application 63/439006 filed on January 13, 2023.
Information Disclosure Statement
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Objection to the Drawings
The drawings are objected to as failing to comply with 37 CFR 1.84(p)(5) because they do not include the following reference sign(s) mentioned in the description:
Para [0050] of the specification references Fig. 4 as showing cyclization of L30 and L40 DNA strands. However, Fig. 4 is limited to a single histogram depicting cyclization of L16 linear ssDNA in conjunction with the H5 splint at various concentrations.
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Objections to the Specification
The use of the term CircLigase in Paras. [0008] and [0025], which is a trade name or a mark used in commerce, has been noted in this application. The term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 7-8, 19, and 21 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.
Claims 7-8, which depend from claim 6, recites the limitation "a” ligating linear ssDNA in line 3, which should the “the” ligating linear ssDNA. There is insufficient antecedent basis for this limitation in the claims as there is a prior recitation of a “ligating linear ssDNA” in the complex of claim 6. Appropriate correction is required.
Claim 19, which depends from claim 18, recites the limitation "T4 ligase" in line 1. There is insufficient antecedent basis for this limitation in the claim as the prior recitation is to T4 DNA ligase. Appropriate correction is required.
Claim 21 recites the limitation “when the DNA ligase is a T4 DNA ligase, the kit further comprises ATP which can be provided separately or combined with the T4 DNA ligase or one or more components of the ligation buffer” which renders the claim indefinite. As claimed, it appears that the ATP is being treated as both an additional component of the kit and also as a compound which can be present in the initially recited components for a ligation buffer which renders the metes and bounds of what is included in the components of the ligation buffer unclear. Appropriate correction is required.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-21 are rejected 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. The claims contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventors, at the time the application was filed, had possession of the claimed invention.
Independent claim 1 encompasses a genus of splint single-stranded DNA (ssDNA) comprising a linear splint sequence and hairpin sequences on each side of the linear splint sequence while the specification only discloses a fixed number of species of splint ssDNA.
Dependent claims 2 and 3 encompass a genus of splint ssDNA where the linear splint sequence comprises at least 10 or at least 12 nucleotides, each of which hybridize with one end of a ligating linear ssDNA.
Dependent claim 4 encompasses a genus of splint ssDNA wherein the hairpin sequences on each side of the linear splint sequence differ from one another.
Dependent claim 5 encompasses specific hairpin sequences of the genus of splint ssDNA.
Dependent claim 6 encompasses a genus of splint ssDNA-ligating liner ssDNA monomeric complexes comprising the splint ssDNA of independent claim 1.
Dependent claims 7 and 8 encompasses a genus of splint ssDNA-ligating liner ssDNA monomeric complexes where the linear splint sequence comprises at least 10 or at least 12 nucleotides, each of which hybridize with one end of a ligating linear ssDNA.
Dependent claim 9 encompasses a genus of splint ssDNA-ligating liner ssDNA monomeric complexes wherein the hairpin sequences on each side of the linear splint sequence differ from one another.
Dependent claim 10 encompasses a genus of splint ssDNA-ligating liner ssDNA monomeric complexes comprising specific hairpin sequences of the genus of splint ssDNA.
Dependent claim 11 encompasses a genus of splint ssDNA-ligating liner ssDNA monomeric complexes comprising a genus of ligating liner ssDNA at least about 16 nucleotides in length.
Dependent claim 12 encompasses a genus of methods of producing a ssDNA circle comprising incubating the genus of splint ssDNA of claim 1 and a genus of ligating linear ssDNA at least about 16 nucleotides in length under hybridizing conditions and a splint ssDNA-ligating liner ssDNA monomeric complex is formed and incubating the splint ssDNA-ligating liner ssDNA monomeric complex with a DNA ligase under ligating conditions in order form a ssDNA circle. As stated supra, specification only discloses a fixed number of species of splint ssDNA.
Dependent claim 13 encompasses a DNA ligase for use in the method of claim 12.
Dependent claim 14 encompasses a concentration of ligating linear ssDNA of about 100 µM for use in the method of claim 12.
Dependent claims 15 and 16 encompasses a genus of ratios of ligating linear ssDNA to splint ssDNA for use in the method of claim 12.
Dependent claims 17-19 encompass a genus of hybridizing and ligating conditions and reagents for use in the method of claim 12.
Dependent claims 20 and 21 encompass kits comprising the splint ssDNA of claim 1 and instructions and reagents for producing a ssDNA circle.
Under the written description guidelines (see MPEP 2163) the Examiner is directed to determine whether one skilled in the art would recognize that the Applicant was in possession of the claimed invention as a whole at the time of filing. The following considerations are critical to this determination.
To satisfy the written description requirement, a patent specification must describe the claimed invention in sufficient detail that one skilled in the art can reasonably conclude that the inventor had possession of the claimed invention. See, e.g., Moba, B.V. v. Diamond Automation, Inc., 325 F.3d 1306, 1319, 66 USPQ2d 1429, 1438 (Fed. Cir. 2003); Vas-Cath, Inc. v. Mahurkar, 935 F.2d at 1563, 19 USPQ2d at 1116. An original claim may lack written description support when (1) the claim defines the invention in functional language specifying a desired result but the disclosure fails to sufficiently identify how the function is performed or the result is achieved or (2) a broad genus claim is presented but the disclosure only describes a narrow species with no evidence that the genus is contemplated. See Ariad Pharms., Inc. v. Eli Lilly & Co., 598 F.3d 1336, 1349-50 (Fed. Cir. 2010) (en banc). The written description requirement is not necessarily met when the claim language appears in ipsis verbis in the specification. "Even if a claim is supported by the specification, the language of the specification, to the extent possible, must describe the claimed invention so that one skilled in the art can recognize what is claimed. The appearance of mere indistinct words in a specification or a claim, even an original claim, does not necessarily satisfy that requirement." Enzo Biochem, Inc. v. Gen-Probe, Inc., 323 F.3d 956, 968, 63 USPQ2d 1609, 1616 (Fed. Cir. 2002).
Accordingly, to satisfy the written description requirement, the specification must describe the claimed invention in sufficient detail that one skilled in the art can reasonably conclude that the inventor had possession of the claimed invention. Vas-Cath, Inc. v. Mahurkar, 935 F.2d 1555, 1562-63, 19 USPQ2d 1111 (Fed. Cir. 1991). See also MPEP 2163.
ACTUAL REDUCTION TO PRACTICE
With regard to claim 1 encompassing a genus of splint single-stranded DNA (ssDNA) comprising a linear splint sequence and hairpin sequences on each side of the linear splint sequence, the specification discloses embodiments of splint ssDNA sequences comprising the hairpin loop sequences of SEQ ID NOs: 1 and 2, and which have single stranded linear splint sequences which allow for binding of a ligating DNA sequence via 4 (H4), 5 (H5), or 6 (H6) nucleotides on either end of the ligating DNA sequence (See Para. [0033], Fig. 2).
