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 .
Priority
This application claims priority to provisional 63/393,022, filed July, 28, 2022.
Election/Restrictions
Applicant’s election without traverse of Species A-F in the reply filed on February 12, 2026 is acknowledged. Applicant has elected the following species: (a) analyte is the biological target type; (b) indirect binding as the detection methodology; (c) mRNA as the specific biological target; (d) rolling circle amplification (RCA) product as the detection technique subspecies; (e) carbazole moiety as the electron donor; and (f) dicyanobenzene moiety as the electron acceptor.
Regarding species election e, the following species have been withdrawn: phenoxazine, diphenylamine, triphenylamine, phenothiazine, diphenylacridine, phenazine, spiroacridine, acridine, and dimethylacridine.
Regarding species election f, the following species have been withdrawn: cyanobenzene, diphenyltriazine, diphenylsulfone, naphthalidmide, heptazine, triazine, dicyanoimidazole, cucuminoid, oxadiazole, benzothiadiazole, pyrimidine, phenylbenzimidazole, dibenzodipyridophenazine, triarylboron, and dicyanopyrazino phenanthrene.
Claim Status
Claims 1, 3, 6, 8, 13, 15, 17, 18, 20, 21, 30, 31, 33, 35, 37, 39, 44, 49, 51, and 71 are pending.
Claims 17 and 51 have been amended.
Claim 44 is withdrawn since mRNA is the elected target type.
Claims 1, 3, 6, 8, 13, 15, 17, 18, 20, 21, 30, 31, 33, 35, 37, 39, 49, 51, and 71 are currently under examination.
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 (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 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 1, 3, 6, 8, 17, 18, 20, 30, 31, 33, 51, and 71 are rejected under 35 U.S.C. 103 as being unpatentable over Ni et al. (Adv. Opt. Master. 2019, 7, 1801519) in view of Sayyadi et al. (Analyst 2019, 144, 766).
Regarding claim 1, the claim recites, “A method of detecting an analyte in a biological sample, the method comprising: contacting the biological sample with a nucleic acid probe conjugated to a thermally activated delayed fluorescence (TADF) emitter, wherein the nucleic acid probe directly or
indirectly binds to the analyte or a product or complex thereof in the biological sample; exciting the biological sample with a pulsed light source such that the biological sample emits autofluorescence and the TADF emitter emits fluorescence; and detecting the fluorescence emitted by the TADF emitter at a location in the biological sample after a time period post excitation.” Ni et al. discloses a TADF compound designated NID-TPP, which comprises a dimethylacridine (DMAC) electron donor and a 1,8-naphthalimide electron acceptor, conjugated to a triphenylphosphonium (TPP+) targeting moiety (p. 2, scheme 1).
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Ni et al. teaches exciting biological samples (HeLa cells) with a pulsed light source, specifically a 405nm pulsed laser, such that the biological sample emits autofluorescence and the TADF emitter emits fluorescence (p. 6, section 2.2, Figure 7). Ni et al. further teaches detecting the fluorescence emitted by the TADF emitter at a location in the biological sample after a time period post excitation, using time-resolved luminescence imaging (TRLI) with a delay time greater than 10 nanoseconds, during which autofluorescence from the biological sample has substantially decayed (p. 6, Figure 7B). Ni et al. demonstrates that the TADF emission can be temporarily resolved from background autofluorescence, noting that autofluorescence from cells decays rapidly (within nanoseconds) while the TADF emission persists on a longer timescale (P. 1, Abstract; p. 6, Figure 7B).
Ni et al. does not explicitly teach contacting the biological sample with a nucleic acid probe conjugated to the TADF emitter, wherein the nucleic acid probe indirectly binds to the analyte.
Sayyadi et al. discloses time-gated luminescent in situ hybridization with a long-lifetime luminescent emitter (the europium chelate BHHTEGST-Eu^3+) covalently conjugated to a DNA oligonucleotide probe via N-hydroxysuccinimide (NHS) ester chemistry to a 5-amino-modified oligonucleotide (p. 3, Section 2.1, Figure 1).
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Sayyadi et al. teaches contacting a biological sample (bacterial cells containing S. aureus) with this nucleic acid probe conjugated to the luminescent emitter, wherein the nucleic acid probe hybridizes to a target analyte (16S rRNA) in the biological sample via in situ hybridization (p. 6, Section 2.2, Figure 4A).
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Sayyadi et al. teaches exciting the biological sample with a pulsed light source (365 nm pulsed UV LED) and detecting the luminescence emitted by the emitter after a time period post excitation using time-gated luminescence (TGL) microscopy with a gate delay that permits autofluorescence to decay before detection (p. 1, Abstract; p. 6, Figure 4, Section 2.2; p. 7). Sayyadi et al. demonstrates that TGL imaging with the europium-conjugated DNA probe achieves a signal-to-noise ratio (SNR) 6-fold higher than conventional Alexa Fluor 488 fluorescence (p. 7-8, Section 2.3, Figures 5-6).
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to substitute the long-lifetime luminescent emitter (the europium chelate BHHTEGST-Eu^3+) of Sayyadi’s et al. nucleic acid probe conjugated detection method with the thermally activated delayed fluorescence (TADF) emitter taught by Ni et al, thereby arriving at a method of detecting an analyte in a biological sample using a nucleic acid probe conjugated to a TADF emitter.
