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 .
DETAILED ACTION
Pursuant to a preliminary amendment filed May 20, 2023 claims 1-8, 16-19 and 22-37 are currently pending in the instant application.
Response to Election/Restriction
Applicant's election of Group IV with traverse, claims 22-35, directed to a method of preparing an RNA-sequence library; and Applicant’s election of Species with traverse as follows:
Species (A): wherein the structure of a hairpin oligonucleotide comprises: 5'-Phos-rA CT-X-AGA TCG GAA GAG CAC ACG AT (SEQ ID NO: 86); LT-AGA CGT GTG CTC TTC CGA TCT (SEQ ID NO: 87) -Z-AG rU-3 '-Phos, wherein X is a barcode of at least 3, 4, 5 or 6 nucleotides, LT is an affinity moiety tagged-Thymine nucleotide, and Z is a sequence of nucleotides that is the reverse complement of the barcode sequence (claim 22);
Species (B): not elected;
Species (C): not elected;
Species (D): not elected;
Species (E): the method of claim 22 further comprising phosphating the 3’-phsphate after ligation and oxidizing 3’ terminal nucleotides (claim 24); and
Species (F): the method of claim 27 further comprising digesting the RNA sequence after reverse transcription (claim 30), in the reply filed May 20, 2026 is acknowledged.
Response to Arguments
Applicant’s arguments filed May 20, 2026 have been fully considered but they are not persuasive. Applicants essentially assert that: (a) the Office Action has focused on differences in the claims and has not sufficiently considered the similarities with regard to ease of searching the claims and species; and search results for the claimed subject matter of any one of Groups I-IV and any one of the species will likely overlap with search results for the claimed subject matter of the other Groups and other species, such that there is not so serious as to require restriction (Applicant Remarks, pg. 10, second full paragraph).
Regarding (a), Applicant did not distinctly and specifically point out the supposed errors in the Examiner’s action as required by 37 CFR 1.111(b). Additionally, as noted in the Office Action mailed April 1, 2026, Groups I-IV lack unity of invention. Moreover, the different Species of hairpin oligonucleotides further comprise an additional component and/or process step, such that each Species recites a completely different structure and/or a different additional step. Thus, the different species have no substantial common core structure, and do not constitute a special technical feature as defined by PCT Rule 13.2, particularly since each of the species does not share a substantially common structural feature, which is the requirement for unity of invention. Therefore, the species therefore lack unity of invention a priori. The restriction is proper.
Claims 1-8, 16-19, 36 and 37 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a non-elected invention, there being no allowable generic or linking claim.
Claims 23, 26-30 and 33-35 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a non-elected species, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on May 20, 2026.
The restriction requirement is still deemed proper and is therefore made FINAL.
The claims will be examined insofar as they read on the elected species.
Therefore, claims 22, 24, 25, 31 and 32 are under consideration to which the following grounds of rejection are applicable.
Priority
The present application filed May 5, 2023 is a 35 U.S.C. 371 national stage filing of International Application PCT/US2021/058258, filed November 5, 2021, which claims the benefit of US Provisional Patent Application 63110605, filed May November 6, 2020.
Claim Objections/Rejections
Claim Objections
Claims 22 and 31 are objected to because of the following informalities: Claims 22 and 31 recite the terms such as "PCR”, “tRNAs” and “piRNAs”, where an abbreviation should be spelled out in the first encounter of the claims.
Appropriate correction is required.
Specification Objection
This disclosure is objected to because it contains an embedded hyperlink and/or other form of
Browser-executable code (e.g., as-filed Specification, paragraphs [0135]; [0136]; and [0137]). Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; it is noted that this can be achieved by amending the hyperlink(s) to remove the web-link and/or http:// recitations. See MPEP § 608.01.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(B) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 22, 24, 25, 31 and 32 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which applicant regards as the invention.
Claim 22 is indefinite for the recitation of the term “the oligonucleotide” such as recited in claim 22, line 3. There is insufficient antecedent basis for the term “the oligonucleotide” in the claim because claim 22, line 2 recites the term “a hairpin oligonucleotide”. The Examiner suggests that Applicant amend the claim to recite, for example, “the hairpin oligonucleotide comprising a 3’-terminal nucleotide.”
