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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 05/22/2026 has been entered.
Claim Status
Claims 9 and 16 are cancelled. Claims 2-3, 10-15, 17-39, 41-42, 44-46, 48-49, 52-63, 65-66, 68-84 were cancelled previously have been cancelled. Claims 1, 4-8, 40,43, 47, 50-51, 64, 67, and 85-88 are pending and under examination. Claims 87 and 88 are new. Claims 1, and 47 have been amended. Claims 1, 47, and 88 are independent claims.
Response to Arguments
Rejections withdrawn
The rejection claims 9 and 16 under 35 U.S.C. 103 as being unpatentable over Chee et al. is withdrawn following the applicant’s amendments and cancellation of the claims.
The rejection of claims 1, 4-9, 16, 40, 43, 47, 50-51, 64, 67 and 85-86 under 35 U.S.C. 103 as being unpatentable over Chee et al. (WO 2017/192633 A1, published Nov. 9, 2017, of record) as applied to claims 1, 4-9, 16, 40, 43, and 85-86 above and included here for reasons supra, in view of Landegren (US 2017/0211133 Al, published Jul. 27, 2017) is withdrawn for claims 47, 50-51, 64, and 67 following the applicant’s amendments to claim 47 and cancellation of claim 9 and 16. The architecture of the Landegren’s splint doesn’t include the composite UMI and/or barcode as required by amended claim 47.
Rejections Maintained
The rejection claims 1, 4-8, 40, 43, and 85-86 under 35 U.S.C. 103 as being unpatentable over Chee et al. is maintained.
Applicant's arguments filed 5/22/2026 have been fully considered but they are not persuasive. Applicant argues that “there is no suggestion in Chee that creating duplicate composite identifiers on both tags would provide any benefit, let alone ‘redundancy,’” and that the Examiner’s rationale is derived solely from Applicant’s own disclosure and thus constitutes impermissible hindsight reconstruction.
As an initial matter, Applicant’s factual premise is incorrect. Chee expressly discloses, in the context of transferring recording tag information to the coding tag (as an alternative to the reverse direction), that “this embodiment may be useful in cases where the recording tag sustains too much damage during Edman degradation process” (see pg. 145 1st para.). This is Chee’s own articulated recognition that capturing the pairing information via the reverse-direction transfer has independent value as a safeguard against loss or degradation of information carried by the other tag, the same type of benefit referenced in the prior rejection. Applicant’s assertion that this rationale “appears nowhere in Chee” is therefore factually inaccurate, and the rejection is not derived from Applicant’s disclosure.
Chee acknowledges that bidirectional transfer is the default mechanism of the constructs in their description of the coding tag starting on the bottom of page 131:
“A coding tag may include a terminator nucleotide incorporated at the 3' end of the 3' spacer sequence. After a binding agent binds to a macromolecule and their corresponding coding tag and recording tags anneal via complementary spacer sequences, it is possible for primer extension to transfer information from the coding tag to the recording tag, or to transfer information from the recording tag to the coding tag. Addition of a terminator nucleotide on the 3' end of the coding tag prevents transfer of recording tag information to the coding tag. It is understood that for embodiments described herein involving generation of extended coding tags, it may be preferable to include a terminator nucleotide at the 3' end of the recording tag to prevent transfer of coding tag information to the recording tag.”
Emphasis added to the affirmative modification necessary to prevent bidirectional information transfer.