A person of ordinary skill in the art would have been motivated to make this substitution for several reasons. First, Sayyadi et al. and Ni et al. are directed to the same field of endeavor and address the same technical problem: detecting a biological target in a sample by means of a long-lifetime luminescent emitter whose emission can be temporally resolved from short-lived cellular autofluorescence using time-gated or time-resolved luminescence microscopy (Sayyadi: Abstract; p.769, TGL microscopy)(Ni: p. 1, col. 2 Introduction; p. 2, col. 1). Both references teach that the operative property of the luminescent emitter is a luminescence lifetime substantially longer than that of cellular autofluorescence, such that the autofluorescence decays during a gate delay before the emitter signal is detected. Sayyadi’s et al. BHHTEGST-Eu^3+ chelate and Ni’s et al. NID TADF emitter both satisfy this functional requirement with luminescence lifetimes in the microsecond range that are well-separated from the nanosecond scale decay of cellular autofluorescence (Ni: p. 5, col. 1)(Sayyadi: p. 769). The POSITA would therefore have recognized Ni’s TADF emitter as a member of the same functional class of long-lifetime luminescent emitters used by Sayyadi et al.
Second, Ni et al. teaches that hydrophilic TADF luminophores offer particular advantages for specific imaging in cells, including better cell compatibility and the ability to support “the targeting of organelles, DNA, proteins, and small molecules of biological interest” (p. 2, col. 2). Ni et al. thus expressly identifies nucleic acid (DNA) targeting as a recognized application of the hydrophilic TADF luminophore class it discloses. A person of ordinary skill in the art reading Sayyadi’s et al. disclosure of an oligonucleotide-conjugated long-lifetime emitter for in situ hybridization would have been motivated to apply Ni’s et al. hydrophilic TADF emitter chemistry in Sayyadi’s et al. probe architecture, both to access the broader application space (nucleic acid targeting) that Ni et al. identifies for TADF emitters and to take advantage of TADF specific benefits such as the two photon excitation capability that Ni et al. demonstrates (p. 7, col. 2; p. 8, col. 1; Figure 9).
Third, the substitution is a simple substitution of one known element for another to obtain predictable results. See KSR INT’l Co. v. Teleflex Inc., 550 U.S> 398, 416 (2007); MPEP 2143(I)(B). Sayyadi et al. already reduced to practice the complete method of detecting an analyte in a biological sample using a nucleic acid probe conjugated to a long-lifetime luminescent emitter, including the conjugation chemistry, the in situ hybridization protocol, the pulsed excitation, and the time-gated detection (p. 768-770). The only modification required to arrive at the claimed invention is replacement of Sayyadi’s et al. europium chelate emitter with Ni’s et al. TADF emitter, with all other elements of Sayyadi’s et al. method retained unchanged.
A person of ordinary skill in the art would have had a reasonable expectation of success in making this substitution. Sayyadi’s et al. conjugation chemistry, NHS ester coupling between a carboxylate functionalized luminescent reporter and a 5’ amino modified oligonucleotide (p. 768) is a general bioconjugation method broadly applicable to any luminescent reporter bearing or modifiable to bear a carboxylate handle. Ni et al. teaches a four-step synthesis of the NID TADF core (p. 2, col. 2; Scheme 1) in which the acridine nitrogen of the DMAC donor unit is available for derivation, as Ni et al. demonstrates by appending a TPP+ group through a nonconjugated methylene linker at that position. The POSITA would have understood from Ni’s et al. own design that the NID TADF core tolerates modification of the substituent at the acridine nitrogen through a nonconjugated linker without disrupting the underlying TADF photo physics (p. 4, col. 2; Figure 3A). Substitution of the TPP+ group with an oligonucleotide via the same nonconjugated linker chemistry (or by an analogous NHS ester compatible carboxylate handle on the NID scaffold) would therefore have been expected to yield an oligonucleotide conjugated TADF emitter retaining the TADF photophysical properties of Ni’s et al. core.
Further, the POSITA would have expected the resulting oligonucleotide conjugated TADF emitter to function in Sayyadi’s et al. time-gated in situ hybridization workflow because the gate-delay detection scheme of Sayyadi et al (p. 769) operates on the same general principle exploited in Ni’s et al. time-resolved luminescence imaging of HeLa cells (Ni, p. 6, col. 1; Figure 7B). The POSITA would therefore have reasonably expected that a TADF emitter conjugated to a nucleic acid probe per Sayyadi’s et al chemistry could be used to detect an analyte in a biological sample by time-resolved luminescence imaging as recited in claim 1.
Regarding claim 3, the claim recites, “The method of claim 1, wherein the time period post excitation is greater than the lifetime of the autofluorescence emitted from the biological sample and less than the lifetime of the fluorescence emitted by the TADF emitter.” As discussed above regarding claim 1, Ni et al. teaches time-resolved luminescence imaging in which the TADF emission is detected after a delay time that allows autofluorescence to decay. Ni et al. teaches that background autofluorescence from cells decays rapidly on a nanosecond timescale, while the TADF emission from NID-TPP persist on a significantly longer timescale due to the delayed fluorescence mechanism involving reverse intersystem crossing (RISC) from the triplet to singlet state (Abstract; p. 2, col. 1). Ni et al. explicitly demonstrates time-resolved luminescence imaging at delay times of 10-50 ns and 20-50 ns (p. 6, Figure 7b), which is greater than the autofluorescence lifetime of cells (approximately 1-2 ns) and less than the TADF emission lifetime of NID-TPP (p. 3-4, Figure 3b; p. 4, Figure 4b). This directly corresponds to the claimed limitation that the time period post excitation is greater than the lifetime of the autofluorescence and less than the lifetime of the TADF fluorescence. Ni et al. does not teach any additional limitation not already addressed in the rejection of claim 1 above, and Sayyadi’s et al. contribution remains as set forth above for claim 1. The motivation to combine and reasonable expectation of success are the same as fully articulated above for claim 1.