Regarding claims 2 and 3 encompassing a genus of splint ssDNA where the linear splint sequence comprises at least 10 or at least 12 nucleotides, each of which hybridize with one end of a ligating linear ssDNA, as stated supra the specification discloses the species of H5 comprising 10 contiguous nucleotides for binding a linear DNA and the species of H6 comprising 12 contiguous nucleotides for binding a linear DNA (See Para. [0033], Fig. 2).
Regarding claims 4 and 5 encompassing a genus of splint ssDNA wherein the hairpin sequences on each side of the linear splint sequence differ from one another, the specification discloses splint ssDNA comprising hairpins wherein one hairpin comprises SEQ ID NO: 1 and the other hairpin comprising SEQ ID NO: 2.
Regarding claim 6 encompassing a genus of splint ssDNA-ligating liner ssDNA monomeric complexes comprising the splint ssDNA of independent claim 1, as stated supra, the specification discloses embodiments of splint ssDNA sequences comprising the hairpin loop sequences of SEQ ID NOs: 1 and 2, and which have single stranded linear splint sequences which allow for binding of a ligating DNA sequence via 4 (H4), 5 (H5), or 6 (H6) nucleotides on either end of the ligating DNA sequence (See Para. [0033], Fig. 2). Further, regarding the ligating linear ssDNA, the specification discloses embodiments of ligating linear ssDNA comprising 12, 14, 16, 18, 20, 30, and 40 nucleotides (See Para. [0033], Fig. 2, Example 4) and monomeric complexes derived from using the splint ssDNA of H6 with ligating linear ssDNA of all lengths (Fig. 2), the splint ssDNA of H5 and H4 with ligating linear ssDNA having 20 nucleotides (Examples 2 and 3), and the splint ssDNA of H5 with ligating linear ssDNA having 16 nucleotides (Fig. 4).
Regarding claims 7 and 8 encompassing a genus of splint ssDNA-ligating liner ssDNA monomeric complexes where the linear splint sequence comprises at least 10 or at least 12 nucleotides, each of which hybridize with one end of a ligating linear ssDNA, as stated supra, the specification discloses the species of H5 comprising 10 contiguous nucleotides for binding a linear DNA and the species of H6 comprising 12 contiguous nucleotides for binding a linear DNA (See Para. [0033], Fig. 2) and monomeric complexes derived from using the splint ssDNA of H6 with ligating linear ssDNA having 12, 14, 16, 18, 20, 30, and 40 nucleotides (Fig. 2) and splint ssDNA of H5 with ligating linear ssDNA having 20 nucleotides (Examples 2 and 3) and 16 nucleotides (Fig. 4).
Regarding claims 9 and 10 encompassing a genus of splint ssDNA-ligating liner ssDNA monomeric complexes wherein the hairpin sequences on each side of the linear splint sequence differ from one another, as state supra the specification discloses splint ssDNA comprising hairpins wherein one hairpin comprises SEQ ID NO: 1 and the other hairpin comprising SEQ ID NO: 2 which are used for all embodiments of ligating linear ssDNA and monomeric complexes.
Regarding claim 11 encompassing a genus of splint ssDNA-ligating liner ssDNA monomeric complexes comprising a genus of ligating liner ssDNA at least about 16 nucleotides in length, as stated supra, specification discloses embodiments of ligating linear ssDNA comprising 12, 14, 16, 18, 20, 30, and 40 nucleotides (See Para. [0033], Fig. 2, Example 4) and monomeric complexes derived from using the splint ssDNA of H6 with ligating linear ssDNA of all lengths (Fig. 2), the splint ssDNA of H5 and H4 with ligating linear ssDNA having 20 nucleotides (Examples 2 and 3), and the splint ssDNA of H5 with ligating linear ssDNA having 16 nucleotides (Fig. 4).
Regarding claim 12 encompassing a genus of methods of producing a ssDNA circle comprising incubating the genus of splint ssDNA of claim 1 and a genus of ligating linear ssDNA at least about 16 nucleotides in length under hybridizing conditions and a splint ssDNA-ligating liner ssDNA monomeric complex is formed and incubating the splint ssDNA-ligating liner ssDNA monomeric complex with a DNA ligase under ligating conditions in order form a ssDNA circle, as stated supra, specification only discloses a fixed number of species of splint ssDNA, a fixed number of ligating linear ssDNA, and a fixed number of splint ssDNA-ligating linear ssDNA monomeric complexes. The specification discloses only use of a T4 DNA ligase and discloses that the splint ssDNA of H6 was not able to produce ssDNA circles for ligating linear ssDNA having less than 16 nucleotides, the splint ssDNA of H4 does not reliably produce ssDNA circles using ligating linear ssDNA having 20 nucleotides (see Examples 2 and 3), and that although the splint ssDNA of H5 is able to produce ssDNA circles using ligating linear ssDNA having 20 nucleotides, production of ssDNA circles using ligating linear ssDNA having 16 nucleotides is heavily dependent on the concentration of DNA (Fig. 4).
Regarding claim 13 encompassing a DNA ligase for use in the method of claim 12, the specification discloses use of T4 DNA ligase (Para. [0035]).
Regarding claim 14 encompassing a concentration of ligating linear ssDNA of about 100 µM for use in the method of claim 12, the specification discloses use of ligating linear ssDNA concentrations between 1-100 µM (i.e., 1, 5, 10, 20, 50, and 100 µM) with the splint ssDNA of H6 (Fig. 3) and H5 and ligating linear ssDNA having 20 nucleotides (Example 3) which are able to produce ssDNA circles. However, when using the splint ssDNA of H5 and ligating linear ssDNA having 16 nucleotides, production of ssDNA circles was only reliable at ligating linear ssDNA concentrations of 10 µM and above (Fig. 4).
Regarding claims 15 and 16 encompassing a genus of ratios of ligating linear ssDNA to splint ssDNA for use in the method of claim 12, the specification discloses use of a 1:1.2 ratio of ligating linear ssDNA to splint ssDNA (Para. [0035]).
Regarding claims 17-19 encompassing a genus of hybridizing and ligating conditions and reagents for use in the method of claim 12, the specification discloses annealing from 95-22 °C in ligation buffer but does not disclose an amount of time, addition of T4 DNA ligase at 100 U/µg per ligating linear ssDNA, and ligation at room temperature (22-25 °C) for 16 hours. (Para. [0035]).
Regarding claims 20 and 21 encompassing kits comprising the splint ssDNA of claim 1 and instructions and reagents for producing a ssDNA circle, as stated supra, the specification discloses a limited number of splint ssDNA which can be used with a limited number of ligating linear ssDNA in order to form ssDNA circles. Further, the specification only discloses use of T4 DNA ligase which is added to a ligation buffer which comprises ATP after annealing (Para. [0035]).
