DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on June 26, 2026 has been entered.
Status of Objections and Rejections
The rejection of claim(s) 2 and 12 is/are obviated by Applicant’s cancellation.
All rejections from the previous office action are withdrawn in view of Applicant’s amendment.
New grounds of rejection are necessitated by the amendments.
Claim Objections
Claim(s) 4, 14, 22, and 24 is/are objected to because of the following informalities:
Claim 4, line 3: “ribonucleic acids (ssRNA)” should be “ribonucleic acids (ssRNAs)”
Claim 14, line 2: “ribonucleic acids (ssRNA)” should be “ribonucleic acids (ssRNAs)”
Claim 22, lines 1-2: “the electrophoretic shift” should be “the electrophoretic mobility shifts”
Claim 24, line 1: “the electrophoretic shift” should be “the electrophoretic mobility shifts”
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claim(s) 1, 3-9, 11, 13-19, and 21-24 is/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 pre-AIA the applicant regards as the invention.
Claim 1 recites “single-stranded nucleic acids” in lines 9 and 10. It is suggested to be “the plurality of single-stranded nucleic acids” in both places.
Subsequent dependent claims 3-9 and 21-22 are rejected due to their dependencies on rejected base claim 1.
Claim 11 recites “single-stranded nucleic acids” in lines 13 and 14. It is suggested to be “the plurality of single-stranded nucleic acids” in both places.
Claim 11 recites “plurality of single-stranded nucleic acids” in line 16. It is suggested to be “the plurality of single-stranded nucleic acids” in both places.
Subsequent dependent claims 13-19 and 23-24 are rejected due to their dependencies on rejected base claim 11.
CLAIM INTERPRETATION
The recited “molecular beacons (MBs)” in the claims are interpreted as hairpin-shaped oligonucleotides (RNA or DNA) that contain an anti-sense hybridization sequence matched to a specific target sequence of nucleotides such as single-stranded RNA or DNA molecule (Specification, PGpub ¶50).
Claim Rejections - 35 USC § 102
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claim(s) 1, 3-5, 8, and 21-24 is/are rejected under 35 U.S.C. 103 as being unpatentable over Chandrasekaran (A.R. Chandasekaran, DNA Nanoswitch Barcodes for Multiplexed Biomarker Profiling, Nano Letters, 2021(21), pp. 469-75) with its Supplemental Info (attached with the journal publication), supported by Chandrasekaran 2019 (A.R. Chandasekaran, Cellular microRNA detection with miRacles: microRNA-activated conditional looping of engineered switches, Science Advances, 2019(5), eaau9443, pp. 1-7) as an evidence for claim 8.
Regarding claim 1, Chandrasekaran teaches a computer-implemented method for detecting one or more single-stranded target nucleic acids in a plurality of single-stranded nucleic acids (bridging para. of pp. 469-470: the use of nanoswitches to a multiplexed DNA barcode system that can be used to detect any combination of up to six different biomarkers; e.g., Fig. 2(b): six different gene fragments; Fig. 3(b): six gene fragments are single-stranded), the method comprising:
incubating molecular beacons (MBs) (Fig. 1: barcoded nanoswitches; Fig. 2(a): the scaffold DNA having intercalating dyes of its backbone oligos and upon the binding of target nucleic acid, the DNA nanoswitch turning from a linear “off” state to a looped “on” state, which can be resolved on an agarose gel; thus, the nanoswitch is deemed to be a molecular beacon) with the plurality of single-stranded nucleic acids (Fig. 3(b): six nanoswitch mixtures and six gene fragments), wherein the MBs are configured to generate a fluorescence signal when bound with a target sequence of the one or more single-stranded target nucleic acids (p. 469, col. 2, para. 2: the signal comes from the intercalation of thousands of dye molecules; Fig. 2(a): upon the binding with the target nucleic acid);
performing gel electrophoresis (p. 474, col. 1, para. 1: use gel electrophoresis for readout) by applying voltage to a gel comprising the incubated MBs and single-stranded nucleic acids (Supplemental, p. 4, last para.: characterization of barcode gels (multiplexing) were run at 55V), thereby obtaining electrophoretic mobility shifts of the incubated MBs and single-stranded nucleic acids (Fig. 3(b)-(d): each target nucleic acid triggers the formation of a specific loop, providing a unique signal; there is lack of cross-reactivity in nanoswitches designed for six different gene fragments, and thus the DNA nanoswitch barcodes detect all possible combinations of the six gene fragments; also see Fig. 2(b): the different locations of electrophoretic bands indicate the electrophoretic mobility shifts on the gel due to the turning from the linear “off” state to the looped “on” state when bound to the target nucleic acid); and
determining, using the electrophoretic mobility shifts of the incubated MBs and the plurality of single-stranded nucleic acids, a quantity of the target sequence (p. 474, col. 1, para. 2: our method provides direct detection without amplification, which makes absolute quantification more straightforward).