Claim 22 is indefinite for the recitation of the term “the sugar component” such as recited in claim 22, lines 3-4. There is insufficient antecedent basis for the term “the sugar component” in the claim.
Claim 24 is indefinite for the recitation of the term “after ligation and oxidizing 3’-terminal nucleotides comprising a 2’,3’-diol” such as recited in claim 24, lines 2-3 because claim 24 depends from instant claim 22, wherein claim 22 does not recite a step of oxidizing the 3’-terminal nucleotides; and the 3’-terminal nucleotides are not recited to comprise a 2’,3’-diol and, thus, the metes and bounds of the claim cannot be determined.
Claim 25 is indefinite for the recitation of the term “demethylating Watson-Crick face methylations on nucleotides of the RNA sequence” such as recited in claim 25, lines 1-2 because claim 25 depends from instant claims 22 and 24, wherein claims 22 and 24 do not recite Watson-Crick face methylations on nucleotides of the RNA sequence and, thus, the metes and bounds of the claim cannot be determined.
Claim 32 is indefinite for the recitation of the term “wherein the method comprises a multiplex method” such as recited in claim 32, lines 1-2 because claim 32 depends from claim 22, wherein claim 22 already recites what the method comprises (e.g., ligating, reverse-transcribing, and amplifying), such that claim 32 cannot recite that the method comprises something different. Moreover, claim 32 does not recite steps that comprise a plurality of RNA sequences, such that it is unclear how the method of preparing a single RNA sequence is a “multiplexed” method and, thus, the metes and bounds of the claim cannot be determined.
Claim 31 is indefinite insofar as it ultimately depends from instant claim 22.
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 24, 25 and 32 are rejected under 35 U.S.C. 112(d) as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 24 recites (in part): “further comprising dephosphorylating the 3’-phosphate after ligation and oxidizing 3’-terminal nucleotides comprising a 2’,3’-diol” such as recited in claim 24, lines 2-3 because claim 24 depends from instant claim 22, wherein claim 22 does not recite a step of oxidizing the 3’-terminal nucleotides; and the 3’-terminal nucleotides are not recited to comprise a 2’,3’-diol. Thus, claim 24 is an improper dependent claim for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 25 recites (in part): “further comprising demethylating Watson-Crick face methylations on nucleotides of the RNA sequence” such as recited in claim 25, lines 1-2 because claim 25 depends from instant claims 22 and 24, wherein claims 22 and 24 do not recite Watson-Crick face methylations on nucleotides of the RNA sequence. Thus, claim 25 is an improper dependent claim for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 32 recites (in part): “wherein the method comprises a multiplex method” such as recited in claim 32, lines 1-2 because claim 32 depends from instant claim 22, wherein claim 22 already recites what the method comprises (e.g., ligating, reverse-transcribing, and amplifying). Moreover, claim 22 does not recite a method comprising a plurality of RNA sequences, the pooling of RNA sequence, etc., such that the preparation of a single RNA sequence is not a multiplexed method. Thus, claim 32 is an improper dependent claim for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Applicant may cancel the claim, amend the claim to place the claim in proper dependent form, rewrite the claim in independent form, or present a sufficient showing that the dependent claim complies with the statutory requirements.
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 –
(b) the invention was patented or described in a printed publication in this or a foreign country or in public use or on sale in this country, more than one year prior to the date of application for patent in the United States.
Claims 22 are rejected under pre-AIA 35 U.S.C. 102(b) as anticipated by Erber et al. (hereinafter “Erber”) (RNA Biology, epub 2019, 17(1), 23-32) as evidenced by Cozen et al. (hereinafter “Cozen”) (Nature Methods, 2015, 12(9), 879-886).