Regarding claim 6, the claim recites, ” The method of claim 1, wherein the biological sample is pulsed with excitation light from the pulsed light source and the fluorescence emitted by the TADF
emitter is detected after a time period post each excitation pulse.” Ni et al. teaches that the biological sample is pulsed with excitation light from a pulsed laser (405 nm pulsed excitation) and that the TADF fluorescence is detected after a time period post each excitation pulse, as this is the fundamental operational principle of the time-resolved luminescence imaging technique employed by Ni et al. (p.6, Section 2.2, Figure 7b). Specifically, Ni et al. describes a time-gated imaging protocol in which a pulsed excitation source is used and luminescence is collected after a defined delay following each pulse (p. 6, Figure 7b). Sayyadi et al. similarly teaches pulsed excitation from a pulsed UV LED source with gated detection after each excitation pulse using a gated auto synchronous luminescence detector (GALD) (Abstract; p. 3; p. 6-7, Section 2.2). The motivation to combine and reasonable expectation of success are the same as fully articulated above for claim 1.
Regarding claim 8, the claim recites, “The method of claim 1, wherein the end point of the time period is at least 10, 25, 50, or 100 nanoseconds post excitation.” Ni et al. teaches time-gated detection with a delay time of greater than 10 nanoseconds post excitation (p. 6, Figure 7b). Ni et al. explicitly demonstrates time-resolved luminescence imaging at delay times of 10 ns and beyond, showing effective suppression of autofluorescence at these time points (p. 6, Figure 7b). Furthermore, a person of ordinary skill in the art would have found it prima facie obvious to optimize the delay time to 25, 50, or 100 nanoseconds post excitation to further suppress autofluorescence while retaining sufficient TADF signal, as Ni teaches that the TADF emission persists well beyond 100 ns and that increasing the delay time improves the signal-to-background ratio (p. 6, Figure 7b; p.4-5, Figure 4-5). The optimization of the time gate delay is a result-effective variable that a person of ordinary skill would routinely optimize. The motivation to combine and reasonable expectation of success are the same as fully articulated above for claim 1.
Regarding claim 17, the claim recites, “The method of claim 1, wherein the TADF emitter is not
encapsulated in or attached to a polymer or a nanoparticle.” Ni et al. teaches the TADF emitter NID-TPP as a molecular compound that is used directly in solution for cellular imaging without encapsulation in a polymer matrix or nanoparticle. Specifically, the NID-TPP compound is dissolved in solution and applied directly to cells for incubation and imaging, not loaded into polymer nanoparticles or a polymer matrix (p. 6, Section 2.2). The motivation to combine and reasonable expectation of success are the same as fully articulated above for claim 1.
Regarding claim 18, the claim recites, “The method of claim 1, wherein the TADF emitter is covalently conjugated to the nucleic acid probe.” While Ni et al. does not teach conjugation to a nucleic acid probe, Sayyadi et al. teaches covalent conjugation of a luminescent emitter to a nucleic acid probe. Specifically, Sayyadi et al. discloses covalent conjugation of the BHHTEGST-Eu^3+ chelate to a DNA oligonucleotide via NHS ester chemistry, wherein the NHS-activated succinimide reactive group of europium chelate BHHTEGST reacts with a 5’-amino-modified oligonucleotide to form a stable covalent amide bond (p. 3, Section 2.1, Figure 1). As discussed in the motivation to combine set forth above for claim 1, it would have been prima facie obvious to a person of ordinary skill in the art to covalently conjugate the TADF emitter of Ni et al. to a nucleic acid probe using the NHS coupling chemistry taught by Sayyadi et al., with a reasonable expectation of success given that NHS ester conjugation to amino-modified oligonucleotides is a well-established and routine technique in the art. Sayyadi et al. confirms successful covalent conjugation, demonstrating >97% purity of the resulting luminescent emitter DNA conjugates (p. 4, Section 2.1). The motivation to combine is the same as fully articulated above for claim 1, and the expectation of success is further supported by the demonstrated efficiency and purity of the covalent conjugation in Sayyadi et al.
Regarding claim 20, the claim recites, “The method of claim 1, wherein the TADF emitter is directly conjugated to the nucleic acid probe through a bond or indirectly conjugated to the nucleic acid
probe through a linker.” Sayyadi et al. teaches direct covalent conjugation of the luminescent emitter (BHHTEGST-Eu^3+) to the DNA oligonucleotide through an amide bond formed via NHS ester chemistry between the succinimide reactive group of BHHTEGST and the 5’-amino group of the modified oligonucleotide (p. 3, Section 2.1, Figure 1). Sayyadi et al. further teaches that prior versions of the chelate used an intermediate linker, such as a tetra-ethylene glycol (TEG) linker, for conjugation (p. 2, Introduction). A person of ordinary skill in the art would have found it obvious, in the combination of Ni et al. and Sayyadi et al. set forth for claim 1, to conjugate the TADF emitter to the nucleic acid probe either directly through a bond or indirectly through a linker as taught by Sayyadi et al., with a reasonable expectation of success for the reasons fully articulated in the rejections of claims 1 and 18 above.