Regarding claim 3, Chandrasekaran teaches wherein the target sequence is a micro ribonucleic acid (miRNA) (p. 469, col. 2, para. 2: detection of microRNAs).
Regarding claim 4, Chandrasekaran teaches wherein the plurality of single-stranded nucleic acids comprise single-stranded ribonucleic acids (ssRNA) (Fig. 4(c): ssRNA; e.g., Fig. 5(a): microRNA 141 and microRNA 30c).
Regarding claim 5, Chandrasekaran teaches wherein the plurality of single-stranded nucleic acids comprise single-stranded deoxyribonucleic acids (ssDNAs) (Fig. 4(c): ssDNA).
Regarding claim 8, Chandrasekaran teaches wherein the gel used in electrophoresis is not stained, and wherein determining the electrophoretic shift further comprises measuring the fluorescent signal (p. 469, col. 2, para. 2: the nanoswitch changes conformation from a linear “off” state to a looped “on” state, providing a distinct signal which comes from the intercalation of thousands of dye molecules, e.g., GelRed; Examiner notes that GelRed dye is a fluorescent dye, and as evidenced by Chandrasekaran 2019, Fig. 4; p. 6, col. 2, para. 2: GelRed were added after incubation, and samples were run in an unstained gel).
Regarding claim 21, Chandrasekaran teaches wherein the quantity is a concentration of the target sequence (Fig. 2(c); p. 470, col. 2, para. 1: we performed sensitivity experiments with decreasing concentration of the DNA and found that the signal could be seen by eye at concentration as low as 50 fM).
Regarding claim 22, Chandrasekaran teaches wherein determining the electrophoretic shift comprises determining a quantity of a first target sequence and a quantity of a second target sequence in the plurality of single-stranded nucleic acids, wherein the second target sequence comprises one or more mutations relative to the first target sequence (Fig. 2(d); p. 471, col. 1, para. 1: the assay is highly specific, able to discriminate even a single nucleotide mismatch in the target sequence; e.g., CF gene targets that contained 1-3 mutations in Fig. 2(d)).
Claim(s) 6 is/are rejected under 35 U.S.C. 103 as being unpatentable over Chandrasekaran in view of Vu (US 2012/0046191).
Regarding claim 6, Chandrasekaran discloses all limitations of claim 1. Chandrasekaran further discloses the nanoswitch barcodes detect all possible combinations of antidig, RNA sequence, DNA sequence, and streptavidin (Fig. 4(c)-(e)). Chandrasekaran does not discloses the method or the system further comprising conjugating single-stranded nucleic acids of the plurality of single-stranded nucleic acids with streptavidin beads.
However, Vu teaches a kit for the separation and detection of a target biomolecule of interest using nanoparticle probes (¶144). The nanoparticle probe includes a specific binding agent that specifically binds the target biomolecule of interest, using a linker, e.g., streptavidin, to link the detectable nanoparticle and the specific binding agent (¶145). Thus, Vu teaches the conjugation between the target biomolecule and the specific binding agent linked to streptavidin nanoparticles.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified Chandrasekaran by incorporating streptavidin nanoparticles for separation and detection of a target biomolecule as taught by Vu because use of nanoparticle probes is a known standard procedure (¶133) so that streptavidin nanoparticles would provide the support to the binding agent for binding the target biomolecule for detection of target sequence of nucleic acids. Applying a known technique to a known method ready for improvement to yield predictable results is prima facie obvious. MPEP 2141(III)(D).