Accordingly, Applicant has not demonstrated possession of species of splint ssDNA beyond those detailed supra, nor ssDNA monomeric complexes comprising ligating linear ssDNA beyond those detailed supra, and did not demonstrate a reduction to practice of a method of making ssDNA circles using any splint ssDNA comprising a linear splint sequence with hairpins on both ends and any ligating linear ssDNA under any conditions. Further, Applicant did not adequately set forth in terms of distinguishing identifying characteristics as evidenced by other descriptions of the invention that are sufficiently detailed to show that Applicant was in possession of the claimed genuses of splint ssDNA, ssDNA monomeric complexes comprising splint ssDNA and ligating linear ssDNA, and methods of making ssDNA circles.
DISCLOSURE OF STRUCTURE
Applicant has provided a general structural framework for a splint ssDNA comprising a linear splint sequence which is able to bind to between 4 and 6 nucleotides on each side of a ligating linear ssDNA sequence and which comprise a hairpin having SEQ ID NO: 1 on one end and a hairpin having SEQ ID NO: 2 on the other end and which have a very specific conformational structure (See Fig. 1). Applicant has further provided several embodiments of ligating linear ssDNA of various lengths. However, only splint ssDNA sequences comprising linear splint sequences which are able to bind to at least 5 continuous nucleotides on each side of a ligating linear ssDNA (i.e., H5 and H6) and ligating linear ssDNA comprising at least 16 nucleotides appear to be able to form monomeric complexes and produce circular ssDNA, under optimized conditions, based on the instant specification. Applicant has provided no examples of a method of making circular ssDNA with splint ssDNA sequences or splint ssDNA-ligating linear ssDNA monomeric complexes other than those having the specifically disclosed structures as detailed supra and with a method other than those disclosed (Para. [0035]). The prior art is silent as to splint ssDNA having the instantly disclosed structure and therefore also to the methods used to make circular ssDNA using a splint ssDNA having the instantly disclosed structure. Furthermore, neither the specification nor the art indicate a relationship between the structure of the claimed genus of splint ssDNA and the ability to form monomeric complexes with linear ligating ssDNA in order to produce circular ssDNA.
SUFFICIENT RELEVANT IDENTIFYING CHARACTERISTICS
As detailed above, a splint ssDNA which forms a specific structure having a linear splint sequence which comprises at least 10 nucleotides and a hairpin having SEQ ID NO: 1 on one end and SEQ ID NO: 2 on the other end which can be used to make circular ssDNA is provided. Further, various ligating linear ssDNA molecules which have sequences at each end which are able bind to at least 5 contiguous nucleotides of the linear splint sequence and which have at least 16 nucleotides in order to be able to form splint ssDNA-ligating linear ssDNA monomeric complexes and circular ssDNA are provided. Although methods of making circular ssDNA are known in the art, the art is silent to use of splint ssDNA having the instantly claimed structure in methods of making circular ssDNA.
If a skilled artisan sought to generate the claimed genus of splint ssDNA comprising a linear splint sequence and a hairpin sequence on each side and splint ssDNA-ligating linear ssDNA monomeric complexes comprising the genus of splint ssDNA, they would need to know which linear splint sequences could be chosen and combined with which specific hairpin sequences/structures which would be able to predictably form monomeric complexes with a specific ligating linear ssDNA and, ultimately form a ssDNA circle. Hence, based on the written description guidelines, the Examiner should conclude that the Applicant was not in possession of the claimed genus of splint ssDNA sequences beyond the sequences as described in the specification nor methods of making a circular ssDNA beyond use of the splint ssDNA as described in the specification under optimized conditions. Because the splint ssDNA is not known, the methods of using the splint ssDNA are also not known. Therefore, it is incumbent on the applicant to provide this nexus between structure and function, in order to be given credit for possession of the claimed genus of methods of making a circular ssDNA.
The breadth of the claims encompass a genus of splint ssDNA comprising any linear splint sequence and any hairpin sequence on each side. The instant specification provides limited guidance and description of any rational in choosing the splint ssDNA that were identified, therefore the skilled artisan would not know what rational approach to take to make modifications with any predictable outcome on formation of splint ssDNA-ligating linear ssDNA monomeric complexes and ultimately production of circular ssDNA. Therefore, it is incumbent on the Applicant to provide this nexus between structure and function, in order to be given credit for possession of a larger genus of splint ssDNA related to those comprising these individually disclosed species. Otherwise, the Written Description guidelines suggest that the applicant is entitled to only the species specifically recited as having this activity. Moreover, even when several species are disclosed, these are not necessarily representative of the entire genus. AbbVie Deutschland GMBH v. Janssen Biotech, 111 USPQ2d 1780, 1790 (Fed. Cir. 2014) (“The ’128 and ’485 patents, however, only describe species of structurally similar antibodies that were derived from Joe-9. Although the number of the described species appears high quantitatively, the described species are all of the similar type and do not qualitatively represent other types of antibodies encompassed by the genus.”). Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus to provide a "representative number” of species. An applicant may show that an invention is complete by disclosure of sufficiently detailed, relevant identifying characteristics which provide evidence that applicant was in possession of the claimed invention, i.e., complete or partial structure, other physical and/or chemical properties, functional characteristics when coupled with a known or disclosed correlation between function and structure, or some combination of such characteristics. Enzo Biochem, 323 F.3d at 964, 63 USPQ2d at 1613.
STATE OF THE ART & QUANTITY OF EXPERIMENTATION
The method of making the claimed invention and the method of using the claimed invention to produce circular ssDNA is not well established. Although use of splint sequences without hairpins for production of circular DNA was known, one of skill in the art would neither expect nor predict the ability of a splint ssDNA comprising a linear splint sequence and a hairpin sequence on each side which can form monomeric complexes with a ligating linear ssDNA and form a circular ssDNA according to the genus of splint ssDNA as broadly as claimed.
The prior art is silent as to splint ssDNA comprising hairpin sequences on each side and their use in production of circular ssDNA. However, Applicant’s instant specification and the contemporaneous art of Paluzzi et al. (2023, Near‐Quantitative Preparation of Short Single‐Stranded DNA Circles. Angewandte Chemie Intl. Ed., 62(16), e202218443) indicates that the ability of a splint ssDNA sequence having a linear splint sequence and hairpin sequences on each end to complex with a ligating linear ssDNA and form a circular ssDNA when incubated with a T4 DNA ligase is unpredictable and is based on several factors including the length of the linear splint sequence, the length of the ligating linear ssDNA to be cyclized, hairpin structure, and the specific conditions under which the method of producing circular ssDNA is performed.