Regarding claim 22, Erber teaches a schematic workflow of the LOTTE-Seq procedure, comprising: (A) a DNA hairpin-oligonucleotide (green) with a 3ʹ-TGGN overhang hybridizes to the tRNA 3ʹ-CCA end (tRNA in blue), wherein the T4 DNA ligase fuses the 3ʹ-end of the CCA terminus to the phosphorylated 5ʹ end of the adapter (ligating a hairpin oligo to an RNA sequence, 22a); (B) the tRNA is reverse transcribed with parts of the hairpin oligonucleotide serving as primer binding site (reverse transcribing the RNA as cDNA, 22b), such that the secondary structure and modified bases can lead to premature RT stops and partial cDNA (yellow); (C) using T4 RNA ligase 1, a 5ʹ-phosphorylated and 3ʹ-blocked second adapter (red) is fused to the 3ʹ-end of the cDNA, leading to the generation of cDNA product with adapters on both sides (red and green); (D) the product is PCR-amplified with indexed primers binding to the adapter overhang sequences (PCR amplification, claim 22c); (E) the cDNA library consisting of full-length as well as prematurely terminated tRNA sequences is analyzed by high-throughput sequencing (a multiplex method; and comprising tRNA, claims 31 and 32) (pg. 25; Figure 1).
Regarding claim 24 (in part), Erber teaches that the tRNA preparation was incubated with snake venom phosphodiesterase (SVPD I) to remove the 3’-CCA ends, wherein the enzyme catalyzes the hydrolysis of 3’-nucleotide phosphates and can easily be adjusted to only remove three nucleotides of the tRNA 3’-end (interpreted as dephosphorylating the 3’-phosphate, claim 24) (pg. 26, col 1, last partial paragraph).
Regarding claim 25, Erber teaches the analysis of the tRNA sequence reads identified a series of stop signals resulting from reverse transcription termination at methylated base positions, wherein ARM-Seq; as well as, DM-Seq compare untreated with enzymatically demethylated samples to identify certain base methylation positions in transcriptome data; and as described LOTTE-Seq reveals strong RT stop signals at these positions (Figure S3) (interpreted as demethylating nucleotides, claim 25) (pg. 28, col 1, last partial paragraph; and col 2, first partial paragraph, lines 1-6), where it is known that ARM-seq to provide sensitive and specific detection of methyl-modified RNAs using RNA-seq, wherein RNA is treated with a dealkylating enzyme, Escherichia coli AlkB, before reverse transcription in library preparation as evidenced by Cozen (pg. 897, col 2, first full paragraph, lines 1-4).
Regarding claim 31, Erber teaches investigating the specificity of the hairpin adapter ligation on tRNAs isolated from total RNA (interpreted as total RNA, and tRNA, claim 31) (pg. 26, col 1, last partial paragraph, lines 1-2).
Regarding claim 32, Erber teaches LOTTE-Seq (long hairpin oligonucleotide based tRNA high-throughput sequencing), specific selection of tRNAs with 3’-CCA end for high-throughput sequencing; and that the LOTTE-Seq strategy renders the approach a powerful and universal tool to analyze the tRNA pool of cells and organisms under different conditions in health and disease (interpreted as a multiplex method, claim 32) (Title; and Abstract, last 3 lines). Erber teaches that high-throughput analysis of the libraries was done as single end run (150 nt) on a MiSeq System (Illumina) with a custom primer designed for Illumina MiSeq analysis (5ʹ-CACTGTCGGTACCGAGCTTGCA TGGAGTCCTA-3ʹ) (interpreted as a multiplex method, claim 32) (pg. 30, col 1, fourth full paragraph). Erber teaches a single gel extraction step in the whole LOTTE-Seq procedure that separates the RT-products from unused hairpin adapter that could interfere within the second ligation step (interpreted as a multiplex method, claim 32) (pg. 24, col 2, first partial paragraph, lines 19-22).
Erber does not specifically exemplify oxidation with periodate (claim 24, in part).
Erber meets all the limitations of the claims and, therefore, anticipates the claimed invention.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and
103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis for
the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102 of this title, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negatived by the manner in which the invention was made.
Claims 22, 24, 25, 31 and 32 are rejected under 35 U.S.C. 103 as being unpatentable over Erber et al. (hereinafter “Erber”) (RNA Biology, epub 2019, 17(1), 23-32) in view of Molly Evans (hereinafter “Evans”) (Dissertation; University of Chicago, Chicago, IL, 2017, 1-117) as evidenced by Cozen et al. (hereinafter “Cozen”) (Nature Methods, 2015, 12(9), 879-886).