Regarding claim 30, the claim recites, “The method of claim 1, wherein the nucleic acid probe hybridizes to the analyte or product thereof.” Sayyadi et al. teaches that the nucleic acid probe (DNA oligonucleotide conjugated to the luminescent emitter) hybridizes directly to the target analyte (16S rRNA of S. aureus) in the biological sample via in situ hybridization (p. 6, Section 2.2, Figure 4A). The DNA probe sequence (SAU69) was specifically designed to be complementary to the ribosomal 16S rRNA of S. aureus (p. 3, Section 2.1). As discussed in the motivation to combine above for claim 1, it would have been obvious to person of ordinary skill in the art to employ the nucleic acid probe hybridization approach of Sayyadi et al with the TADF emitter of Ni et al. The motivation and expectation of success are the same as fully articulated above for claim 1.
Regarding claim 31, the claim recites, “The method of claim 1, wherein the nucleic acid probe is
conjugated to a binding moiety that directly or indirectly binds to the analyte or product thereof.” Sayyadi et al. teaches that the nucleic acid probe is itself the binding moiety that directly binds to the analyte (16S rRNA) through hybridization (p. 6, Section 2.2, Figure 4). In the combination of Ni et al. and Sayyadi at el., the nucleic acid probe serves as the binding moiety that directly binds to the target analyte. Furthermore, the specification of the present application at [0016] indicates that the nucleic acid probe can be conjugated to a binding moiety that directly or indirectly binds to the analyte or product thereof, and notes that the binding moiety can comprise an antibody. Sayyadi et al. teaches that the oligonucleotide probe sequence is designed to be complementary to and hybridize with the target rRNA sequence (p. 3, Section 2.1, Figure 1; p. 6, Section 2.2, Figure 4B,C), and the probe thus functions as a binding moiety that directly binds the analyte. It would have been prima facie obvious to a person of ordinary skill in the art to configure the nucleic acid probe of the Ni et al. and Sayyadi et al. combination to serve as a binding moiety for the analyte as taught by Sayyadi et al. The motivation and expectation of success are the same as fully articulated above for claim 1.
Regarding claim 33, the claim recites, “The method of claim 1, wherein the analyte comprises an mRNA.” Ni et al. teaches imaging in biological cells (HeLa cells) (p. 6, Section 2.2, Figure 7), and Sayyadi et al. teaches in situ hybridization detection of a nucleic acid analyte (16S rRNA) in bacterial cells (p. 6, Section 2.2). While Sayyadi specifically targets rRNA rather than mRNA, detecting mRNA by in situ hybridization is one of the most common and well-established applications of fluorescence in situ hybridization (FISH) technology in the biological arts (Levsky et al., p. 2833). A person of ordinary skill in the art would have found it obvious to apply the method of Ni et al. in view of Sayyadi et al. to detect mRNA as the analyte, because mRNA detection by in situ hybridization using labeled oligonucleotide probes was well-known at the time of the invention. The substitution of one nucleic acid analyte target (mRNA) for another (rRNA) in an in situ hybridization assay involves merely selecting a probe with an appropriate complementary sequence and would have been obvious to try with a reasonable expectation of success, as both rRNA and mRNA are detectable by nucleic acid probe hybridization in fixed cells. The motivation to combine Ni et al. and Sayyadi et al. remains as fully articulated above for claim 1.
Regarding claim 51, the claim recites, “The method of claim 1, wherein the product of the nucleic acid probe is generated in situ in the biological sample.” Sayyadi et al. teaches that the nucleic acid probe hybridizes to and detects its target (16S rRNA) in situ within bacterial cells fixed on a substrate (p. 6, Section 2.2, Figure 4A). The entire detection methodology of Sayyadi et al. is an in situ hybridization technique in which the probe-target complex (the product of the nucleic acid probe interaction) is both generated and detected in situ within the biological sample. As discussed in the motivation to combine above for claim 1, it would have been obvious to combine the TADF emitter of Ni et al. with the in situ hybridization detection approach of Sayyadi et al. The motivation and expectation of success are the same as fully articulated above for claim 1.
Regarding claim 71, the claim recites, “The method of claim 1, wherein the biological sample is a cell or tissue sample.” Ni et al. teaches imaging in cells, specifically HeLa cells (p. 6, Section 2.2, Figure 7), and Sayyadi et al. teaches in situ hybridization in bacterial cells (S. aureus) (p. 6, Section 2.2, Figure 4). The biological sample in both references is a cell sample. Thus, the combination of Ni et al. in view of Sayyadi et al. renders obvious a method wherein the biological sample is a cell sample. The motivation to combine and reasonable expectation of success are the same as fully articulated above for claim 1.
Claims 13 and 15 are rejected under 35 U.S.C. 103 as being unpatentable over Ni et al. (Adv. Opt. Master. 2019, 7, 1801519) in view of Sayyadi et al. (Analyst 2019, 144, 766) as applied to claims 1, 3, 6, 8, 17, 18, 20, 30, 31, 33, 51, and 71 above and further in view of Ishimatsu et al. (Angew. Chem. Int. Ed. 2014, 53, 6993).
Regarding claim 13, the claim recites, “The method of claim 1, wherein the TADF emitter comprises at least one electron donor which is a carbazole, phenoxazine, diphenylamine, triphenylamine, phenothiazine, diphenylacridine, phenazine, spiroacridine, acridine, or dimethylacridine moiety.” Ni et al. teaches a TADF emitter (NID-TPP) comprising a dimethylacridine (DMAC) electron donor moiety (p. 2, Scheme 1), which is one of the species recited in claim 13. However, the elected species is a carbazole moiety as the electron donor.