As an initial matter, Applicant’s splint ssDNA comprising a linear splint sequence and a hairpin sequence on each side of the linear splint sequence appears to require a very specific structural conformation in order for proper functional ability of making circular ssDNA (See Fig. 1). The splint ssDNA sequence appears to require a single stranded linear splint sequence which remains in an open conformation and includes double stranded hairpins at each end which form “borders”. The splint ssDNA must be arranged in a specific conformation such that the linear splint sequence is located within a cavity or depression between the double stranded hairpin borders. Applicant’s exemplary splint ssDNA sequences all comprise hairpin sequences which differ from the hairpin sequence on the other end (i.e., SEQ ID NO: 1 and 2) and have single stranded linear splint sequences having at least enough nucleotides for a T4 DNA ligase to recognize upon the splint ssDNA binding with a ligating linear ssDNA. The instant specification indicates that, typically, T4 DNA ligase requires at least 12 base pairs (bp) on each side of the nick to be ligated due to side of the T4 DNA ligase (Para. [0024]) and Applicant’s exemplary embodiments all include sequences which allow for equal numbers of bp binding to the linear splint sequence by each side of the ligating linear ssDNA (See Figs. 1 and 2). Applicant’s disclosure suggests that presence of the hairpins increases the stability of the splint ssDNA-ligating linear DNA complex which allows for splint sequences “significantly shorter than 6 bps” which still facilitate DNA ligation and also that the hairpins allow intermolecular repulsion which results in increased intramolecular cyclization (Para. [0026]). Thus, the instantly claimed splint ssDNA requires a specific structural configuration for functional ability.
Regarding the length of the linear splint sequence, the instant specification and Paluzzi et al. disclose that splint ssDNA sequences comprising linear splint sequences which had fewer than 10 contiguous nucleotides (i.e., H4) were not effective at forming stable monomeric complexes with ligating linear ssDNA having 20 nucleotides (Specification Example 2; Paluzzi et al. Pg. 3, bridging para. and right col., full para.) and were therefore not able to reliably form circular ssDNA when incubated with a T4 DNA ligase, instead forming 5’-adenylpyrophosphorylated intermediate products which are indicative of inefficient cyclization (Para. [0045]). Therefore, based on the instant specification and Applicant’s contemporaneous art, it appears that the ability of splint ssDNA to form monomeric complexes with a ligating linear ssDNA and generate circular ssDNA in the presence of a T4 DNA ligase is unpredictable and requires binding of at least 5 bps on either side of the linear splint sequence with the ends of a ligating linear ssDNA.
Regarding the length of the ligating linear ssDNA to be cyclized, the instant specification and Paluzzi et al. discloses that ligating linear ssDNA strands shorter than 16 nucleotides were not able to be cyclized and instead resulted in formation of longer DNA strands first which were cyclized (See specification Example 4; Paluzzi et al. Pg. 4, left col., middle para.). Further, when splint ssDNA which bind five bp on either side of the ligating linear ssDNA (i.e., H5) were combined with ligating linear ssDNA having 16 nucleotides (i.e., L16), the ability to predictably cyclize ssDNA was dependent on the concentration of ligating linear ssDNA (see specification Fig. 4; Paluzzi et al. Fig. 4b). Thus, the ability of a splint ssDNA to form a monomeric complex with a ligating linear ssDNA and form circular ssDNA in the presence of T4 DNA ligase is also unpredictable and depends on the length of the ligating linear ssDNA as well as the specific combination of splint ssDNA and ligating linear ssDNA.
Regarding the hairpin sequences, Applicant’s exemplary hairpins have a four bp stem, the minimal four nucleotide loop, and sequences which differ from each other. Since, based on Applicant’s specification, presence of the hairpins results repulsive forces between hairpins which favor intramolecular interaction, a skilled artisan would recognize that changes to the hairpin sequence could also change hairpin conformation and resultant repulsive forces. Applicant’s contemporaneous art indicates that reductions of the hairpin loop size in H6 splint ssDNA sequences when combined with ligating linear ssDNA having 20 nucleotides result in increased formation of longer DNA strands which are then cyclized by a T4 DNA ligase (Paluzzi et al., Pg. 3, left col., 1st para and Supp. Fig 5a-c), indicating that functional ability to form monomeric complexes and form circular ssDNA is dependent upon hairpin structure and therefore would not be predictable for any hairpin sequence. Further, Applicant’s exemplary embodiments utilize hairpin sequences which differ from one another which a skilled artisan would recognize contributes to conformational structure. As detailed supra, it appears that the splint ssDNA requires a specific conformational structure which is required for functional effect. Applicant has provided no guidance as to how the hairpin sequences were generated or which factors must be considered in order to create the required repulsive forces between hairpins which favor intramolecular interactions. If, for example, Applicant’s claims encompass the hairpin sequences on either end of the splint ssDNA were the same, wherein the splint ssDNA could form a circular-type structure based on complementary pairing between the opposite ends of the splint ssDNA sequence which would result in a non-functional splint ssDNA.
Regarding the specific conditions under which the method of producing circular ssDNA is performed, based on the instant specification, although concentration of ligating linear DNA does not affect the circular ssDNA formation when using an H6 splint ssDNA and ligating linear ssDNA having 20 nucleotides (See Fig. 3), formation of circular DNA is dependent upon ligating linear ssDNA concentration when using a shorter H5 splint ssDNA and a shorter ligating linear ssDNA having 16 nucleotides (Fig. 4). Paluzzi et al. discloses that, when using a combination of the H6 splint and ligating linear ssDNA having 20 nucleotides, DNA concentrations of 250 µM and above resulted in increased production and cyclizing of longer DNA strands (Pg. 4, right col., 1st para. and Supp. Fig 9a-b). Paluzzi et al. ultimately concludes that the method of making ssDNA circles is effective for ssDNA as short as 16 nucleotides at concentrations up to 100 µM (Pg. 4, right col, last para.). Therefore, the functional ability of a splint ssDNA to form splint ssDNA-ligating liner ssDNA monomeric complexes and subsequently form ssDNA circles is not predictable for all splint ssDNA and ligating linear ssDNA at all concentrations.
Applicant has claimed a genus of splint ssDNA which can form monomeric complexes with a genus of ligating linear ssDNA and subsequently form ssDNA circles in the presence of a DNA ligase, yet the specification has only managed to identify very specific splint ssDNA sequences that can be used as in combination with specific ligating linear ssDNA under specific parameters to form ssDNA circles. Independent of how these specific splint sequences were arrived upon by Applicant, a splint ssDNA requires a particular structure and conformation, ligating linear ssDNA of a particular length, and specific DNA concentrations in order to predictably form monomeric complexes and subsequently circular ssDNA. Because Applicant has provided no manner a priori to predict which splint ssDNA and which ligating linear ssDNA be chosen and used to make a ssDNA circle, the genus of splint ssDNA claimed by Applicant cannot be predictably made or used by the ordinary artisan.
