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 .
Please note: The examiner handling this application has changed. The new examiner on this case is Kailey Cash (kailey.cash@uspto.gov) in AU1683. Any correspondence relating to the instant application should be directed to this examiner.
Election/Restrictions
Applicant’s election without traverse of Group II (claims 24-25 and 28) in the reply filed on 3/2/2026 is acknowledged.
The Examiner notes that in the original Requirement for Restriction/Election of 9/30/2025, claim 41 was erroneously included in Group II. Claim 41, which depends from claim 33, should have been included in Group I (drawn to a kit and including claim 33). Applicant has appropriately withdrawn claim 41, as indicated below as well. Applicant has cancelled claim 39 which was presented in Group II with claims 24-25 and 28.
Claims 1-2, 7, 11, 24-25, 28, 33, 41-52 are pending.
Claims 1-2, 7, 11, 33, and 41 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 3/2/2026.
Claims 24-25, 28, and 42-52 are being examined on the merits.
Nucleotide and/or Amino Acid Sequence Disclosures
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Items 1) and 2) provide general guidance related to requirements for sequence disclosures.
37 CFR 1.821(c) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.821(a) must contain a "Sequence Listing," as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.821 - 1.825. This "Sequence Listing" part of the disclosure may be submitted:
In accordance with 37 CFR 1.821(c)(1) via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter "Legal Framework") as an ASCII text file, together with an incorporation-by-reference of the material in the ASCII text file in a separate paragraph of the specification as required by 37 CFR 1.823(b)(1) identifying:
the name of the ASCII text file;
ii) the date of creation; and
iii) the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(1) on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation-by-reference of the material in the ASCII text file according to 37 CFR 1.52(e)(8) and 37 CFR 1.823(b)(1) in a separate paragraph of the specification identifying:
the name of the ASCII text file;
the date of creation; and
the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(2) via the USPTO patent electronic filing system as a PDF file (not recommended); or
In accordance with 37 CFR 1.821(c)(3) on physical sheets of paper (not recommended).
When a “Sequence Listing” has been submitted as a PDF file as in 1(c) above (37 CFR 1.821(c)(2)) or on physical sheets of paper as in 1(d) above (37 CFR 1.821(c)(3)), 37 CFR 1.821(e)(1) requires a computer readable form (CRF) of the “Sequence Listing” in accordance with the requirements of 37 CFR 1.824.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed via the USPTO patent electronic filing system as a PDF, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the PDF copy and the CRF copy (the ASCII text file copy) are identical.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed on paper or read-only optical disc, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the paper or read-only optical disc copy and the CRF are identical.
Specific deficiencies and the required response to this Office Action are as follows:
Specific deficiency – Nucleotide and/or amino acid sequences appearing in the drawings are not identified by sequence identifiers in accordance with 37 CFR 1.821(d) (see Figure 9). Sequence identifiers for nucleotide and/or amino acid sequences must appear either in the drawings or in the Brief Description of the Drawings.
Required response – Applicant must provide:
Replacement and annotated drawings in accordance with 37 CFR 1.121(d) inserting the required sequence identifiers;
AND/OR
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required sequence identifiers into the Brief Description of the Drawings, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter.
Specification
The specification is objected to as failing to provide proper antecedent basis for the claimed subject matter. See 37 CFR 1.75(d)(1) and MPEP § 608.01(o). Correction of the following is required: the specification does not provide antecedent basis for the terms “Terbium” or “Samarium” as recited in claim 46. Europium (Eu) is mentioned several times throughout the specification (e.g., pg 28 and 39). Terbium and Samarium do not appear in the disclosure at all. The specification recites “Lanthanide” as the donor which excites a fluorophore in a FRET pair, with the example of Europium as a specific lanthanide (pg 28). However, the only species of the generic “lanthanide” mentioned in the specification is Europium.
Claim Objections
Claims 24-25, 28, 46, 49, and 52 are objected to because of the following informalities:
Claim 24, line 5 reads “a complementary 3’ and 5’ stem sequences” and should read “[[a]] complementary 3’ and 5’ stem sequences”.
