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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 7/13/2026 has been entered. It is noted that this entry enters the claims originally submitted on 6/10/2026.
Applicant’s arguments and amendments have been thoroughly reviewed and considered. Claims 1-8, 10-13, 20-22, and 43-47 are pending and are examined on the merits herein.
Response to Applicant’s Amendments
Nucleotide and/or Amino Acid Sequence Disclosure Objections
The specification was objected to for a defective Sequence Incorporation by Reference paragraph. In light of Applicant’s amendments to the specification submitted 6/10/2026, this objection has been withdrawn.
35 USC 112(b) Rejections
Claims 1-8, 10-13, 20-22, and 43-47 were rejected for various indefiniteness issues. In light of Applicant’s amendments to the claims submitted 6/10/2026, these rejections have been withdrawn, though see new grounds of rejection below.
35 USC 103 Rejections
Claims 1-3, 6-7, 10-13, 20-22, 43-44, and 47 were rejected under 35 U.S.C. 103 as being unpatentable over Halbert et al. (US 2016/0003835 A1) in view of Feagin et al. (WO 2019/236548 A1).
Claims 4 and 45 were rejected under 35 U.S.C. 103 as being unpatentable over Halbert et al. (US 2016/0003835 A1), in view of Feagin et al. (WO 2019/236548 A1), and further in view of Domenyuk et al. (US 2020/0376022 A1).
Claims 5 and 46 were rejected under 35 U.S.C. 103 as being unpatentable over Halbert et al. (US 2016/0003835 A1), in view of Feagin et al. (WO 2019/236548 A1), in view of Domenyuk et al. (US 2020/0376022 A1), and further in view of Santala et al. (Journal of Immunological Methods, 2004).
Claim 8 was rejected under 35 U.S.C. 103 as being unpatentable over Halbert et al. (US 2016/0003835 A1), in view of Feagin et al. (WO 2019/236548 A1), and further in view of Wanekaya et al. (US 2012/0088232 A1).
Applicant’s arguments and amendments have been thoroughly reviewed and considered. These rejections have been maintained. See “Response to Applicant’s Arguments” below.
Response to Applicant’s Arguments
Regarding the 35 USC 103 Rejections, Applicant argues that the combination of references does not teach the ordered method of claim 1, particularly steps (c)-(e) (Remarks, pages 9-10 and 11, para. 2). Applicant argues against para. 26 of the Final Rejection mailed 4/21/2026, specifically in that the Examiner allegedly takes official notice of what a person of ordinary skill in the art would have found obvious, that the rejection uses impermissible hindsight to draw from the teachings of the instant specification, and does not provide a reasonable expectation of success (Remarks, page 10). Applicant states that similar arguments apply for para. 27 of the Final Rejection, which discusses providing aptamer targets via fluid (Remarks, page 11, para. 1). Applicant similarly argues that the combination of Halbert in view of Feagin allegedly relies only on reasoning found in the Applicant's disclosure (page 11).
The reasoning provided in para. 26 of the Final Rejection does not rely on official notice, but rather utilizes the teachings of Halbert that already describe wells, as stated in para. 25 of the rejection. Specifically, Halbert teaches that wells can be used as substrates, and can be physical separations on an array. As the targets of Halbert are tethered to a substrate, it would thus be prima facie obvious that this substrate could be a well. Then, as the target/aptamer methods of Halbert described in para. 25 state that the aptamers bind to the tethered targets, this binding would also occur in the well, and the subsequent method steps described by Halbert and recited in the rejection would then continue to proceed in said wells. In further discussing this obviousness combination of the teachings of Halbert, reasoning is provided that would motivate the ordinary artisan to use said wells ("By utilizing wells, aptamer pools could easily be flowed or placed over the attached targets, and the well would provide a relatively easy surface to wash and remove unbound aptamers"). As Halbert already teaches the use of wells, there would be a reasonable expectation of success.