Ishimatsu et al. teaches TADF emitter molecules comprising carbazole as the electron donor moiety. Specifically, Ishimatsu et al. discloses a series of donor-acceptor TADF compounds including 2CzPN (1,2-bis(carbazole-9-yl)-4,5-dicyanobenzene), 4CzPN, 4CzIPN (1,2,3,5-tetrakis(carbazole-9-yl)-4,6-dicyanobenzene), and 4CzTPN, all of which comprise carbazole moieties as the electron donor (p. 6993, Figure 1; p. 6994). Ishimatsu et al. confirms that these carbazole-containing TADF emitters exhibit thermally activated delayed fluorescence with small singlet-triplet energy gaps (ΔE_ST) that facilitate efficient reverse intersystem crossing (p. 6994).
The combination of Ni et al. and Sayyadi et al., as set forth for claim 1 above, does not teach a TADF emitter comprising a carbazole electron donor moiety (the elected species). Ishimatsu et al. supplies this missing limitation. It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to substitute the dimethylacridine donor moiety in the TADF emitter of Ni et al. with a carbazole donor moiety as taught by Ishimatsu et al. A person of ordinary skill in the art would have been motivated to make this substitution because Ishimatsu et al. demonstrates that carbazole-based TADF emitters exhibit efficient thermally activated delayed fluorescence (p. 6994), and because the substitution of one known electron donor moiety for another in a donor-acceptor TADF framework represents a simple substitution of one known element for another to yield predictable results. Both dimethylacridine and carbazole are well-known electron donor groups in the TADF art, and a person of ordinary skill in the art would have had a reasonable expectation of success in preparing a TADF emitter with a carbazole donor for use in the time-resolved imaging method of Ni et al. in view of Sayyadi et al., given that Ishimatsu et al. demonstrates that carbazole-based TADF compounds exhibit the requisite small ΔE-ST and efficient TADF emission (p. 6994) and that the synthesis of carbazole-containing donor-acceptor TADF molecule was established in the art.
Regarding claim 15, the claim recites, “The method of claim 1, wherein the TADF emitter comprises at least one electron acceptor which is a cyanobenzene, dicyanobenzene, diphenyltriazine,
diphenylsulfone, naphthalimide, heptazine, triazine, dicyanoimidazole, curcuminoid, oxadiazole,
benzothiadiazole, pyrimidine, phenylbenzimidazole, dibenzodipyridophenazine, triarylboron, or
dicyanopyrazino phenanthrene moiety.” Ni et al. teaches a TADF emitter comprising a 1,8-naphthalimide electron acceptor moiety (p. 2, Scheme 1), which corresponds to the “naphthalimide” species recited in claim 15. However, the elected species is a dicyanobenzene moiety as the electron acceptor. Ni et al. does not specifically teach a TADF emitter comprising a dicyanobenzene electron acceptor.
Ishimatsu et al. teaches TADF emitter molecules comprising dicyanobenzene as the electron acceptor moiety. Specifically, the compounds 2CzPN, 4CzIPN, and 4CzTPN disclosed by Ishimatsu et al. each comprise a dicyanobenzene core as the electron acceptor (p. 6993, Figure 1). Ishimatsu et al. demonstrates that these dicyanobenzene-based TADF emitters exhibit efficient thermally activated delayed fluorescence and confirms the role of the dicyanobenzene acceptor in establishing the small ΔE_ST required for TADF (p. 6994).
The combination of Ni et al. and Sayyadi et al. does not teach a TADF emitter comprising a dicyanobenzene electron acceptor (the elected species).
Ishimatsu et al. supplies this missing limitation. It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to employ a TADF emitter comprising a dicyanobenzene electron acceptor, as taught by Ishimatsu et al., in the time-resolved imaging method of Ni et al. in view of Sayyadi et al. The substitution of one known electron acceptor moiety (naphthalimide, as in Ni et al.) for another (dicyanobenzene, as in Ishimatsu et al.) in a TADF emitter framework would have been obvious as a simple substitution of known elements with a reasonable expectation of success, given that Ishimatsu et al. demonstrates that dicyanobenzene-based TADF emitters exhibit the delayed fluorescence properties necessary for time-resolved imaging applications. See KSR, 550 U.S. at 416. A person of ordinary skill would have had a reasonable expectation of success because Ishimatsu et al. demonstrates efficient TADF emission from dicyanobenzene-acceptor compounds (p. 6994), confirming that such emitters possess the requisite photophysical properties (small ΔE_ST, efficient RISC, delayed fluorescence) for time-resolved luminescence imaging.
Claim 21 is rejected under 35 U.S.C. 103 as being unpatentable over Ni et al. (Adv. Opt. Master. 2019, 7, 1801519) in view of Sayyadi et al. (Analyst 2019, 144, 766) as applied to claims 1, 3, 6, 8, 17, 18, 20, 30, 31, 33, 51, and 71 above and further in view of Crucho et al. (Front. Chem. 2020, 8, 404).
Regarding claim 21, the claim recites, “The method of claim 1, wherein the detection of the fluorescence emitted by the TADF emitter is performed with the biological sample in a buffer comprising an oxygen scavenger.” Ni et al. teaches time-resolved imaging of TADF emitters in biological cells but does not explicitly teach performing the detection in a buffer comprising an oxygen scavenger. Sayyadi et al. similarly does not explicitly teach the use of an oxygen scavenger buffer.