Claim(s) 7, 11, 13-15, 17-18, and 23-24 is/are rejected under 35 U.S.C. 103 as being unpatentable over Chandrasekaran in view of Li (WO 2018/227426), supported by Chandrasekaran 2019 as an evidence for claim 18.
Regarding claim 7, Chandrasekaran discloses all limitations of claim 1. Chandrasekaran further discloses the microRNA biomarkers are in blood, but fails to teach prior to incubation: obtaining blood of a patient; and isolating ssRNA from red blood cells (RBCs) of the blood of the patient.
However, Li teaches the sample is a target nucleic acid, e.g., a biological sample including blood, which is obtained from a subject (¶296). The blood may be components of blood, e.g., white blood cells, red blood cells, platelets (¶296). The example of nucleic acids include isolated RNA of any sequence (¶106).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified Chandrasekaran by incorporating steps of obtaining blood of a patient and isolating the target nucleic acid from red blood cells of the blood as taught by Li because it is known in the art for isolating target nucleic acid for sample analysis by gel electrophoresis, and applying a known technique to a known method ready for improvement to yield predictable results is prima facie obvious. MPEP 2141(III)(D). Here, the claimed limitations are obvious because 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 yielded nothing more than predictable results. MPEP 2143(I)(A).
Regarding claim 11, Chandrasekaran teaches a system to perform a computer implemented method for detecting one or more single-stranded target nucleic acids in a plurality of single-stranded nucleic acids (bridging para. of pp. 469-470: the use of nanoswitches to a multiplexed DNA barcode system that can be used to detect any combination of up to six different biomarkers; e.g., Fig. 2(b): six different gene fragments; Fig. 3(b): six gene fragments are single-stranded), the method comprising:
incubating molecular beacons (MBs) (Fig. 1: barcoded nanoswitches; Fig. 2(a): the scaffold DNA having intercalating dyes of its backbone oligos and upon the binding of target nucleic acid, the DNA nanoswitch turning from a linear “off” state to a looped “on” state, which can be resolved on an agarose gel; thus, the nanoswitch is deemed to be a molecular beacon) with the plurality of single-stranded nucleic acids (Fig. 3(b): six nanoswitch mixtures and six gene fragments), wherein the MBs are configured to generate a fluorescence signal when bound with a target sequence of the one or more single-stranded target nucleic acids (p. 469, col. 2, para. 2: the signal comes from the intercalation of thousands of dye molecules; Fig. 2(a): upon binding with the target nucleic acid);
performing gel electrophoresis (p. 474, col. 1, para. 1: use gel electrophoresis for readout) by applying voltage to a gel comprising the incubated MBs and single-stranded nucleic acids (Supplemental, p. 4, last para.: characterization of barcode gels (multiplexing) were run at 55V), thereby obtaining electrophoretic mobility shifts of the incubated MBs and single-stranded nucleic acids (Fig. 3(b)-(d): each target nucleic acid triggers the formation of a specific loop, providing a unique signal; there is lack of cross-reactivity in nanoswitches designed for six different gene fragments, and thus the DNA nanoswitch barcodes detect all possible combinations of the six gene fragments; also see Fig. 2(b): the different locations of electrophoretic bands indicate the electrophoretic mobility shifts on the gel due to the turning from the linear “off” state to the looped “on” state when bound to the target nucleic acid); and
determining, using the fluorescent signal and the electrophoretic mobility shifts of the incubated MBs and plurality of single-stranded nucleic acids, a quantity of the target sequence (p. 474, col. 1, para. 2: our method provides direct detection without amplification, which makes absolute quantification more straightforward).