Regarding obviousness, MPEP 2144 I states, "The rationale to modify or combine the prior art does not have to be expressly stated in the prior art; the rationale may be expressly or impliedly contained in the prior art or it may be reasoned from knowledge generally available to one of ordinary skill in the art, established scientific principles, or legal precedent established by prior case law." Thus, Halbert does not need to provide an explicit motivation to use their target/aptamer methods with wells, and the Examiner has based their determination on the teachings of the reference and knowledge generally available to one of ordinary skill in the art. Regarding impermissible hindsight, MPEP 2145 X (a) states, ""[a]ny judgment on obviousness is in a sense necessarily a reconstruction based on hindsight reasoning, but so long as it takes into account only knowledge which was within the level of ordinary skill in the art at the time the claimed invention was made and does not include knowledge gleaned only from applicant’s disclosure, such a reconstruction is proper." In re McLaughlin, 443 F.2d 1392, 1395, 170 USPQ 209, 212 (CCPA 1971)." Nothing in the combination of teachings presented in para. 26 of the Final Rejection relied on teachings from the instant specification, and Applicant has not provided any specific teachings in the instant specification to further this point, nor has Applicant particularly pointed out what knowledge used in para. 26 of the Final Rejection the ordinary artisan would have allegedly lacked that would cast doubt upon the obviousness rejection.
Similar reasoning was used in para. 27 of the Final Rejection. Specifically, Halbert teaches microfluidics in conjunction with array-based assays (paras. 229 and 233). As established above, well assays may be used in Halbert (paras. 60 and 198). Para. 208 in particular states that, “…in certain embodiments, arrays are situated within microwell plates having any number of wells. In such embodiments, the bottoms of the wells may serve as surfaces for the formation of arrays.” Thus, as wells may be present in arrays, and the arrays of Halbert may be used in conjunction with microfluidic devices, it would be prima facie obvious to use microfluidics with wells. The Examiner also provides motivation to this point in the Final Rejection (para. 27, “This would allow for a more efficient introduction process that ensures that each well receives a similar amount and flow of fluid, and thus would ensure that similar amounts of aptamers are introduced into each well. This would reduce user error and create a more efficient methodology.”). The Examiner does not use official notice or improper hindsight reasoning regarding this combination of the teachings of Halbert.
Thus, these arguments are not considered persuasive.
Regarding the combination of Halbert in view of Feagin, the rationale of which is provided in para. 31 of the Final Rejection, similar reasoning was used to arrive at the obviousness determination. Namely, this rationale utilizes the teachings of the references with the skill, creativity, and knowledge of the ordinary artisan. MPEP 2141.03 I states, "'A person of ordinary skill in the art is also a person of ordinary creativity, not an automaton.' KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 421, 82 USPQ2d 1385, 1397 (2007). "[I]n many cases a person of ordinary skill will be able to fit the teachings of multiple patents together like pieces of a puzzle." Id. at 420, 82 USPQ2d 1397. Office personnel may also take into account "the inferences and creative steps that a person of ordinary skill in the art would employ." Id. at 418, 82 USPQ2d at 1396." Thus, it is the Examiner's position that the statements of motivation in para. 31 ("Specifically, both Halbert and Feagin teach aptamers with fixed sequences that can act as primer binding sites. Halbert does not particularly limit the amplification that may be performed in the methods of their invention (e.g. para. 39), and utilizing the bridge amplification teachings for aptamers of Feagin, the ordinary artisan would recognize that by having the primers for amplification attached to the well substrate, this would eliminate the need for the addition of primers later, particularly in free solution, thus simplifying the method. This also allows for amplification of the aptamers to occur on the wells, eliminating the need to capture aptamer candidates and amplify them outside of the wells, which would save on time and equipment. Keeping initial amplification on the wells also provides the efficiency benefits for sequencing described above in Feagin. These reasons would all be motivating to the ordinary artisan.") are conclusions that ordinary artisan would be capable of reaching.