Crucho et al. teaches that oxygen is a significant quencher of TADF emission in biological imaging applications. Specifically, Crucho discloses that the triplet excited states involved in the TADF mechanism are susceptible to quenching by molecular oxygen, which can significantly reduce the delayed fluorescence intensity and lifetime of TADF emitters in aqueous and biological environments (p. 2, Introduction, Col. 2). Crucho et al. explicitly states that the delayed fluorescence component “is extremely sensitive to the presence of oxygen” (p. 2, Introduction, Col. 2). Crucho et al. demonstrates this oxygen sensitivity experimentally by comparing emission spectra of TADF-loaded nanoparticles under aerated and degassed conditions, showing dramatically increased emission intensity upon removal of oxygen (p. 6, Figures 3 and 5). Crucho et al. notes that this oxygen quenching problem presents a challenge for the use of TADF emitters in biological imaging (p. 1; Abstract).
The combination of Ni et al. and Sayyadi et al., as applied to claim 1, does not teach performing the detection of TADF fluorescence in a buffer comprising an oxygen scavenger.
Crucho et al. identifies the problem (oxygen quenching of TADF triplet states) that motivates the claimed solution. It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to perform the TADF detection step of the method of Ni et al. in view of Sayyadi et al. in a buffer comprising an oxygen scavenger. A person of ordinary skill in the art would have been motivated to do so because Crucho et al. teaches that oxygen quenching of TADF triplet states is a recognized problem in biological imaging (p. 2, Introduction), and the use of oxygen scavenger systems (such as enzymatic oxygen scavenger buffers comprising protocatechuic acid and protocatechuate dioxygenase, or glucose oxidase/catalase systems) to reduce dissolved oxygen in fluorescence imaging buffers was well-known and routinely practiced in the fluorescence microscopy art at the time of the invention. The present specification at paragraphs [0015], [0064], and [0090]-[0093] describes various oxygen scavenger buffer compositions, confirming that such buffers were known in the art. A person of ordinary skill in the art, aware of the oxygen sensitivity of TADF emitters as taught by Cruhco et al., would have been motivated to employ an oxygen scavenger buffer during detection to preserve the TADF delayed fluorescence signal. A person of ordinary skill would have had a reasonable expectation of success given that oxygen scavenger buffers were commercially available and widely used in single-molecule fluorescence imaging applications, and Crucho et al. confirms that removal of oxygen dramatically improves TADF emission (p. 6, Figures 3 and 5).
Claims 35, 37, and 39 are rejected under 35 U.S.C. 103 as being unpatentable over Ni et al. (Adv. Opt. Master. 2019, 7, 1801519) in view of Sayyadi et al. (Analyst 2019, 144, 766) as applied to claims 1, 3, 6, 8, 17, 18, 20, 30, 31, 33, 51, and 71 above and further in view of Choi et al. (Nat. Biotechnol. 2010, 28, 1208).
Regarding claim 35, the claim recites, “The method of claim 33, wherein the nucleic acid probe
hybridizes to a primary probe or a product or complex thereof, wherein the primary probe hybridizes to the mRNA.” As discussed above regarding claim 33, Ni et al. in view of Sayyadi et al. renders obvious a method of detecting mRNA in a biological sample using a TADF emitter conjugated to a nucleic acid probe with pulsed excitation and time-gated detection. However, Ni et al. in view of Sayyadi et al. does not explicitly teach an indirect detection scheme in which the nucleic acid probe (conjugated to the TADF emitter) hybridizes to a primary probe or product/complex thereof, wherein the primary probe hybridizes to the mRNA.
Choi et al. teaches a method of in situ detection of mRNA using a multi-probe architecture in which primary probes (termed “probe sets”) hybridize directly to target mRNA sequences, and secondary detection components hybridize to the primary probes or their products. Specifically, Choi et al. discloses that RNA probe sets are designed to hybridize to target mRNA in fixed cells and tissues, with each probe set carrying an initiator sequence that triggers hybridization chain reaction (HCR) amplification upon binding to the target (p. 1208, Abstract; p. 1209, Figure 1c-e). Fluorescently labeled hairpin probes then hybridize to the initiator sequences of the primary probes (or their HCR amplification products), generating a detectable signal at the site of the target mRNA (p. 1209, Figure 1d).
The combination of Ni et al. and Sayyadi et al. does not teach the indirect architecture recited in claim 35. Choi et al. supplies this missing limitation.
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to incorporate the multi-probe detection architecture of Choi et al. into the time-resolved detection method of Ni et al. in view of Sayyadi et al. A person of ordinary skill in the art would have been motivated to do so because Choi et al. teaches that using primary probes that hybridize to the target mRNA, followed by hybridization of labeled detection probes to the primary probes or their amplification products, provides significant signal amplification and enables multiplexed detection of multiple mRNA targets simultaneously (p. 1208, Abstract).
A person of ordinary skill in the art would have recognized that combining the signal amplification advantages of Choi’s et al. multi-probe architecture with the autofluorescence-reduction advantages of TADF-based time-resolved detection from Ni et al. in view of Sayyadi et al. would yield a method with improved sensitivity and specificity for mRNA detection in situ. See KSR, 550 U.S. at 416. A person of ordinary skill would have had a reasonable expectation of success given that the probe hybridization and signal amplification steps are independent of the detection modality (fluorescence vs. time-resolved fluorescence). And substituting a TADF-labeled probe for conventional fluorophore-labeled probe in the detection step would not alter the upstream hybridization and amplification chemistry.