Claim 24, line 14 reads “a hairpin loop structures” and should read “a hairpin loop structure[[structures]]”.
Claim 24, line 17-18 reads “the stem region of the Target Initiation probe opens” and should read “the stem [[region]]portion of the Target Initiation probe opens” to maintain consistent claim terminology with “a double stranded stem portion” of the Target Initiation probe as defined in lines 5-6.
Claim 24, line 18-19 reads “permits hybridisation to the loop region of the first Chain Loop probe” and should read “permits hybridisation to the loop [[region]] of the first Chain Loop probe” to maintain consistent claim terminology.
Claims 24 and 25 are missing a period at the end of the claim with the amendments that have been made.
Claim 28, line 1 reads “A method of claim 24” and should read “The[[A]] method of claim 24” to maintain consistent claim formatting.
Claim 28, line 2 reads “two or more sets of Target initiation probes” and should read “two or more sets of Target [[initiation]]Initiation probes” to maintain consistent claim term formatting.
Claim 28, line 4 reads “one or more target nucleic acid sequences” and should read “[[one]]two or more target nucleic acid sequences” to remain consistent with the rest of the claim.
Claim 46, lines 2, 6, and 10 have a letter notation followed by a period (e.g., “a).”). The period is superfluous and should be removed.
Claim 46 ends with a semi-colon and not a period. Appropriate correction to end the claim with a period is required.
Claim 49 reads “wherein the hybridising step a) is carried out at a temperature higher than the hybridisation reaction of step b)” and should read “wherein the [[hybridizing]]hybridisation reaction of step a) is carried out at a temperature higher than the hybridisation reaction of step b)” to maintain consistent claim terminology with claim 24 and throughout the claim itself.
Claim 52 reads “a tumour sample selected from clinical, veterinary, food or environmental source” and should read “a tumour sample selected from a clinical, veterinary, food or environmental source”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112b - Indefiniteness
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 25, 42-43, 45-48, and 50-52 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 25 recites the limitation "the hybridising buffer" in lines 4-5. There is insufficient antecedent basis for this limitation in the claim.
Claim 42 recites the limitation "the set of four or more hairpin loop nucleic acid probes" in line 4. There is insufficient antecedent basis for this limitation in the claim. For the purposes of examination, this is being interpreted to mean that there are at least four hairpin loop nucleic acid probes used in the method of claim 24, including the Target Initiation probe. However, further clarification is required.
Claim 43 recites the limitation "the target loop of another Chain Loop probe" in line 3. There is insufficient antecedent basis for this limitation in the claim. There is only one “target loop” defined in claim 24, from which claim 43 depends, and that is the loop within the Target Initiation probe and not within a Chain Loop probe. For purposes of examination this is interpreted as the loop structure within the Chain Loop probe. However, further clarification is required.
Claim 45 recites the limitation "the acceptor fluorophore and donor fluorophore" in line 3. There is insufficient antecedent basis for this limitation in the claim.
Claim 46 recites the limitation "the reporter time resolved FRET pair" in line 1. There is insufficient antecedent basis for this limitation in the claim. No FRET pairs are defined in claim 24, from which claim 46 depends. For purposes of examination, this is being interpreted as the signal generating component of the Chain Loop probes as defined in claim 24. Meaning, the signal generating component of the Chain Loop probes is a reporter time resolved FRET pair selected from a)-c). However, further clarification is required.
Regarding claim 46, the phrase "preferably" (lines 3, 7, and 11) renders the claim indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
Claim 47 recites the limitation "the final Chain Loop probe" in line 1. There is insufficient antecedent basis for this limitation in the claim. For purposes of examination this will be interpretated as whichever Chain Loop probe is the last in the polymer chain, however further clarification is required.
Claim 48 recites “wherein the Gibbs free energy of binding of the Target Initiation probe and of one or more Chain Loop probes is negative under respective hybridisation conditions and the Tm of the stem formation is higher than the effective hybridising temperature of the Chain Loop probes”. It is unclear which stem formation is higher than the effective hybridising temperature of the Chain Loop probes. Is it that the stem formation of the Chain Loop probes is higher than the hybridising temperature of the Chain Loop probes or is it that the Tm of the stem formation of the Target Initiation probe is higher that the effective hybridising temperature of the Chain Loop probes? There are multiple probes with stems mentioned in this claim and some clarification is required regarding which “stem” is being discussed. For the purposes of examination this is being interpreted to mean that the stem of the Chain Loop probe has a higher Tm than the effective hybridisation Tm of the Chain Loop probe. However, further clarification is required.