Regarding impermissible hindsight in this scenario, Applicant points to para. 29 of the instant specification, which does note simplification and streamlining associated with their disclosed invention. However, the Examiner does not reference this paragraph in combining these references. MPEP 2144 II states, "'Indeed, we have repeatedly held that an implicit motivation to combine exists not only when a suggestion may be gleaned from the prior art as a whole, but when the ‘improvement’ is technology-independent and the combination of references results in a product or process that is more desirable, for example because it is stronger, cheaper, cleaner, faster, lighter, smaller, more durable, or more efficient. Because the desire to enhance commercial opportunities by improving a product or process is universal—and even common-sensical—we have held that there exists in these situations a motivation to combine prior art references even absent any hint of suggestion in the references themselves.'" Thus, as combining Halbert and Feagin in the manner suggested by the Examiner would result in a method that saves time and equipment and provides efficiency benefits, there is an implicit motivation that would be known to the ordinary artisan, and this is not considered impermissible hindsight reasoning.
Thus, these arguments are not considered persuasive.
Finally, in considering the combination of Halbert in view of Feagin in light of Applicant’s amendments to the claims and the recited ordered steps of claim 1, Applicant argues that the teachings of Feagin in particular do not recite the claimed order (Remarks, page 11, para. 2). However, Feagin, as stated in para. 31 of the Final Rejection, is used to add to the method of Halbert, where Halbert teaches attaching targets to well substrates, providing a fluidic pool of aptamer candidates to the target (where the aptamers contain adapters), washing away unbound aptamers, and generally performing PCR amplification and high-throughput sequencing to identify a selected group of aptamers (see paras. 25-29 of the Final Rejection).
Halbert is noted to not teach that primers are in a well or attached to a substrate before the addition of the aptamers, nor does the reference teach the use of bridge amplification or direct use of the well surface for amplification.
Feagin then teaches aptamers with adapters on their ends (thus making them similar to the aptamers of Halbert) and the formation of clusters of aptamers on a solid support generated via the use of bridge amplification, which occurs by hybridizing the adapters on the aptamers to sequences (which act analogously to the claimed primers) on the solid support. After this amplification, sequencing occurs on the support (see para. 30 of the Final Rejection).
In the Examiner’s discussion of the teachings of Feagin, paras. 60-63 are mainly contemplated. Applicant discusses Feagin’s use of non-natural nucleotides, but these are not required by all embodiments of the reference (note that paras. 65-66 discuss particular embodiments, and do not limit the entirety of the invention. Para. 78 of the reference also states that non-natural aptamers/nucleotides are present in some embodiments of their invention.). Even if the non-natural nucleotides were required by Feagin, which the Examiner does not assert, such an addition would still be encompassed by the method of instant claim 1, which comprises the listed steps, and so can include additional steps/modifications. These additional steps would not change the fact that the reference teaches amplifying aptamer candidates on a solid surface via bridge amplification, followed by sequencing, and this much is stated by Applicant on page 11 of their Remarks (“…Feagin's disclosure, which states that clusters of aptamer candidates are generated (see Feagin at [0059]-[0060], [0063]-[0064]), during which a non-natural nucleotide is introduced (see Feagin at [0065]-[0066]); then antisense oligonucleotides in the clusters are sequenced (see Feagin at [0061]),” emphasis added).
In combining Halbert in view of Feagin, the amplification and sequencing generally described by Halbert are performed as the described bridge amplification and subsequent sequencing of Feagin. As noted above in the response to Applicant’s arguments concerning the rationale behind the combination of Halbert in view of Feagin, this obviousness determination is considered proper. Given this, the combination of Halbert in view of Feagin teaches each limitation of instant claim 1 in the ordered manner provided by the claim.
Thus, these arguments are not considered persuasive.
Regarding claim 22, it is noted that this claim is a system claim that contains all of the components of claims 1 and 6, with the addition of a buffer. As noted in para. 39 of the Final Rejection, such a buffer is taught by Halbert. Thus, all the limitations of this claim are taught by Halbert in view of Feagin as they relate to this teaching and the rejections of claims 1 and 6 presented in the Final Rejection and asserted in the Examiner’s response above.
As none of Applicant’s arguments are considered persuasive, the 35 USC 103 Rejections of record are maintained.