Regarding claim 37, the claim recites, “The method of claim 35, wherein the nucleic acid probe
hybridizes to a barcode sequence in the primary probe or product or complex thereof.” Choi et al. teaches that the primary probes carry unique initiator (barcode) sequences that are orthogonal to one another, enabling multiplexed detection of different mRNA targets. Specifically, Choi et al. discloses that each probe set carries a distinct initiator sequence that serves as a unique identifier (barcode) for the corresponding target mRNA, and that detection probes (fluorescent HCR hairpins) hybridize to these specific initiator/barcode sequences (p. 1209, Figure 1c-e; p. 1210; p. 1212). This teaches that the detection probe (analogous to the claimed nucleic acid probe) hybridizes to a barcode sequence in the primary probe or product/complex thereof. It would have been prima facie obvious for the same reasons set forth above regarding claim 35 to incorporate this barcode sequence feature into the method of Ni et al. in view of Sayyadi et al., as it enables multiplexed detection capability. The motivation and reasonable expectation of success are the same as fully articulated above for claim 35.
Regarding claim 39, the claim recites, “The method of claim 33, wherein the nucleic acid probe
hybridizes to an intermediate probe or a product or complex thereof, wherein the intermediate
probe hybridizes to a primary probe or a product or complex thereof, and wherein the primary
probe hybridizes to the mRNA.” Choi et al. teaches a multi-layered probe architecture for mRNA detection in which a primary probe hybridizes to the target mRNA, and an intermediate amplification step occurs before the labeled detection probe binds. Specifically, Choi et al. discloses a two-stage protocol in which: (1) primary probes hybridize to target mRNA (detection stage), and (2) HCR amplification generates extended products from the initiator sequences on the primary probes, to which fluorescent hairpin probes ultimately hybridize (amplification stage) (p. 1209, Figure 1c-e). The HCR amplification products serve as intermediate probes or products that bridge the primary probe and the final labeled detection probe. The fluorescently labeled hairpin probes (analogous to the claimed nucleic acid probe) hybridize to the HCR amplification products (intermediate products), which themselves are generated by hybridization to the initiator sequences on the primary probes, which hybridize to the mRNA.
The combination of Ni et al. and Sayyadi et al. does not teach the three-tier probe hierarchy recited in claim 39. Choi et al. supplies that limitation. It would have been prima facie obvious to a person of ordinary skill in the art to employ this multi-layered probe architecture of Choi et al. in the method of Ni et al. in view of Sayyadi et al. for the same reasons set forth above regarding claim 35, particularly because the multi-step amplification provides enhanced signal amplification for detecting low-abundance mRNA targets (p. 1208, Abstract; p. 1210, Figure 2m). The motivation and reasonable expectation of success are the same as fully articulated above for claim 35.
Claim 49 is rejected under 35 U.S.C. 103 as being unpatentable over Ni et al. (Adv. Opt. Master. 2019, 7, 1801519) in view of Sayyadi et al. (Analyst 2019, 144, 766) as applied to claims 1, 3, 6, 8, 17, 18, 20, 30, 31, 33, 51, and 71 above and further in view of Choi et al. (Nat. Biotechnol. 2010, 28, 1208). and Larsson et al. (Nat. Methods 2004, 1, 227).
Regarding claim 49, the claim recites, “The method of claim 39, wherein the product or complex of the intermediate probe is selected from the group consisting of: a rolling circle amplification (RCA) product, a complex comprising an initiator and an amplifier for hybridization chain reaction (HCR), a complex comprising an initiator and an amplifier for linear oligonucleotide hybridization chain reaction (LO-HCR), a primer exchange reaction (PER) product, and a complex comprising a pre-amplifier and an amplifier for branched DNA (bDNA).” As discussed above regarding claim 39, Ni et al. in view of Sayyadi et al. and Choi et al. renders obvious a method of detecting mRNA using a multi-layered probe architecture in which a nucleic acid probe hybridizes to an intermediate probe or product/complex thereof. Choi et al. teaches that the intermediate product/complex can comprise an initiator and an amplifier for HCR, which is one of the species recited in claim 49 (p. 1209, Figure 1a-d). However, the elected species is a rolling circle amplification (RCA) product.
Larsson et al. teaches in situ detection of individual nucleic acid molecules using RCA. Specifically, Larsson et al. discloses a method in which padlock probes are circularized on target nucleic acid sequences and subsequently amplified by RCA using Φ29 DNA polymerase, generating localized RCA products (concatemeric DNA molecules) that remain tethered at the site of the target nucleic acid in the cell (p. 227, Abstract; p. 227-228, Results, Figure 1). Larsson et al. further teaches that the RCA products are detected by hybridization of fluorescently labeled oligonucleotide probes to repetitive tag sequences within the RCA product (p. 228, Figure 1; p. 230, Table 1). In the context of claim 39, the RCA product of Larsson et al. serves as the product/complex of the intermediate probe (padlock probe), to which the labeled detection probe (analogous to the nucleic acid probe conjugated to the TADF emitter) hybridizes.