The teachings of Erber as applied to claims 22, 24, 25, 31 and 32 are described supra.
Erber does not specifically exemplify oxidation with periodate (claim 24, in part).
Regarding claim 24 (in part), Evans teaches in Figure 1.2, examples of cDNA library preparation for RNA high-throughput sequencing, wherein: (A) RNA is first fragmented; sequential ligation of a 3' and 5' adapter is used to add the primer sequences required for Illumina sequencing; and the adapters are used for first and second strand cDNA synthesis and PCR amplification to create the sequencing library; and (B) RNA fragments are randomly primed (interpreted as library preparation, claim 22) (pg. 4, Figure 1.2). Evans teaches in Figure 2.1, charged DM-tRNA-Seq method, where in (A) and (B), periodate oxidation and β-elimination can differentiate between charged and uncharged tRNAs prior to sequencing, wherein periodate selectively oxidizes the 3' end of uncharged tRNA (A), while the 3' end of charged tRNA is protected (B), such that treatment with high pH causes β-elimination of the oxidized nucleotide and deacylation of charged tRNA, such that after end repair with T4 PNK, charged tRNAs will end in –CCA while uncharged tRNAs will end in –CC (C); and using modified DM-tRNA-seq to determine charging fractions, wherein tRNA is first treated with demethylase to remove common tRNA modifications (m1G, m1A, m3 (C) that impair reverse transcription (interpreted as periodate oxidation, claim 24) (pg. 20, Figure 2.1). Figure 2.1 is shown below:
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Greyscale
Evans teaches that tRNA microarrays can be used to determine charging fraction, wherein periodate oxidation of the 3' end is specific to uncharged tRNAs, effectively inactivating the 3' end for any further enzymatic reaction, such that after deacylation and subsequent ligation to fluorescent probes, the level of tRNA charging can be ascertained; and by comparing untreated sample (total tRNA) to periodate treated sample (charged tRNA), the charged fraction of tRNA can be determined (pg. 18, second full paragraph, lines 1-5).
It is prima facie obvious to combine prior art elements according to known methods to yield predictable results; the court held that, "…a conclusion that a claim would have been obvious is that all the claimed elements were known in the prior art and one skilled in the art could have combined the elements as claimed by known methods with no change in their respective functions, and the combination would have yielded nothing more than predictable results to one of ordinary skill in the art. KSR International Co. v. Teleflex Inc., 550 U.S. ___, ___, 82 USPQ2d 1385, 1395 (2007); Sakraida v. AG Pro, Inc., 425 U.S. 273, 282, 189 USPQ 449, 453 (1976); Anderson’s-Black Rock, Inc. v. Pavement Salvage Co., 396 U.S. 57, 62-63, 163 USPQ 673, 675 (1969); Great Atlantic & P. Tea Co. v. Supermarket Equipment Corp., 340 U.S. 147, 152, 87 USPQ 303, 306 (1950)”. Therefore, in view of the benefits of selective oxidation of charged and uncharged tRNAs as exemplified by Evans, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of preparing an RNA-sequence library from 2-4 mg of total RNA using the LOTTE-Seq method including tRNA incubation with snake venom phosphodiesterase as disclosed by Erber to include one or more steps of the DM-tRNA-seq method including periodate oxidation as taught by Evans with a reasonable expectation of success in selecting the appropriate oxidative reagents and/or conditions for the selective oxidation of 3’-terminal nucleotides; in producing a dialdehyde; and/or in reverse transcribing tRNAs into cDNA, such that complete information with respect to nucleoside modifications is obtained.
Thus, in view of the foregoing, the claimed invention, as a whole, would have been obvious to one of ordinary skill in the art at the time the invention was made. Therefore, the claims are properly rejected under 35 USC §103(a) as obvious over the art.
Conclusion
Claims 22, 24, 25, 31 and 32 are rejected.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to AMY M BUNKER whose telephone number is (313) 446-4833. The examiner can normally be reached on Monday-Friday (6am-2:30pm).
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Heather Calamita can be reached on (571) 272-2876. The fax phone number for the organization where this application or proceeding is assigned is (571) 273-8300.
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/AMY M BUNKER/Primary Examiner, Art Unit 1684
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