Claim Objections
Claim 22 is objected to because of the following informalities: in the newly amended detection circuitry component, for clarity in line 3, it is recommended to amend “and then to sequence” to “and then sequencing.” Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 46 is 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 46 is rejected because it requires the cleaving of the first moiety from the primer “before performing the sequencing.” In claim 22, from which this claim depends, the detection circuitry is for sequencing any aptamer candidates “using operations including performing bridge amplification…and then to sequence any cluster of amplicons.” Thus, the sequencing of the aptamer candidates via the detection circuitry appears to include the use of bridge amplification. This bridge amplification would require the first and second primers to remain attached to the wells, as bridge amplification is performed on a solid substrate. However, in claim 46, it is not clear if “the sequencing” is referring to the overall sequencing of the detection circuitry (which would include bridge amplification), or the sequencing of the cluster of amplicons specifically (which would not include bridge amplification). In the former scenario, it does not appear that the bridge amplification and subsequent sequencing would be capable of functioning with the cleaving of claim 46, thus rendering the scope of the claim indefinite. It will be interpreted as though the latter scenario, where the sequencing of claim 46 only refers to the sequencing of the cluster of amplicons, is Applicant’s intention.
It is noted that if Applicant incorporates the Examiner’s suggestion described in the objection to claim 22 above, this 112(b) issue may still persist due to the use of the phrasing “detection circuitry to sequence any aptamer candidates” in claim 22. It is recommended to have the detection circuitry limitation in claim 22 use language such as “detection circuitry to perform operations including bridge amplification to generate a cluster of amplicons of any aptamer candidate within each of the wells and then sequencing any cluster of amplicons within each of the wells to identify any aptamers that are selective for the single target,” or similar phrasing. Such an amendment would likely aid in overcoming the indefiniteness related to “the sequencing” in claim 46 as well.
Claim Interpretation
It is noted that in the instant specification, an aptamer is defined as an oligonucleotide that has a tertiary structure causing that oligonucleotide to be selective for a target, and when an aptamer is selective for a target, this means that the aptamer couples to that particular target and not another target. An “aptamer candidate” is an oligonucleotide that may potentially be selective for a target (para. 51).
In newly amended claim 1, step (e) now specifies that only selective aptamer candidates are to be amplified within the wells. In the previous step, any aptamer candidates that are not coupled to the target (and thus are not selective for the target) are removed from the wells, thus leaving only coupled/selective aptamer candidates. Thus, step (e) would naturally only amplify selective aptamer candidates, as these would be the only aptamer candidates left in the wells.
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, 6-7, 10-13, 20-22, 43-44, and 47 are rejected under 35 U.S.C. 103 as being unpatentable over Halbert et al. (US 2016/0003835 A1) in view of Feagin et al. (WO 2019/236548 A1).
Halbert teaches methods for aptamer pools that bind to biomarkers of interest (Abstract). The aptamers are nucleic acid molecules having specific binding affinity to molecules through interactions other than classic Watson-Crick base pairing, and specifically can be DNA or RNA (paras. 7 and 21; instant claim 21). This specific binding affinity is considered to be selective as defined by the instant specification (see the “Claim Interpretation” above). This is further evidenced by para. 8 of Halbert, which states, “Aptamers…are capable of specifically binding to selected targets…” Halbert teaches method in which a target is tethered to a substrate and is contacted by a pool of aptamer candidates, and the aptamers which preferentially bind to the tethered target are isolated (paras. 56-57). Figure 13A and para. 125 show that this selection can involve a washing step to remove aptamer oligonucleotides unbound to a target. A similar method is shown in Figure 15B and para. 127. In particular, the method of para. 127 and Figure 15B recites identical interactions of the aptamer and target as in instant claim 1. The reference teaches the use of wells, particularly as substrates (e.g. paras. 27, 60, 101, 103, 112, and 198), and so it would be possible to tether the targets to a well substrate. Halbert also teaches that a plurality of wells may be used (paras. 208 and 421). The wells may act as physical separations and may be used with arrays (para. 208).
Thus, though Halbert does not explicitly teach the use of wells with the target/aptamer methods described above, it would have been prima facie obvious for the ordinary artisan to use a well substrate with these methods of Herbert, attach a target to said well substrate, and then analyze a pool of aptamer candidates to obtain and analyze aptamers as described in instant claim 1. By utilizing wells, aptamer pools could easily be flowed or placed over the attached targets, and the well would provide a relatively easy surface to wash and remove unbound aptamers.