Chandrasekaran does not disclose the system comprising: at least one computer hardware processor; and at least one non-transitory computer-readable storage medium storing processor-executable instructions that, when executed by the at least one computer hardware processor, cause the at least one computer hardware processor to perform its computer implemented method.
However, Li teaches a system (Fig. 26) comprising at least one computer hardware processor (Fig. 26; ¶278: computer processor 2605); and
at least one non-transitory computer-readable storage medium storing processor-executable instructions (Fig. 26; ¶278: memory 2610; ¶276: machine executable code) that, when executed by the at least one computer hardware processor, cause the at least one computer hardware processor to perform a computer implemented method (¶276) for detecting one or more nucleic acids comprising a target sequence of nucleotides (¶209: a method for sample analysis; ¶110: a target nucleic acid).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified Chandrasekaran by incorporating a computer hardware processor and storage medium storing instructions that causes the process to perform the computer implemented method as taught by Li because it enables the operation automation under computer control system for implementing the programmed method on the computer, and applying a known technique to a known method ready for improvement to yield predictable results is prima facie obvious. MPEP 2141(III)(D). Here, the claimed limitations are obvious because 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 yielded nothing more than predictable results. MPEP 2143(I)(A).
Regarding claim 13, Chandrasekaran teaches wherein the target sequence is a micro ribonucleic acid (miRNA) (p. 469, col. 2, para. 2: detection of microRNAs).
Regarding claim 14, Chandrasekaran teaches wherein the plurality of single-stranded nucleic acids comprise single-stranded ribonucleic acids (ssRNA) (Fig. 4(c): ssRNA; e.g., Fig. 5(a): microRNA 141 and microRNA 30c).
Regarding claim 15, Chandrasekaran teaches wherein the plurality of single-stranded nucleic acids comprise single-stranded deoxyribonucleic acids (ssDNAs) (Fig. 4(c): ssDNA).
Regarding claim 17, Chandrasekaran and Li disclose all limitations of claim 11. Chandrasekaran further discloses the microRNA biomarkers are in blood, but fails to teach prior to incubation: obtaining blood of a patient; and isolating ssRNA from red blood cells (RBCs) of the blood of the patient.
However, Li teaches the sample is a target nucleic acid, e.g., a biological sample including blood, which is obtained from a subject (¶296). The blood may be components of blood, e.g., white blood cells, red blood cells, platelets (¶296). The example of nucleic acids include isolated RNA of any sequence (¶106).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified Chandrasekaran by incorporating steps of obtaining blood of a patient and isolating the target nucleic acid from red blood cells of the blood as taught by Li because it is known in the art for isolating target nucleic acid for sample analysis by gel electrophoresis, and applying a known technique to a known method ready for improvement to yield predictable results is prima facie obvious. MPEP 2141(III)(D). Here, the claimed limitations are obvious because 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 yielded nothing more than predictable results. MPEP 2143(I)(A).
Regarding claim 18, Chandrasekaran teaches wherein the gel used in electrophoresis is not stained, and wherein determining the electrophoretic shift further comprises measuring the fluorescent signal (p. 469, col. 2, para. 2: the nanoswitch changes conformation from a linear “off” state to a looped “on” state, providing a distinct signal which comes from the intercalation of thousands of dye molecules, e.g., GelRed; Examiner notes that GelRed dye is a fluorescent dye, and as evidenced by Chandrasekaran 2019, Fig. 4; p. 6, col. 2, para. 2: GelRed were added after incubation, and samples were run in an unstained gel).
Regarding claim 22, Chandrasekaran teaches wherein the quantity is a concentration of the target sequence (Fig. 2(c); p. 470, col. 2, para. 1: we performed sensitivity experiments with decreasing concentration of the DNA and found that the signal could be seen by eye at concentration as low as 50 fM).
Regarding claim 24, Chandrasekaran teaches wherein determining the electrophoretic shift comprises determining a quantity of a first target sequence and a quantity of a second target sequence in the plurality of single-stranded nucleic acids, wherein the second target sequence comprises one or more mutations relative to the first target sequence (Fig. 2(d); p. 471, col. 1, para. 1: the assay is highly specific, able to discriminate even a single nucleotide mismatch in the target sequence; e.g., CF gene targets that contained 1-3 mutations in Fig. 2(d)).