Not knowing, absent further experimentation and screening, which splint ssDNA function in combination with which ligating linear ssDNA and which do not, when, as set forth above, formation of a monomeric complex of splint ssDNA and ligating linear ssDNA and effective cyclization is vary based on the length of linear splint sequences, length of the ligating linear ssDNA, shape of the hairpin, and specific reaction conditions, leads to one having no predictability or expectation of success for the ability of any splint ssDNA having any linear splint sequence and a hairpin at each end to form a monomeric complex with any ligating linear ssDNA and function to produce a ssDNA when incubated with a DNA ligase. Such random experimentation to identify at a later time what splint ssDNA structure, splint sequence, ligating linear ssDNA, or variant or modification is or is not functional and is embraced by Applicant’s claims is undue experimentation. Furthermore, functionally defined genus claims (i.e., formation of monomeric complexes and circular ssDNA) can be inherently vulnerable to invalidity challenge for lack of written description support, especially in technology fields that are highly unpredictable, where it is difficult to establish a correlation between structure and function for the whole genus or to predict what would be covered by the functionally claimed genus. See ABBVIE DEUTSCHLAND GMBH & 2 CO. v. JANSSEN BIOTECH, INC., Appeals from the United States District Court for the District of Massachusetts in Nos. 09-CV-11340-FDS, 10-CV-40003-FDS, and 10-CV-40004-FDS, Judge F. Dennis Saylor, IV. See also Ariad, 598 F.3d at 1351 (“[T]he level of detail required to satisfy the written description requirement varies depending on the nature and scope of the claims and on the complexity and predictability of the relevant technology.”); see also Centocor Ortho Biotech, Inc. v. Abbott Labs., 636 F.3d 1341, 1352 (Fed. Cir. 2011) (noting the technical challenges in developing fully human antibodies of a known human protein).
CONCLUSION
Therefore, the Examiner concludes that there is insufficient written description of the instantly claimed genus of splint ssDNA and methods of producing a circular ssDNA using the claimed genus of splint ssDNA. Specifically, there is limited description of the structure-function relationship between the claimed genus of splint ssDNA and their ability to form splint ssDNA-ligating linear ssDNA monomeric complexes and produce circular ssDNA when incubated with a DNA ligase and the Examiner further concludes a skilled artisan would find the specification inadequately describes the claimed genus of splint ssDNA which can be subsequently used in a method of making circular ssDNA.
Claim Rejections - 35 USC § 112(a) – Scope of Enablement
Claims 1-21 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for splint ssDNA having a linear splint sequence comprising at least 10 or at least 12 contiguous nucleotides, half of which can hybridize to each end of a ligating linear ssDNA, and having hairpin sequences which differ from one another on each end of the splint the hairpins having SEQ ID NO: 1 and SEQ NO: 2 in order to form monomeric complexes with ligating linear ssDNA having at least 16 nucleotides and which can be used to generate ssDNA circles in the presence of a T4 DNA ligase at ligating linear ssDNA concentrations up to 100 µM, does not reasonably provide enablement for any splint ssDNA comprising any linear splint sequence and a hairpin sequence on each end which can form monomeric complexes with any ligating linear ssDNA and be used in a method of making ssDNA circles. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
SCOPE OF THE INVENTION
The breadth of the claims encompasses a genus of splint ssDNA comprising a linear splint sequence and hairpins on each end which can form monomeric complexes with ligating linear ssDNA and be used in a method of making ssDNA circles in the presence of a DNA ligase. As discussed supra, the specification fails to describe the genus of splint ssDNA and would require undue experimentation to discover these splint ssDNA. The specification only discloses and provides guidance for splint ssDNA having a linear splint sequence comprising at least 10 or at least 12 contiguous nucleotides, half of which can hybridize to each end of a ligating linear ssDNA, and having hairpin sequences which differ from one another on each end of the splint the hairpins having SEQ ID NO: 1 and SEQ NO: 2 in order to form monomeric complexes with ligating linear ssDNA having at least 16 nucleotides and which can be used to generate ssDNA circles in the presence of a T4 DNA ligase at ligating linear ssDNA concentrations up to 100 µM.
Independent claim 1 encompasses a genus of splint single-stranded DNA (ssDNA) comprising a linear splint sequence and hairpin sequences on each side of the linear splint sequence while the specification only discloses a fixed number of species of splint ssDNA.
Dependent claims 2 and 3 encompass a genus of splint ssDNA where the linear splint sequence comprises at least 10 or at least 12 nucleotides, each of which hybridize with one end of a ligating linear ssDNA.
Dependent claim 4 encompasses a genus of splint ssDNA wherein the hairpin sequences on each side of the linear splint sequence differ from one another.
Dependent claim 5 encompasses specific hairpin sequences of the genus of splint ssDNA.
Dependent claim 6 encompasses a genus of splint ssDNA-ligating liner ssDNA monomeric complexes comprising the splint ssDNA of independent claim 1.
Dependent claims 7 and 8 encompasses a genus of splint ssDNA-ligating liner ssDNA monomeric complexes where the linear splint sequence comprises at least 10 or at least 12 nucleotides, each of which hybridize with one end of a ligating linear ssDNA.
Dependent claim 9 encompasses a genus of splint ssDNA-ligating liner ssDNA monomeric complexes wherein the hairpin sequences on each side of the linear splint sequence differ from one another.
Dependent claim 10 encompasses a genus of splint ssDNA-ligating liner ssDNA monomeric complexes comprising specific hairpin sequences of the genus of splint ssDNA.
Dependent claim 11 encompasses a genus of splint ssDNA-ligating liner ssDNA monomeric complexes comprising a genus of ligating liner ssDNA at least about 16 nucleotides in length.
Dependent claim 12 encompasses a genus of methods of producing a ssDNA circle comprising incubating the genus of splint ssDNA of claim 1 and a genus of ligating linear ssDNA at least about 16 nucleotides in length under hybridizing conditions and a splint ssDNA-ligating liner ssDNA monomeric complex is formed and incubating the splint ssDNA-ligating liner ssDNA monomeric complex with a DNA ligase under ligating conditions in order form a ssDNA circle. As stated supra, specification only discloses a fixed number of species of splint ssDNA.
Dependent claim 13 encompasses a DNA ligase for use in the method of claim 12.
Dependent claim 14 encompasses a concentration of ligating linear ssDNA of about 100 µM for use in the method of claim 12.
Dependent claims 15 and 16 encompasses a genus of ratios of ligating linear ssDNA to splint ssDNA for use in the method of claim 12.
Dependent claims 17-19 encompass a genus of hybridizing and ligating conditions and reagents for use in the method of claim 12.
Dependent claims 20 and 21 encompass kits comprising the splint ssDNA of claim 1 and instructions and reagents for producing a ssDNA circle.
The factors to be considered in determining whether undue experimentation is required are summarized In re Wands 858 F.2d 731, 8 USPQ2nd 1400 (Fed. Cir, 1988). The Court in Wands states: “Enablement is not precluded by the necessity for some 'experimentation.'” Clearly, enablement of a claimed invention cannot be predicated on the basis of quantity of experimentation required to make or use the invention. “Whether undue experimentation is needed is not a single simple factual determination, but rather is a conclusion reached by weighing many factual considerations.” (Wands, 8 USPQ2d 1404). The factors to be considered in determining whether undue experimentation is required include: (1) the quantity of experimentation necessary, (2) the amount or direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skill of those in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claims. While all of these factors are considered, a sufficient amount for a prima facie case is discussed below.