Additionally, the teachings provided above do not explicitly state that the aptamer candidates are provided to the bound targets via a fluid. However, Halbert teaches the use of microfluidic devices with the planar substrates and array-based assays of their invention (para. 229 and 233). As established above, well assays may be used in Halbert (paras. 60 and 198). Para. 208 in particular states that, “…in certain embodiments, arrays are situated within microwell plates having any number of wells. In such embodiments, the bottoms of the wells may serve as surfaces for the formation of arrays.” Thus, it would also be prima facie obvious to use microfluidics to introduce aptamers candidates into the wells of Halbert, which can act as an array. This would allow for a more efficient introduction process that ensures that each well receives a similar amount and flow of fluid, and thus would ensure that similar amounts of aptamers are introduced into each well. This would reduce user error and create a more efficient methodology.
In methods of identifying the aptamer oligonucleotides, amplification and sequencing of the aptamers can be performed (paras. 39-40 and 55-56). Halbert teaches that aptamer candidates may contain fixed sequences that are hybridization sites for PCR primers (para. 145), where preferably, the candidates have both a randomized portion and fixed portions necessary for efficient amplification, where the fixed sequences are 5’ and 3’ terminal sequences (para. 146). In para. 298, Halbert teaches that aptamers can also have 5’ leader and 3’ tail sequences that can facilitate primer binding. Example 1 and paras. 444-448 of the reference describe a method in which a target is affixed to a solid substrate along with aptamer candidates that contain 5’ and 3’ primer tails, and during aptamer selection, the aptamers can be amplified via primer binding to said tails. Para. 198 also notes specifically that PCR can occur in wells on arrays. These 5’ and 3’ sequences on the aptamers are considered analogous to the claimed first and second adapters of the aptamers.
However, Halbert does not appear to teach that primers are in a well or attached to a substrate before the addition of the aptamers, nor does the reference teach the use of bridge amplification or direct use of the well surface for amplification.
Feagin teaches methods for screening aptamers to find those that have a binding affinity to a target molecule (Abstract). Specifically, the reference teaches the formation of clusters of aptamers on a solid support, where the aptamers can have adapter sequences at each end (para. 59). In para. 60, such cluster generation may be carried out via bridge amplification, where the aptamers are hybridized to short sequences on the solid support that contain sequences complementary to the first and section adapters (i.e. two distinct short sequences, one complementary to the first adapter and another complementary to the second, acting analogously to the claimed primers). Then, amplification and sequencing may occur (para. 61). Para. 63 of the reference notes that these methods may be performed within a flow cell that utilizes wells, where cluster generation creates many aptamers in each well that contain the same sequence. The reference teaches that their aptamer selection methods are efficient (paras. 56 and 72), and notes that performing clustering in wells specifically saves time during sequencing processes (para. 101).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to combine the teachings of Halbert and Feagin to arrive at the invention of instant claim 1. Specifically, both Halbert and Feagin teach aptamers with fixed sequences that can act as primer binding sites. Halbert does not particularly limit the amplification that may be performed in the methods of their invention (e.g. para. 39), and utilizing the bridge amplification teachings for aptamers of Feagin, the ordinary artisan would recognize that by having the primers for amplification attached to the well substrate, this would eliminate the need for the addition of primers later, particularly in free solution, thus simplifying the method. This also allows for amplification of the aptamers (via the adapter primer binding sites) to occur on the wells, eliminating the need to capture aptamer candidates and amplify them outside of the wells, which would save on time and equipment. Keeping initial amplification on the wells also provides the efficiency benefits for sequencing described above in Feagin. These reasons would all be motivating to the ordinary artisan. There would be a reasonable expectation of success as the methods of Feagin are specifically noted to work with aptamers of the same structure as in Halbert, and are also stated to specifically work with wells in a flow cell.
Therefore, claims 1 and 21 are prima facie obvious over Halbert in view of Feagin.
Regarding claims 2-3 and 43-44, para. 640 of Halbert teaches that when analytes (i.e. targets of interest) are attached to a substrate, it may be through biotin-streptavidin binding. Para. 338 notes that targets may be attached to substrates through a linker, where said linker may be a biotinylated nucleic acid. Therefore, it would be prima facie obvious that the target of Halbert in view of Feagin could be linked to a biotinylated nucleic acid that binds to a streptavidin coated substrate (i.e. streptavidin coated wells).