Claim 50 recites the limitations "the first hairpin loop probe, the second hairpin loop probe, the third hairpin loop probe, the fourth hairpin loop probe". There is insufficient antecedent basis for these limitations in the claim. There are no probe designations (first, second, third, etc.) in claim 24, from which claim 49 depends. For the purposes of examination, the first hairpin probe is being interpreted as the Target Initiation probe and the second, third, and fourth hairpin probes are Chain Loop probes. However, further clarification is required.
Claim 51 recites the limitations "the (n+1)th hairpin loop probe” and “the nth hairpin loop probe”. There is insufficient antecedent basis for these limitations in the claim. Claim 24, from which claim 51 depends defines a first Chain Loop probe, a second Chain Loop probe, and subsequent Chain Loop probes. The differing claim language makes it unclear if these are the same Chain Loop probes as defined in claim 24 and also, given that the first and second Chain Loop probes are specifically and separately defined, whether these terms would apply to the first, the second, the subsequent, or all of the Chain Loop probes defined in claim 24. Clarification is required.
Claim 51 recites “wherein the hairpin sequence of the (n+1)th hairpin loop probe is capable of annealing to the complementary stem region sequence of the nth hairpin loop probe”. It is unclear which hairpin loop probe is being discussed here. Both the Target Initiation probe and the Chain Loop probes are hairpin probes, but the Target Initiation probe contains a sequence complementary to the target analyte while the Chain Loop probes comprise signal generating components, therefore making them very different types of hairpin loop probes. Is this referring to all hairpin loop probes in claim 24 or a specific type of hairpin loop probe? Clarification is required.
Claim 52 recites “wherein the target nucleic acid in the sample is an RNA or a DNA from an infectious agent or a tumour sample selected from clinical, veterinary, food or environmental source”. The wording of this claim is unclear. One interpretation is that the sample is “an RNA or a DNA from an infection agent” OR is “a tumour sample selected from clinical veterinary, food or environmental source”. The other interpretation is that that sample is “an RNA or a DNA from an infectious agent or a tumour sample”, either of which can be collected from clinical, veterinary, food, or environmental sources. This ambiguity makes the scope of the claim unclear. For the purposes of examination, the latter interpretation is being considered, in which the RNA or DNA target nucleic acid can be from an infectious agent or from a tumour sample, either of which can be collected from a clinical, veterinary, food, or environmental source. However, further clarification is required.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 24, 42, 44, and 52 are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Riemer (Riemer et al., US 2021/254155 A1, EFD of 2/14/2020).
Claim 24: Riemer teaches a method for the detection of a target substance (such as a nucleic acid) in a sample through detection of a signal (Abstract, paragraph [0007, 0018]). Riemer teaches hybridization of a primary probe (reads on Target Initiation probe) that binds specifically to a target segment of a substance (nucleic acid) which then allows the hybridization of at least one secondary probe which includes a segment that is complementary to the primary probe (paragraph [0009]). At least three probe types are hybridized together and matched in a way to create a chain of probes that are then detected through the formation of a signal (such as through the use of detectably labeled probes (paragraph [0009-0010]). Riemer teaches that the probes are nucleic acids and allow for specific base pairing of probe to the target and to each other to form a polymer chain (paragraph [0012]) and Riemer teaches that the probes are hairpin probes (paragraph [0016]). Riemer teaches that the primary probe (Target Initiation probe) does not contain a detectable label (reads on a signal generating component) but that each that each subsequent probe type (reads on Chain Loop probes) contains a detectable tag (paragraph [0017]). As illustrated in Figure 1 from Riemer below, the Target Initiation probe (primary probe, 6), is a hairpin nucleic acid comprising complementary 3’ and 5’ stem sequences forming a double stranded stem portion and a single stranded target loop portion, which comprises a sequence complementary to the target nucleic acid (5 in the figure below). Riemer then teaches a set of Chain Loop probes (secondary, tertiary, and quaternary probes; 9, 11, and 13, respectively) which are then able to hybridize to the Target Initiation probe by the 5’ or 3’ stem sequence and then to each prior Chain loop probe via the exposed 5’ or 3’ stem sequence. Each Chain Loop probe has a signal generating component (8, detectable tag). The hybridization of all probes together forms a probe chain (reads on polymer) that generates a signal (paragraph [0039]).