The office has analyzed the specification in direct accordance to the factors outlined in In re Wands. MPEP 2164.04 states: "[W]hile the analysis and conclusion of a lack of enablement are based on factors discussed in MPEP 2164.01(a) and the evidence as whole, it is not necessary to discuss each factor in written enablement rejection." These factors will be analyzed, in turn, to demonstrate that one of ordinary skill in the art would have had to perform "undue experimentation" to make and/or use the invention and therefore, Applicant's claims are not enabled commensurate with the scope of the invention.
ACTUAL REDUCTION TO PRACTICE
The specification does not provide guidance for or a working example for use of splint ssDNA that have linear splint sequences shorter than 10 contiguous nucleotides and having hairpin sequences other than those which differ from one another and have SEQ ID NO: 1 and SEQ NO: 2 in formation of a monomeric complex with ligating linear ssDNA shorter than 16 nucleotides in a method of making a ssDNA circle with a DNA ligase other than T4 DNA ligase and at ligating linear DNA concentrations beyond 100µM. The absence of working examples directed to use of splint ssDNA sequence beyond the disclosed embodiments necessitates further experimentation. Additionally, no working examples were provided that utilize splint ssDNA having linear splint sequences having less than 10 contiguous or more than 12 contiguous nucleotides for hybridizing with each end of a ligating linear ssDNA, there are no working examples of formation of circular ssDNA using ligating linear ssDNA having less than 16 nucleotides, and no working examples of the method using a DNA ligase other than T4 DNA ligase and at a ligating linear ssDNA concentration above 100µM. Therefore, the specification does not provide sufficient guidance on how to make and use the full genus of splint ssDNA in formation of monomeric complexes with ligating linear ssDNA in a method of making ssDNA circles.
With regard to claim 1 encompassing a genus of splint single-stranded DNA (ssDNA) comprising a linear splint sequence and hairpin sequences on each side of the linear splint sequence, the specification discloses embodiments of splint ssDNA sequences comprising the hairpin loop sequences of SEQ ID NOs: 1 and 2, and which have single stranded linear splint sequences which allow for binding of a ligating DNA sequence via 4 (H4), 5 (H5), or 6 (H6) nucleotides on either end of the ligating DNA sequence (See Para. [0033], Fig. 2).
Regarding claims 2 and 3 encompassing a genus of splint ssDNA where the linear splint sequence comprises at least 10 or at least 12 nucleotides, each of which hybridize with one end of a ligating linear ssDNA, as stated supra the specification discloses the species of H5 comprising 10 contiguous nucleotides for binding a linear DNA and the species of H6 comprising 12 contiguous nucleotides for binding a linear DNA (See Para. [0033], Fig. 2).
Regarding claims 4 and 5 encompassing a genus of splint ssDNA wherein the hairpin sequences on each side of the linear splint sequence differ from one another, the specification discloses splint ssDNA comprising hairpins wherein one hairpin comprises SEQ ID NO: 1 and the other hairpin comprising SEQ ID NO: 2.
Regarding claim 6 encompassing a genus of splint ssDNA-ligating liner ssDNA monomeric complexes comprising the splint ssDNA of independent claim 1, as stated supra, the specification discloses embodiments of splint ssDNA sequences comprising the hairpin loop sequences of SEQ ID NOs: 1 and 2, and which have single stranded linear splint sequences which allow for binding of a ligating DNA sequence via 4 (H4), 5 (H5), or 6 (H6) nucleotides on either end of the ligating DNA sequence (See Para. [0033], Fig. 2). Further, regarding the ligating linear ssDNA, the specification discloses embodiments of ligating linear ssDNA comprising 12, 14, 16, 18, 20, 30, and 40 nucleotides (See Para. [0033], Fig. 2, Example 4) and monomeric complexes derived from using the splint ssDNA of H6 with ligating linear ssDNA of all lengths (Fig. 2), the splint ssDNA of H5 and H4 with ligating linear ssDNA having 20 nucleotides (Examples 2 and 3), and the splint ssDNA of H5 with ligating linear ssDNA having 16 nucleotides (Fig. 4).
Regarding claims 7 and 8 encompassing a genus of splint ssDNA-ligating liner ssDNA monomeric complexes where the linear splint sequence comprises at least 10 or at least 12 nucleotides, each of which hybridize with one end of a ligating linear ssDNA, as stated supra, the specification discloses the species of H5 comprising 10 contiguous nucleotides for binding a linear DNA and the species of H6 comprising 12 contiguous nucleotides for binding a linear DNA (See Para. [0033], Fig. 2) and monomeric complexes derived from using the splint ssDNA of H6 with ligating linear ssDNA having 12, 14, 16, 18, 20, 30, and 40 nucleotides (Fig. 2) and splint ssDNA of H5 with ligating linear ssDNA having 20 nucleotides (Examples 2 and 3) and 16 nucleotides (Fig. 4).
Regarding claims 9 and 10 encompassing a genus of splint ssDNA-ligating liner ssDNA monomeric complexes wherein the hairpin sequences on each side of the linear splint sequence differ from one another, as state supra the specification discloses splint ssDNA comprising hairpins wherein one hairpin comprises SEQ ID NO: 1 and the other hairpin comprising SEQ ID NO: 2 which are used for all embodiments of ligating linear ssDNA and monomeric complexes.
Regarding claim 11 encompassing a genus of splint ssDNA-ligating liner ssDNA monomeric complexes comprising a genus of ligating liner ssDNA at least about 16 nucleotides in length, as stated supra, specification discloses embodiments of ligating linear ssDNA comprising 12, 14, 16, 18, 20, 30, and 40 nucleotides (See Para. [0033], Fig. 2, Example 4) and monomeric complexes derived from using the splint ssDNA of H6 with ligating linear ssDNA of all lengths (Fig. 2), the splint ssDNA of H5 and H4 with ligating linear ssDNA having 20 nucleotides (Examples 2 and 3), and the splint ssDNA of H5 with ligating linear ssDNA having 16 nucleotides (Fig. 4).