Regarding claim 6, it is noted that “detection circuitry” is not defined in the instant specification, and so is considered any technology that is within or attached to the substrate that allows sequencing data to be obtained. In Halbert, Example 17 shows an aptamer selection protocol. In Step 8, aptamers are tested against particular targets (paras. 812-814). Wells are used (para. 815). After the binding of the aptamer to the target and washing steps, sequencing is performed within the wells (para. 825). The sequencing results from the reactions in these wells can then be obtained and analyzed (paras. 823-830). Thus, Halbert teaches that their wells can be capable of use with sequencing technologies. It would therefore be prima facie obvious to use such a well array in the method of Halbert in view of Feagin described above, as this would prevent the need to remove the aptamer/target complex from the wells, cutting down on method steps and resources required. It is noted that the bridge amplification described by Halbert in view of Feagin already occurs in wells, and so would occur in this well array also.
Regarding claims 7 and 47, as noted above in the rejection of claim 1, Halbert and Feagin teach the use of microfluidic devices. Halbert teaches that such devices can control the flow of fluid, and describes designs of such devices that allow fluid to flow into multiple pluralities of channels and reaction sites (e.g. wells) before isolating each site and performing the desired reaction (para. 235). It would thus be prima facie obvious to design the microfluidic device in the combination of teachings of Halbert in view of Feagin described above using the teachings and already described microfluidic devices of Halbert, as such inventions have established success being used together.
Regarding claims 10-13, Halbert teaches that negative or positive selection of aptamers can be performed (Example 9). The focus of the negative selection is to remove aptamers that bind non-target antigen components of the final assay. The positive selection is to identify aptamers that bind the desired target (para. 494). These selections involve multiple rounds of aptamer recovery and PCR using standard methods (i.e. para. 494). Though Example 9 does not specify that wells are used, para. 351 notes the use of wells with selection methods, and para. 350 notes that multiple rounds of positive selection in particular can provide improved stringency of selection. Thus, the ordinary artisan would be motivated to perform the selection methods described by Halbert in the context of the method of Halbert in view of Feagin described above in the rejection of claim 1 – namely performing multiple rounds of positive selection on the aptamers in the plurality of wells (see para. 350), where aptamers are recovered and undergo PCR between rounds. As the purpose of the selection is to ensure that only aptamers that bind the desired target remain, it would be logical to do this before the bridge amplification of Halbert in view of Feagin, so that only the aptamers that have successfully been through the rounds of positive selection, and thus are most likely to truly be good aptamers for a target, would be used. The aptamer recovering is noted by Halbert to specifically not include the target (i.e. it would involve decoupling the target and the aptamer, para. 349). As the purpose of this selection is to recover and amplify the aptamers for additional exposure to the target, it would be prima facie obvious for the PCR to occur away from the wells so that targets or other reaction components are not inadvertently amplified, which would affect results. Halbert teaches standard PCR methodologies, and so the ordinary artisan would recognize a thermocycler could be used, for example.
Regarding claim 20, the instant specification states that spacers “may provide suitable distance between the oligonucleotide subsequence 171", 172", 173", 174" and capture primer adapter 311 or 312 such that capture primer adapter 311 may not inhibit the oligonucleotide subsequence from suitably coupling to target 160. Illustratively, spacer 300 may include 5 or more nucleotides, 10 or more nucleotides, or 15 or more nucleotides,” (para. 68). No specific definition is provided for this term, and there is no specific requirement for the sequences/structure of the spacers. Thus, any nucleic acid sequence can be considered to contain a spacer.
In Halbert, the primer tail adaptors are taught to be 5-10 nucleotides long (para. 444). In Feagin, these adaptor sequences can be 5-40 nucleotides long (para. 59). Thus, these adaptors can themselves can be considered to contain spacer sequences of a minimum amount as exemplified by the instant specification (e.g. 5 nucleotides) and still contain at least 5 nucleotides of adaptor sequence, while still meeting the adaptor requirements of the references. The use of such adaptors in Halbert in view of Feagin would therefore meet the requirements of instant claim 20 in view of the interpretation of spacers provided above.