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Riemer teaches detection of an optical signal for the detection of the target nucleic acid (paragraph [0039]).
Claim 42: Riemer teaches that the quaternary probe may have a sequence that is complementary to the tertiary probe AND to the secondary probe. This reads on the secondary probe having each strand of the stem loop sequence region being capable of being hybridized by a distinct single-stranded hairpin loop sequence of a different hairpin loop probe (both the tertiary and quaternary probes). Riemer teaches that this allows formation of a branched structure during polymerization (paragraph [0038]).
Claim 44: Riemer teaches that the Chain Loop probes comprise one or more groups capable of generating a signal through fluorescence (paragraph [0010, 0017]).
Claim 52: Riemer teaches detecting DNA or RNA associated with pathogenicity-specific sequences (reads on from an infectious agent; paragraph [0018]). Riemer teaches analyzing samples from foodstuffs, clinical, or environmental sources for analysis (paragraph [0027]).
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 25, 28, 43, 45, 47, and 50 are rejected under 35 U.S.C. 103 as being unpatentable over Riemer (Riemer et al., US 2021/254155 A1, EFD of 2/14/2020) in view of Söderberg (Söderberg et al., WO 2015/118029 A1; cited on IDS of 7/20/2023).
Claim 25: The teachings of Riemer as they apply to claim 24, from which this claim depends, are detailed above. Relevant to the instantly rejected claim, Riemer teaches performing hybridization between hairpin probes and a target substance and between subsequent hairpin probes. Riemer additionally teaches the use of a fluorescent signal to detect the presence of the target substance.
Riemer does not teach that the hybridization reaction is carried out in the presence of formamide. However, the use of formamide in hybridization chain reactions, such as those taught by Riemer, is known in the art as taught by Söderberg.
Söderberg teaches using formamide in the hybridization reaction buffer (pg 56, ln 17-24). It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Riemer to include formamide in the reaction buffer, as taught by Söderberg. One would be motivated to do so given the assertion by Söderberg that the inclusion of formamide significantly increases the fluorescent signal of the resulting probe chains (pg 56, ln 17-24). One would have a reasonable expectation of success given that Söderberg is also performing a hybridization chain reaction for detection of optical signal and thus detection of a target analyte in a sample (pg 42), a methodology utilizing similar hybridization/polymerization of hairpin probe chains as that employed by Riemer.
Claim 28: Söderberg teaches using multiple sets of HCR probes for the detection of two or more target analytes (specifically, nucleic acid sequences; pg 33-34).
Claim 43: Söderberg teaches formation of a dendrimer species using multiple species of HCR monomers (hairpin loop probes) in which non-linear (or branched) amplification can be achieved through the use of a monomer that contains an additional “tag” sequence that can act as an initiator for further HCR (pg 29, ln 35-pg 30, ln 2). This reads on “a further nucleic acid sequence extending from the 5’ or 3’ stem sequence”. The claim does not specify that the further nucleic acid sequence must be unpaired or single-stranded. Söderberg teaches that this allows for exponential amplification (pg 29, ln 26-30).