Regarding claim 12 encompassing a genus of methods of producing a ssDNA circle comprising incubating the genus of splint ssDNA of claim 1 and a genus of ligating linear ssDNA at least about 16 nucleotides in length under hybridizing conditions and a splint ssDNA-ligating liner ssDNA monomeric complex is formed and incubating the splint ssDNA-ligating liner ssDNA monomeric complex with a DNA ligase under ligating conditions in order form a ssDNA circle, as stated supra, specification only discloses a fixed number of species of splint ssDNA, a fixed number of ligating linear ssDNA, and a fixed number of splint ssDNA-ligating linear ssDNA monomeric complexes. The specification discloses only use of a T4 DNA ligase and discloses that the splint ssDNA of H6 was not able to produce ssDNA circles for ligating linear ssDNA having less than 16 nucleotides, the splint ssDNA of H4 does not reliably produce ssDNA circles using ligating linear ssDNA having 20 nucleotides (see Examples 2 and 3), and that although the splint ssDNA of H5 is able to produce ssDNA circles using ligating linear ssDNA having 20 nucleotides, production of ssDNA circles using ligating linear ssDNA having 16 nucleotides is heavily dependent on the concentration of DNA (Fig. 4).
Regarding claim 13 encompassing a DNA ligase for use in the method of claim 12, the specification discloses use of T4 DNA ligase (Para. [0035]).
Regarding claim 14 encompassing a concentration of ligating linear ssDNA of about 100 µM for use in the method of claim 12, the specification discloses use of ligating linear ssDNA concentrations between 1-100 µM (i.e., 1, 5, 10, 20, 50, and 100 µM) with the splint ssDNA of H6 (Fig. 3) and H5 and ligating linear ssDNA having 20 nucleotides (Example 3) which are able to produce ssDNA circles. However, when using the splint ssDNA of H5 and ligating linear ssDNA having 16 nucleotides, production of ssDNA circles was only reliable at ligating linear ssDNA concentrations of 10 µM and above (Fig. 4).
Regarding claims 15 and 16 encompassing a genus of ratios of ligating linear ssDNA to splint ssDNA for use in the method of claim 12, the specification discloses use of a 1:1.2 ratio of ligating linear ssDNA to splint ssDNA (Para. [0035]).
Regarding claims 17-19 encompassing a genus of hybridizing and ligating conditions and reagents for use in the method of claim 12, the specification discloses annealing from 95-22 °C in ligation buffer but does not disclose an amount of time, addition of T4 DNA ligase at 100 U/µg per ligating linear ssDNA, and ligation at room temperature (22-25 °C) for 16 hours. (Para. [0035]).
Regarding claims 20 and 21 encompassing kits comprising the splint ssDNA of claim 1 and instructions and reagents for producing a ssDNA circle, as stated supra, the specification discloses a limited number of splint ssDNA which can be used with a limited number of ligating linear ssDNA in order to form ssDNA circles. Further, the specification only discloses use of T4 DNA ligase which is added to a ligation buffer which comprises ATP after annealing (Para. [0035]).
STATE OF THE ART & QUANTITY OF EXPERIMENTATION
The method of making the claimed invention and the method of using the claimed invention to produce circular ssDNA is not well established. Although use of splint sequences without hairpins for production of circular DNA was known is routine and conventional, one of skill in the art would neither expect nor predict the ability of a splint ssDNA comprising a linear splint sequence and a hairpin sequence on each side which can form monomeric complexes with a ligating linear ssDNA and form a circular ssDNA according to the genus of splint ssDNA as broadly as claimed.
The prior art is silent as to splint ssDNA comprising hairpin sequences on each side and their use in production of circular ssDNA. However, Applicant’s instant specification and the contemporaneous art of Paluzzi et al. (2023, Near‐Quantitative Preparation of Short Single‐Stranded DNA Circles. Angewandte Chemie Intl. Ed., 62(16), e202218443) indicates that the ability of a splint ssDNA sequence having a linear splint sequence and hairpin sequences on each end to complex with a ligating linear ssDNA and form a circular ssDNA when incubated with a T4 DNA ligase is unpredictable and is based on several factors including the length of the linear splint sequence, the length of the ligating linear ssDNA to be cyclized, hairpin structure, and the specific conditions under which the method of producing circular ssDNA is performed.
As an initial matter, Applicant’s splint ssDNA comprising a linear splint sequence and a hairpin sequence on each side of the linear splint sequence appears to require a very specific structural conformation in order for proper functional ability of making circular ssDNA (See Fig. 1). The splint ssDNA sequence appears to require a single stranded linear splint sequence which remains in an open conformation and includes double stranded hairpins at each end which form “borders”. The splint ssDNA must be arranged in a specific conformation such that the linear splint sequence is located within a cavity or depression between the double stranded hairpin borders. Applicant’s exemplary splint ssDNA sequences all comprise hairpin sequences which differ from the hairpin sequence on the other end (i.e., SEQ ID NO: 1 and 2) and have single stranded linear splint sequences having at least enough nucleotides for a T4 DNA ligase to recognize upon the splint ssDNA binding with a ligating linear ssDNA. The instant specification indicates that, typically, T4 DNA ligase requires at least 12 base pairs (bp) on each side of the nick to be ligated due to side of the T4 DNA ligase (Para. [0024]) and Applicant’s exemplary embodiments all include sequences which allow for equal numbers of bp binding to the linear splint sequence by each side of the ligating linear ssDNA (See Figs. 1 and 2). Applicant’s disclosure suggests that presence of the hairpins increases the stability of the splint ssDNA-ligating linear DNA complex which allows for splint sequences “significantly shorter than 6 bps” which still facilitate DNA ligation and also that the hairpins allow intermolecular repulsion which results in increased intramolecular cyclization (Para. [0026]). Thus, the instantly claimed splint ssDNA requires a specific structural configuration for functional ability.
Regarding the length of the linear splint sequence, the instant specification and Paluzzi et al. disclose that splint ssDNA sequences comprising linear splint sequences which had fewer than 10 contiguous nucleotides (i.e., H4) were not effective at forming stable monomeric complexes with ligating linear ssDNA having 20 nucleotides (Specification Example 2; Paluzzi et al. Pg. 3, bridging para. and right col., full para.) and were therefore not able to reliably form circular ssDNA when incubated with a T4 DNA ligase, instead forming 5’-adenylpyrophosphorylated intermediate products which are indicative of inefficient cyclization (Para. [0045]). Therefore, based on the instant specification and Applicant’s contemporaneous art, it appears that the ability of splint ssDNA to form monomeric complexes with a ligating linear ssDNA and generate circular ssDNA in the presence of a T4 DNA ligase is unpredictable and requires binding of at least 5 bps on either side of the linear splint sequence with the ends of a ligating linear ssDNA.
Regarding the length of the ligating linear ssDNA to be cyclized, the instant specification and Paluzzi et al. discloses that ligating linear ssDNA strands shorter than 16 nucleotides were not able to be cyclized and instead resulted in formation of longer DNA strands first which were cyclized (See specification Example 4; Paluzzi et al. Pg. 4, left col., middle para.). Further, when splint ssDNA which bind five bp on either side of the ligating linear ssDNA (i.e., H5) were combined with ligating linear ssDNA having 16 nucleotides (i.e., L16), the ability to predictably cyclize ssDNA was dependent on the concentration of ligating linear ssDNA (see specification Fig. 4; Paluzzi et al. Fig. 4b). Thus, the ability of a splint ssDNA to form a monomeric complex with a ligating linear ssDNA and form circular ssDNA in the presence of T4 DNA ligase is also unpredictable and depends on the length of the ligating linear ssDNA as well as the specific combination of splint ssDNA and ligating linear ssDNA.