Regarding claim 22, this claim is a system claim that contains all of the components of instant claim 6, in that there is a well substrate, targets and primers coupled in the well, a fluid of aptamer candidates introduced into the wells, and detection circuitry for sequencing the aptamers, where the structure of each component is stated to be the same as those in claim 6. Claim 22 also specifically notes that to remove aptamers that are not coupled to targets in the wells, a buffer must be used. The use of such a buffer for this purpose is taught by Halbert (e.g. para. 464). Thus, Halbert in view of Feagin teaches the methods of claims 1 and 6 as described above, as well as the additional limitations of claim 22, and so also teaches the system of claim 22.
Claims 4 and 45 are rejected under 35 U.S.C. 103 as being unpatentable over Halbert et al. (US 2016/0003835 A1), in view of Feagin et al. (WO 2019/236548 A1), and further in view of Domenyuk et al. (US 2020/0376022 A1).
Halbert in view of Feagin teaches the methods of claim 2 and 43 as described above, where streptavidin is covalently attached to the substrate (para. 126 and Figure 14). However, Halbert generally teaches that streptavidin may be attached to other components (e.g. a reporter in para. 184). Feagin also teaches the use of labeled/tagged primers and targets (e.g. paras. 9, 41, 67, and 75)
It is noted that regarding claims 4 and 45, if the well-attached first or second primer of Halbert in view of Feagin was attached to the target via biotin/streptavidin binding, the ordinary artisan would recognize that this would mean that the aptamer would be capable of binding to the target and primer sequence at the same time, as these two components would be in close proximity.
Domenyuk teaches methods and compositions for oligonucleotides that bind to targets of interest (Abstract). This can involve the use of aptamers (paras. 7-8 and 16). Figure 2A shows that a target for detection (in this example, a disease vesicle), can be attached to a capture agent bound to a substrate, where the capture agent can be an aptamer (para. 30). The reference teaches that the oligonucleotides of their invention can be modified to alter desired characteristics, where one such listed modification is to include streptavidin on an oligonucleotide (paras. 327-328). Domenyuk also teaches that to recover oligonucleotide probes that are bound to targets in a sample, a capture oligonucleotide can be used, where the captured oligonucleotide may be captured to a substrate (para. 571). The reference also teaches that aptamer candidates can contain biotin moieties, which can be used for capturing/anchoring the aptamers (para. 669).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Domenyuk, along with ordinary skill, knowledge, and creativity in the art, to arrive at the inventions of claims 4 and 45. Specifically, Domenyuk teaches that oligonucleotides can be attached to a substrate and can contain a streptavidin moiety on the end, as well as the use of biotin for capturing and anchoring. Halbert in view of Feagin also already teaches primer sequences that are attached to well surfaces and the biotin labeling of targets. As noted above, the ordinary artisan would recognize the benefit of attaching the primer and target to one another, as it would more easily allow for aptamer binding to both components. This would likely increase aptamer binding to appropriate targets, as the adapter on the aptamer would bind with the primer, and would ensure that the aptamer and target are in close proximity. This would increase the accuracy of the aptamer selection process. There would be a reasonable expectation of success as biotin/streptavidin binding is well-known in the art as evidenced by Halbert and Domenyuk, and the primers of Halbert in view of Feagin are already attached to a substrate.
Thus, claims 4 and 45 are prima facie obvious over Halbert, in view of Feagin, and further in view of Domenyuk.
Claims 5 and 46 are rejected under 35 U.S.C. 103 as being unpatentable over Halbert et al. (US 2016/0003835 A1), in view of Feagin et al. (WO 2019/236548 A1), in view of Domenyuk et al. (US 2020/0376022 A1), and further in view of Santala et al. (Journal of Immunological Methods, 2004).
Regarding claims 5 and 46, Halbert, in view of Feagin, and further in view of Domenyuk render obvious the methods of claims 4 and 45, where a primer is bound to streptavidin, and the streptavidin is bound to a biotin-labeled target.