Claim 45: Söderberg teaches labeling the hairpin loop probes for detection using FRET pairs in a way that detection of the signal is dependent on conformation of the hairpin loop probe (pg 38, ln 14-20). This can be achieved by placement of a fluorophore and quencher in close proximity on the same probe, wherein hybridization into the polymer chain relieves said quenching and generates signal. Alternatively, the FRET pair can be between monomers, such that the donor and acceptor are on separate HCR monomers and when hybridized into the polymer chain can generate a signal (pg 38, ln 14-35). Söderberg does not explicitly teach that the donor and acceptor fluorophores are within the same monomer. However, inclusion of a donor and acceptor fluorophore in the same monomer at a distance that prevents excitation within the closed configuration but allows for generation of signal upon hybridization into the polymer probe chain, is an obvious variation of the FRET method as taught by Söderberg that would produce the predicted result of lowering background signal when unbound but allowing for an increase in fluorescence upon generation of the open configuration and hybridization into a growing polymer probe chain, as taught by Söderberg.
Claim 47: Söderberg teaches that hairpin loop probes may have a sequence complementary to another hairpin loop probe that is within their stem region (pg 8-9). Söderberg does not explicitly teach that the last Chain Loop probe in a probe chain is the one that has the complementary nucleic acid in the stem of the molecule, but this is an obvious variation, with predictable results of effects on hybridization, of the stem loop structure, as taught by Söderberg.
Claim 49: Söderberg teaches performing the initial hybridisation reaction between the Target Initiation probe at 37ºC and then the subsequent amplification with labeled probes (Chain Loop probes) at room temperature (Söderberg defines room temperate as between 18-30ºC (pg 35, ln 14), less than the hybridisation reaction of the initial hybridisation between Target Initiation probe and target sequence; pg 54, ln 19-22).
Claim 50: Söderberg teaches using hairpin probes that have a stem length of 18-30 nucleotides (a total of 36-60 nucleotides) and a loop length of 17-19 nucleotides (pg 19, ln 11-15). Therefore, Söderberg teaches hairpin probes that are a total length of 120 nucleotides or less.
Claim 46 is rejected under 35 U.S.C. 103 as being unpatentable over Riemer (Riemer et al., US 2021/254155 A1, EFD of 2/14/2020) in view of Söderberg (Söderberg et al., WO 2015/118029 A1; cited on IDS of 7/20/2023) and Cho (Cho et al., Cell Chemical Biology 2020).
The teachings of Riemer as they apply to claim 24, from which this claim depends, are detailed above. Relevant to the instantly rejected claim, Riemer teaches performing hybridization between hairpin probes and a target substance and between subsequent hairpin probes. Riemer teaches that the primary probe (Target Initiation probe) does not contain a detectable label (reads on a signal generating component) but that each that each subsequent probe type (reads on Chain Loop probes) contains a detectable tag (reads on a signal generating component; paragraph [0017]).
Riemer does not teach that the signal generating component is a FRET pair or that it is a time resolved FRET pair. However, use of time resolved FRET pairs on hairpin loop probes is known in the art, as taught by Söderberg.
Claim 46: Söderberg teaches labeling the hairpin loop probes for detection using FRET pairs in a way that detection of the signal is dependent on conformation of the hairpin loop probe (pg 38, ln 14-20). This can be achieved by placement of a fluorophore and quencher in close proximity on the same probe, wherein hybridization into the polymer chain relieves said quenching and generates signal. Alternatively, the FRET pair can be between monomers, such that the donor and acceptor are on separate HCR monomers and when hybridized into the polymer chain can generate a signal (pg 38, ln 14-35). Söderberg teaches using fluorescent signals to detect fluorescence in real time (pg 42, ln 30-35).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Riemer to include a time resolved FRET pair, as taught by Söderberg. One would be motivated to use FRET pairs for labeling of the hairpin loop probes given the teaching by Söderberg that this allows for detection of the target nucleic acid through the conformational change of the hairpin loop probe (whose conformational change is dependent upon the presence of the target nucleic acid; pg 38, ln 14-35). One would be motivated to monitor this change in fluorescence in a time resolved manner given the assertion by Söderberg that this allows for “spectra generated in this way can be resolved, for example, using "fits" of pre-selected fluorescent moieties such as dyes, to form peaks representative of each signalling moiety (i.e. fluorophore). The areas under the peaks can be determined which represents the intensity value for each signal, and if required, expressed as quotients of each other” and may allow for determination of quantity of the target nucleic acid as well as the presence or absence (pg 43, ln 1-15). One would have a reasonable expectation of success given that Söderberg successfully applied this measuring methodology to the detection of nucleic acids using a hybridization chain reaction similar to that employed by Riemer.