The combination of Ni et al., Sayyadi et al., and Choi et al. does not teach a RCA product as the elected species of intermediate probe product. Larsson et al. supplies this missing limitation. It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to substitute the HCR amplification method of Choi et al with the RCA amplification method of Larsson et al. in the method of Ni et al. in view of Sayyadi et al. and Choi et al. A person of ordinary skill in the art would have been motivated to make this substitution because Larson teaches that RCA provides highly localized signal amplification for in situ detection of individual nucleic acid molecules with high spatial resolution (p. 227, Abstract), and because RCA and HCR are both well-established signal amplification strategies for in situ nucleic acid detection that were recognized as interchangeable alternatives in the art. See KSR, 550 U.S. at 416. A person of ordinary skill in the art would have had a reasonable expectation of success given that both amplification methods generate localized products that can be detected by hybridization of labeled oligonucleotide probes. Larsson et al. demonstrates that RCA products produce strong, discrete fluorescent signals when detected by fluorophore-labeled probe hybridization in fixed cells (p. 229, Figure 3a), confirming that the RCA amplification and detection-by-hybridization strategy is compatible with the overall in situ detection workflow of the Ni et al./Sayyadi et al./Choi et al. combination. Substituting the RCA product for the HCR product as the intermediate product to which the TADF-conjugated nucleic acid probe hybridizes would predictably yield a method for detecting mRNA in situ with signal amplification and autofluorescence reduction.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
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Claims 1, 3, 6, 8, 13, 15, 17, 18, 20, 21, 30, 31, 33, 35, 37, 39, 44, 49, 51, and 71 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-7, 18, 19, 21, 24-26, 32-36, and 23 of copending Application No. 18/360,135 (reference application).
Although the claims at issue are not identical, they are not patentably distinct from each other because the copending application claims methods of analyzing biological samples using detectably labeled probes comprising thermally activated delayed fluorescence (TADF) emitters with time-resolved detection at one or more locations in the biological sample. The instant claims are directed to a species or obvious simplification of the genus claimed in the copending application.
Regarding instant claim 1, claims 1, 15, and 24 of the copending application (18/360,135) share the same limitations of contacting a biological sample with a probe that directly or indirectly binds to an analyte or product thereof, where the probe comprises a TADF emitter as the detectable label, and detecting the fluorescence emitted by the TADF emitter at a location in the biological sample at the time after excitation/stimulus. Claims 32-34 of the ‘135 application recite that the analyte is a nucleic acid analyte and that the detectably labeled probes bind to primary or intermediate probes, rendering the “nucleic acid probe: recitation of instant claims an obvious species within the ‘135 probe genus. The “pulsed light source” and detection “after a time period post excitation” are the expected mode of operation for time-gated detection of a TADF emitter having a long signal emission lifetime relative to autofluorescence, which are the same detection scheme defined by the t1/t2 intervals of ‘135. The recitation of sample autofluorescence is inherent to exciting a biological sample at fluorophore excitation wavelengths and does not add any patentably distinct step.
Regarding instant claims 3 and 8, claims 1, 18, and 19 of copending ‘135 share the central limitations of the detection window to lie temporally between the decay of the short-lived emission and the persistence of the long-lived TADF emission.
Regarding instant claim 6, claim 1 of copending ‘135 share the central limitations of the delivery of pulsed excitation followed by time-gated detection.
Regarding instant claims 13 and 15, claim 25 of copending ‘135 application share the central limitations of the following donor moieties (carbazole, phenoxazine, triphenylamine, acridine, and dimethylacridine) and acceptor moieties (cyanobenzene dicyanobenzene, diphenyltriazine, diphenylsulfone, naphthalimide, and dicyanopyrazino).
Regarding instant claim 17, claim 24 and 26 of ‘135 share the central limitations of the detectable labels comprise a TADF emitter.
Regrading instant claims 30, 31, 33, 35, 37, 39, 44, and 71, claims 1, 32, 33, 34, and 36 of ‘135 share the following similarities. Claim 1 of the ‘135 application recites that each detectably labeled probe is configured to directly or indirectly bind to a different analyte or product thereof, which encompasses both hybridization-based binding (claim 30 of ‘137) and binding through a conjugated binding moiety (claim 31 of ‘137). Claim 32 of the ‘135 application recites that each analyte is independently a nucleic acid analyte, which encompasses mRNA (claim33 of ‘137) and is broader than but includes the mRNA species. Claim 33 of ‘135 recites that each detectably labeled probe binds to a primary probe that directly binds to the corresponding analyte, or binds to an intermediate probe that binds directly or indirectly to a primary probe that directly binds to the analyte, which reads directly on claims 35 and 39 of ‘137. Claim 34 of ‘135 recites primary and intermediate probes comprising 3’/5’ overhangs with barcode sequences, which encompasses the barcode hybridization of instant claim 37 of ‘137. Claim 36 of ‘135 recites that the biological sample is an FFPE, frozen , or fresh tissue sample, which encompasses the cell or tissue sample of instant claim 71 of ‘137.
Regarding instant claim 49, claim 35 of ‘135 share the central limitations of the product of each analyte is a (RCA) rolling circle amplification product.
Regarding instant claim 51, claim 35 and 43 of ‘135 share the central limitations of the product generated in situ in the biological sample.
Claim 21 is rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 and 24 of copending Application No. 18/360,135 in view of Crucho et al. (Front. Chem. 2020, 8, 404).
Patent ‘135 does not require the use of an oxygen scavenger buffer (instant claim 21).The teachings of Crucho as they relate to this claim is given previously in this office action and are fully incorporated here.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Conclusion
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/BREANNA MARIE TAVERNINI/
Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682