However, none of these references teach that the streptavidin is removed from the oligonucleotide before sequencing occurs. Feagin teaches cleavage of sequences between an aptamer and primer sequence (e.g. para. 64 and 123), but this does not involve moiety cleavage. Halbert and Domenyuk also generally teach cleavable chemical linkages (e.g. Halbert paras. 211 and 612 and Domenyuk paras. 127 and 571).
Santala teaches methods for more efficiently removing targets from substrates (Abstract). Particularly, the reference notes that in washing methods to remove unbound or loosely bound targets from a substrate, the efficiency of the washing may be lowered due to tight moiety binding (page 159, column 2, para. 1). In this reference, streptavidin is linked to a bead via a linker, where the streptavidin then binds to a biotinylated target (Figure 1). Santala teaches the cleavage of this DNA linker to remove the streptavidin from the bead, rather than cleaving the streptavidin and biotin apart (Figure 1). Such cleavage can be done with a nuclease. By removing targets from beads in this manner, Santala teaches that high total recovery was achieved with low nonspecific background noise (page 162, column 2, para. 1).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Santala in the method of Halbert, in view of Feagin, and further in view of Domenyuk to cleave the streptavidin from the primer described above in the rejection of claims 4 and 45 before aptamer sequencing, and additionally before bridge amplification. As the biotin/streptavidin linkage connects the primer and the target, this binding would make it difficult for the primer sequence to participate in bridge amplification. By cleaving the streptavidin from the primer, the biotin/streptavidin/target complex could be moved, leaving the primers and aptamers and resulting in more efficient bridge amplification with less background noise. This efficiency would then extend to the sequencing of the clustered amplification products, and would be motivating to the ordinary artisan. There would be a reasonable expectation of success as the cleavage method of Santala specifically works on a bond between DNA and streptavidin, where the streptavidin is bound to biotin.
Thus, claims 5 and 46 are prima facie obvious over Halbert, in view of Feagin, in view of Domenyuk, and further in view of Santala.
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Halbert et al. (US 2016/0003835 A1), in view of Feagin et al. (WO 2019/236548 A1), and further in view of Wanekaya et al. (US 2012/0088232 A1).
Regarding claim 8, Halbert teaches that their aptamers may be generated via the SELEX process (para. 144). This process “is based on the unique insight that nucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (i.e., form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric.” Feagin also teaches that their aptamers can be developed with SELEX (para. 62). Halbert also defines aptamers as nucleic acids that have specific binding affinity through interactions other than Watson-Crick base pairing (para. 7). Thus, tertiary structure is important to aptamers generally. Halbert also teaches scenarios in which aptamer folding and rearrangement can occur (paras. 289 and 613-614). Halbert also teaches that aptamers may contain reporter elements, such as a fluorescent label (e.g. paras. 22, 24, and 113).
However, neither Halbert nor Feagin specifically teach that aptamers may change tertiary structure when coupled to a target.
Wanekaya teaches the use of aptamers for detecting components or molecules in a sample (Abstract). Specifically, the reference teaches that when an aptamer binds to a target molecule, the tertiary structure of said aptamer may be altered. This could in turn result in changes to reporter signals present on the aptamer (paras. 17-20).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Wanekaya to design aptamers that would undergo confirmational tertiary structure changes when bound to a target in the method of Halbert in view of Feagin. Wanekaya teaches that this type of confirmational change can involve changes to aptamer reporter signals, and thus could provide a clear indication to a user when target binding has occurred. By utilizing such a method in the methods of Halbert in view of Feagin, it could be noted when no aptamer binding has occurred, and would thus prevent a waste of resources and time from performing washing steps that are not necessary. This addition would also note when a large amount of aptamer binding has occurred, which would provide an early indicator of a promising aptamer candidate. As aptamers that undergo confirmational changes generally and the addition of reporters to aptamers are both well-known in the art, as evidenced by Halbert and Wanekaya, there would be a reasonable expectation of success.
Thus, claim 8 is prima facie obvious over Halbert, in view of Feagin, and further in view of Wanekaya.
Conclusion
No claims are currently allowable.
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/FRANCESCA FILIPPA GIAMMONA/Examiner, Art Unit 1681
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