Riemer in view of Söderberg do not teach that the FRET pair is Europium and a fluorophore that is capable of being excited by light emitted from Europium. However, use of Europium and a fluorophore pair for biological sensing is known in the art, as taught by Cho.
Cho teaches that lanthanides (such as Eu) can participate in a process analogous to FRET in which the lanthanide transfers energy to a fluorophore (Luminescence Resonance Energy Transfer, pg 925, col 2, paragraph 2). Cho describes an example of a Eu-fluorophore pair in which Eu is on one end of a dsDNA molecule and the fluorophore to excite is Cy5 (Figure 4A).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Riemer in view of Söderberg to use an Eu-fluorophore pair as taught by Cho. One would be motivated to use Eu-fluorophore pair for FRET reporting given the assertion by Cho that their “line-like emission peaks…achieve J values that are about an order of magnitude greater than those of fluorescent donors”, have much greater quantum yields, and are longer-lived than common fluorophores (Luminescence Resonance Energy Transfer, pg 925, col 2, paragraphs 2-3). One would have a reasonable expectation of success given that Cho demonstrates that Eu and its fluorophore pair can be successfully attached to DNA (Figure 4A) and that this pair can be used in time-resolved assays (Photometric Assays, pg 927, col 2, paragraph 2).
Claims 48 and 51 are rejected under 35 U.S.C. 103 as being unpatentable over Riemer (Riemer et al., US 2021/254155 A1, EFD of 2/14/2020) in view of Thrippleton (Thrippleton et al., US 8,993,239 B2) and Green (Green et al., Biophysical Journal 2006).
The teachings of Riemer as they apply to claim 24, from which this claim depends, are detailed above. Relevant to the instantly rejected claim, Riemer teaches performing hybridization between hairpin probes and a target substance and between subsequent hairpin probes.
Claim 48: Riemer does not teach that the Gibbs free energy of binding of the Target Initiation probe and of one or more Chain Loop probes is negative under respective hybridisation conditions or that the Tm of the stem formation is higher than the effective hybridising temperature of the Chain Loop probes. However, these features of probe design are known in the art, as taught by Thrippleton and Green.
Thrippleton teaches the use of molecular beacons which are in the form of hairpin loops when not bound to a target sequence (col 3, ln 39-54). Thrippleton teaches that the Gibbs free energy of hairpin loop probes (or molecular beacons) should be negative under hybridization conditions (col 4, ln 52-58).
Green teaches hybridisation temperatures below that of the melting temperatures of the hairpin’s stem structures (Introduction, paragraph 3).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Riemer to ensure that the hairpin loops have Gibbs free energy of binding that are negative and to ensure that the melting temperature of the probe stems formation is higher than that of the effective hybridising temperature, as taught by Thrippleton and Green, respectively. One would be motivated to design the hairpin loop probes to have a negative Gibbs free energy of binding given the teaching by Thrippleton that this ensures that the probes spontaneously form their secondary structures (the hairpin loop) in the absence of initiation sequences (col 4, ln 52-58). One would be motivated to ensure that the melting temperature of the stem of the hairpin loop probe is higher than the effective hybridising temperature of the probe given the teaching by Green that this enables a “long-lived fuel” to fuel a chain reaction via releasing the stored energy in the loop of the probes through catalyzing hybridisation (Introduction, paragraph 3). One would have a reasonable expectation of successfully implementing these design parameters given that Thrippleton and Green discuss optimizing these parameters for desired outputs and thermodynamic control of the reaction.
Claim 51: Green teaches a hybridization between hairpin loop sequences with a delta G of -11.3 kcal/mol (which is at least about -10 kcal/mol – 1.5 kcal/mol; Table 1).
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KAILEY E CASH whose telephone number is (571)272-0971. The examiner can normally be reached Monday-Friday 8:30am-6pm ET.
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/KAILEY ELIZABETH CASH/Examiner, Art Unit 1683
/STEPHEN T KAPUSHOC/Primary Examiner, Art Unit 1683
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