Regarding the hairpin sequences, Applicant’s exemplary hairpins have a four bp stem, the minimal four nucleotide loop, and sequences which differ from each other. Since, based on Applicant’s specification, presence of the hairpins results repulsive forces between hairpins which favor intramolecular interaction, a skilled artisan would recognize that changes to the hairpin sequence could also change hairpin conformation and resultant repulsive forces. Applicant’s contemporaneous art indicates that reductions of the hairpin loop size in H6 splint ssDNA sequences when combined with ligating linear ssDNA having 20 nucleotides result in increased formation of longer DNA strands which are then cyclized by a T4 DNA ligase (Paluzzi et al., Pg. 3, left col., 1st para and Supp. Fig 5a-c), indicating that functional ability to form monomeric complexes and form circular ssDNA is dependent upon hairpin structure and therefore would not be predictable for any hairpin sequence. Further, Applicant’s exemplary embodiments utilize hairpin sequences which differ from one another which a skilled artisan would recognize contributes to conformational structure. As detailed supra, it appears that the splint ssDNA requires a specific conformational structure which is required for functional effect. Applicant has provided no guidance as to how the hairpin sequences were generated or which factors must be considered in order to create the required repulsive forces between hairpins which favor intramolecular interactions. If, for example, Applicant’s claims encompass the hairpin sequences on either end of the splint ssDNA were the same, it is possible that instead of forming hairpins, wherein the splint ssDNA could form a circular-type structure based on complementary pairing between the opposite ends of the splint ssDNA sequence which would result in a non-functional splint ssDNA.
Regarding the specific conditions under which the method of producing circular ssDNA is performed, based on the instant specification, although concentration of ligating linear DNA does not affect the circular ssDNA formation when using an H6 splint ssDNA and ligating linear ssDNA having 20 nucleotides (See Fig. 3), formation of circular DNA is dependent upon ligating linear ssDNA concentration when using a shorter H5 splint ssDNA and a shorter ligating linear ssDNA having 16 nucleotides (Fig. 4). Paluzzi et al. discloses that, when using a combination of the H6 splint and ligating linear ssDNA having 20 nucleotides, DNA concentrations of 250 µM and above resulted in increased production and cyclizing of longer DNA strands (Pg. 4, right col., 1st para. and Supp. Fig 9a-b). Paluzzi et al. ultimately concludes that the method of making ssDNA circles is effective for ssDNA as short as 16 nucleotides at concentrations up to 100 µM (Pg. 4, right col, last para.). Therefore, the functional ability of a splint ssDNA to form splint ssDNA-ligating liner ssDNA monomeric complexes and subsequently form ssDNA circles is not predictable for all splint ssDNA and ligating linear ssDNA at all concentrations.
In fact, as detailed supra, the state of the art teaches that use of methods of making circular ssDNA using splint DNA is not a highly successful technique or has highly variable results. Additionally, the prior art of Cui et al. (2018, Terminal hairpin in oligonucleotide dominantly prioritizes intramolecular cyclization by T4 ligase over intermolecular polymerization: an exclusive methodology for producing ssDNA rings. Nucleic Acids Res., 46(22), e132-e132) discloses that methods of synthesizing circular DNA “have not been sufficiently established” and generally are formed by use of T4 DNA ligase and a short oligonucleotide splint which is complementary to the 5’ and 3’ ends of the linear DNA to be cyclized (Pg. 1, right col., 1st para.). Cui et al. discloses that large scale production of circular DNA is hampered by formation of polymerized linear DNA byproducts and that in order to select for desired circular DNA, techniques such as low concentration of linear DNA, slow addition of linear DNA, and diluted T4 DNA ligase have been used but these techniques are not compatible with rapid or large scale production (Pg. 1-2, bridging para.; see also Fig. 1). Therefore, Cui et al. discloses that use of splint DNA in order to generate circular DNA from linear ssDNA without generating undesirable polymerized DNA byproducts is unpredictable and highly variable.
Consequently, there is ample reason to conclude that there would be a high degree of unpredictability in a method of using the instantly claimed splint ssDNA in a method of making ssDNA circles. Since the prior art at the effective filing date of the present application did not provide guidance for use of the full genus of splint ssDNA having the instantly disclosed structure for formation of monomeric complexes with any ligating linear ssDNA in a method of making ssDNA circles, it is incumbent upon the instant specification to do so.
The physiological art is recognized as unpredictable (MPEP 2164.03). As set forth in In re Fisher, 166 USPQ 18 (CCPA 1970), compliance with 35 USC 112(a) requires: “That scope of claims must bear a reasonable correlation to scope of enablement provided by specification to persons of ordinary skill in the art; in cases involving predictable factors, such as mechanical or electrical elements, a single embodiment provides broad enablement in the sense that, once imagined, other embodiments can be made without difficulty and their performance characteristics predicted by resort to known scientific laws; in cases involving unpredictable factors, such as most chemical reactions and physiological activity, scope of enablement varies inversely with degree of unpredictability of factors involved.” Moreover, the courts have also stated that reasonable correlation must exist between scope of exclusive right to patent application and scope of enablement set forth in the patent application (27 USPQ2d 1662 Ex parte Maize!.). In view of the foregoing, due to the lack of sufficient guidance provided by the specification regarding the issues set forth above, the state of the relevant art, and the breadth of the claims, it would have required undue experimentation for one skilled in the art to make and use the instant broadly claimed invention.
CONCLUSION
In conclusion, since the art teaches that success of said method is prone to influence by multiple factors, and is highly unpredictable with respect to the ability to make ssDNA circles using any splint ssDNA, and ligating linear ssDNA at any concentration, and the specification does not provide ample guidance with respect to achieving the unexpected results, one would be burdened with undue experimentation to use the claimed invention in a method of making ssDNA circles.
In conclusion, given the breadth of the claims and the limited scope of the specification, an undue quantity of experimentation is required to make and use the invention beyond the scope of splint ssDNA having a linear splint sequence comprising at least 10 or at least 12 contiguous nucleotides, half of which can hybridize to each end of a ligating linear ssDNA, and having hairpin sequences which differ from one another on each end of the splint the hairpins having SEQ ID NO: 1 and SEQ NO: 2, which can form monomeric complexes with ligating linear ssDNA having at least 16 nucleotides and which can be used to generate ssDNA circles in the presence of a T4 DNA ligase at ligating linear ssDNA concentrations up to 100 µM.
Conclusion
No claims are allowed.
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/ERIN V PAULUS/Examiner, Art Unit 1631
/ARTHUR S LEONARD/Examiner, Art Unit 1631
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