Claim(s) 9 is/are rejected under 35 U.S.C. 103 as being unpatentable over Chandrasekaran in view of Hochstrasser (US 2002/0150569).
Regarding claim 9, Chandrasekaran discloses all limitations of claim 1. Chandrasekaran does not discloses where the gel electrophoresis comprises applying a first voltage for a first period of time and a second voltage for a second period of time.
However, Hochstrasser teaches running gel electrophoresis at a voltage which is increased linearly from 300 to 3500 V during 3 hours, then for 3 additional hours at 3500 V, and finally for 17 hours at 5000 V (¶18). Thus, Hochstrasser teaches the gel electrophoresis is performed by applying a first voltage for a first period of time and a second voltage for a second period of time.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified Chandrasekaran for running the gel electrophoresis by applying a first voltage for a first period of time and a second voltage for a second period of time as taught by Hochstrasser because it is a suitable technique for voltage application of electrophoresis. Applying a known technique to a known method ready for improvement to yield predictable results is prima facie obvious. MPEP 2141(III)(D).
Claim(s) 16 is/are rejected under 35 U.S.C. 103 as being unpatentable over Chandrasekaran in view of Li, and further in view of Vu.
Regarding claim 16, Chandrasekaran and Li disclose all limitations of claim 11. Chandrasekaran further discloses the nanoswitch barcodes detect all possible combinations of antidig, RNA sequence, DNA sequence, and streptavidin (Fig. 4(c)-(e)). Chandrasekaran and Li do not discloses the method or the system further comprising conjugating single-stranded nucleic acids of the plurality of single-stranded nucleic acids with streptavidin beads.
However, Vu teaches a kit for the separation and detection of a target biomolecule of interest using nanoparticle probes (¶144). The nanoparticle probe includes a specific binding agent that specifically binds the target biomolecule of interest, using a linker, e.g., streptavidin, to link the detectable nanoparticle and the specific binding agent (¶145). Thus, Vu teaches the conjugation between the target biomolecule and the specific binding agent linked to streptavidin nanoparticles.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified Chandrasekaran and Li by incorporating streptavidin nanoparticles for separation and detection of a target biomolecule as taught by Vu because use of nanoparticle probes is a known standard procedure (¶133) so that streptavidin nanoparticles would provide the support to the binding agent for binding the target biomolecule for detection of target sequence of nucleic acids. Applying a known technique to a known method ready for improvement to yield predictable results is prima facie obvious. MPEP 2141(III)(D).
Claim(s) 19 is/are rejected under 35 U.S.C. 103 as being unpatentable over Chandrasekaran in view of Li, and further in view of Hochstrasser.
Regarding claim 19, Chandrasekaran and Li disclose all limitations of claim 11. Chandrasekaran and Li do not discloses where the gel electrophoresis comprises applying a first voltage for a first period of time and a second voltage for a second period of time.
However, Hochstrasser teaches running gel electrophoresis at a voltage which is increased linearly from 300 to 3500 V during 3 hours, then for 3 additional hours at 3500 V, and finally for 17 hours at 5000 V (¶18). Thus, Hochstrasser teaches the gel electrophoresis is performed by applying a first voltage for a first period of time and a second voltage for a second period of time.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified Chandrasekaran and Li for running the gel electrophoresis by applying a first voltage for a first period of time and a second voltage for a second period of time as taught by Hochstrasser because it is a suitable technique for voltage application of electrophoresis. Applying a known technique to a known method ready for improvement to yield predictable results is prima facie obvious. MPEP 2141(III)(D).
Response to Arguments
Applicant’s arguments has/have been considered but are moot because the arguments do not apply to any of the references being used in the current rejection.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CAITLYN M SUN whose telephone number is (571)272-6788. The examiner can normally be reached on M-F: 8:30am - 5:30pm.
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/C. SUN/Primary Examiner, Art Unit 1795
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