To the extent Applicant argues that Chee presents the two directions of transfer as alternative embodiments rather than as a combined, simultaneous bidirectional operation, the Examiner agrees that Chee’s disclosure frames the reverse direction embodiment as useful when the primary (forward-direction) information may be compromised rather than as a feature to be layered onto the forward direction for its own sake. This distinction, however, does not defeat the obviousness rationale. A motivation to combine or modify prior art teachings need not be expressly stated in the reference itself, nor must it mirror the applicant’s own stated purpose for the combination; it is sufficient that the rationale have “rational underpinning” grounded in the knowledge of one of ordinary skill in the art, the nature of the problem to be solved, or the predictable use of known elements according to their established functions. See KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). Here Chee’s own disclosure established that the annealed recording tag/coding tag junction is inherently competent to support primer extension in either direction from its complementary sequences, and that a terminator nucleotide must be affirmatively incorporated to suppress transfer in one of the two directions (see Chee pg. 131 last para.). Bidirectional information transfer is accordingly not a modification requiring an independent motivation drawn from Chee’s stated purpose; it is the unmodified default behavior of the disclosed structure absent the optional blocking modification and selection of the unmodified embodiment, one of two predictable outcomes expressly identified by Chee itself (block one direction or do not), involves the exercise of no more than ordinary skill. For at least this reason, the rejection is not the product of impermissible hindsight, and Chee’s express reference to preserving information against damage or loss (cited above) provides further corroborating support for why a person of ordinary skill would have recognized the benefit of the composite information being independently recoverable from either tag.
The applicant’s argument that “the claimed invention requires that during step (b), information is transferred bidirectionally such that both the polypeptide nucleic acid tag and the moiety nucleic acid tag independently acquire the composite UMI/barcode before dissociation,” this is not the case as the claim has been amended by the Applicant to remove the portion stating “or ligating the polypeptide nucleic acid tag and the moiety nucleic acid to form a composite UMI and/or barcode, wherein the composite UMI and/or barcode comprises both the polypeptide UMI and/or barcode and the moiety UMI and/or barcode” and therefore each tag independently acquiring the composite UMI/barcode is no longer a limitation of the claim. Furthermore, the ligation-based methods are no longer a component of claim 1 either and Applicant’s argument pertaining to the ligation-based methods of Chee are also moot.
The Applicant’s argument that step (c) of new claim 88 requires steps not part of the current rejection is moot as new claim 88 was not part of the rejection.
Accordingly, the rejection claims 1, 4-9, 16, 40, 43, and 85-86 under 35 U.S.C. 103 as being unpatentable over Chee et al. is maintained.
New Objections
Claim Objections
Claim 47 is objected to because of the following informalities: typographical error.
The claim repeatedly recites “-staid” which should be removed all together. The previous claim set dated 10/24/25 had “.
Appropriate correction is required.
New Rejections
Claim Rejections - 35 USC § 112
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 1, 7, and 8 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 recites the limitation "the composite UMI and/or barcode" in steps c) and d). There is insufficient antecedent basis for this limitation in the claim. The portion of claim 1 that provided antecedent basis for this limitation was removed from claim 1 step b) in the most recent amendment.
Claim 7 recites the limitation "the composite UMI and/or barcode" in line 4. There is insufficient antecedent basis for this limitation in the claim.
Claim 8 recites the limitation "the composite UMI and/or barcode" in line 3. There is insufficient antecedent basis for this limitation in the claim.
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 text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 1, 4-8, 40,43, 47, 50-51, 64, 67, and 85-88 are rejected under 35 U.S.C. 103 as being unpatentable over Chee (WO 2017/192633 A9, published Nov. 9, 2017, on IDS 07/19/2021) in view of Peterson et al. (US 2018/0208975 A1, published Jul. 26, 2018).
Chee discloses a method in which a recording tag associated with a polypeptide and a coding tag associated with binding agent (itself a polypeptide) anneal via complementary sequence to form a linked structure, and primer extension transfers identifying information between the two tags (see Chee para. spanning pgs. 131-132). Chee’s disclosure established that absent an affirmatively incorporated terminator nucleotide blocking one 3’ end, the annealed junction is inherently competent to support primer extension bidirectionally, each tag’s free 3’-OH end serves as a primer using the other tag as a template.
Peterson discloses multi-analyte intermolecular region (IMR)/ unique molecular identifier (UMI) proximity probe architecture in which nucleic acid tags conjugated to binding agents for DNA, RNA, and protein targets are brought into proximity, hybridize, and undergo bidirectional polymerase extension so that each tag acquires a copy of the other’s barcode sequence.
In regards to claim 1, Chee teaches “a method for assessing identity and spatial relationship between a polypeptide and a moiety in a sample, wherein the moiety comprises another polypeptide or a polynucleotide” (see Chee, pg. 1 line 12, pg. 4 line 17-22).
In regards to the limitation “a) forming a linking structure between a site of a polypeptide in a sample and a site of a moiety in the sample, the linking structure comprising a polypeptide nucleic acid tag attached to the site of the polypeptide and a moiety nucleic acid tag attached to the site of the moiety, wherein the polypeptide nucleic acid tag and the moiety nucleic acid tag are attached to each other, the polypeptide nucleic acid tag comprises a polypeptide unique molecule identifier (UMI) and/or barcode, and the moiety nucleic acid tag comprises a moiety UMI and/or barcode;” Chee’s recording tag (attached to the site of the polypeptide via the binding event/attachment chemistry disclosed therein) and coding tag (attached to the site of the moiety, i.e., the binding agent/antibody) anneal to one another via complementary sequences, forming the linking structure. Each tag comprises a UMI and/or barcode sequence (see Chee Fig. 3).
In regards to limitation “b) transferring information bidirectionally between the polypeptide nucleic acid tag and the moiety nucleic acid tag”, Chee teaches embodiments transferring information in each direction (coding tag to recording tag as the primary embodiment (see Chee Fig. 3), and recording tag to coding tag in a separate embodiment (see Chee pg. 145 1st para.)), combined with Chee’s own teaching that the unmodified annealed junction (without the optional terminator nucleotide) supports extension from either 3’ end (see Chee para. spanning pgs. 131-132). Therefore Chee teaches instance where extending either tag or both tags is beneficial and teaches that bidirectional extension is the default when using complementary sequences between tags as the primer location. It would have been obvious to one of ordinary skill in the art to omit the optional terminator-nucleotide blocking modification, yielding the default bidirectional behavior of Chee’s own disclosed structure, particularity in light of Chee’s own recognition of the value of capturing information via the reverse direction as a hedge against loss of information from the primary direction. Peterson corroborates that bidirectional extension between two annealed complementary-end oligonucleotide probes was a known, predictably successful technique for proximity-based analyte detection generally, supporting a reasonable expectation of success (see Peterson Fig. 2).
In regards to limitation “c) breaking the linking structure via dissociating the polypeptide from the moiety and dissociating the polypeptide nucleic acid tag from the moiety nucleic acid tag, while maintaining attachment between the polypeptide and the polypeptide nucleic acid tag, and maintaining attachment between the moiety and the moiety nucleic acid tag, wherein the polypeptide nucleic acid tag comprises the composite UMI and/or barcode and the moiety nucleic acid tag comprises the composite UMI and/or barcode;” Chee discloses releasing the bound binding agent from the polypeptide following information transfer (see Chee Figs. 32-33). Applying this disclosed release step to the bidirectionally-extended tag pair of limitation b), rather than to Chee’s unidirectionally extended pair, results in dissociation of the polypeptide from the moiety and the two tags from one another, with the recording tag remaining attached to the polypeptide and the coding tag remaining attached to the moiety/binding agent, each independently carrying the sequence information generated during the bidirectional extension step.
In regards to step “d) determining a sequence of the polypeptide nucleic acid tag and at least a partial sequence of the polypeptide, and assessing the moiety tag and at least a partial identity of the moiety, wherein the assessed portions of the polypeptide tag and the moiety tag comprise the shared unique molecule identifier (UMI) and/or barcode indicates that the site of the polypeptide and the site of the moiety in the sample are in spatial proximity” Chee teaches sequencing /readout of the extended recording tag and under the combination above, corresponding sequencing of the extended coding tag, each carrying the composite sequence, with successful paring of the two composite sequences evidencing that the recording tag site and coding tag site were in spatial proximity sufficient for annealing and extension to occur (e.g., “identifying information on the recording tags comprising barcodes can be used to map the extended coding tag or di-tag sequence reads back to the originating macromolecule”) (see Chee pg. 60, last para.; pg. 91, ¶ 1, pg. 145, ¶1).
Thus the limitations of amended claim 1 are either expressly taught or made obvious by Chee, and the claim is unpatentable over Chee.
In regards to claim 4, as discussed above in regards to claim 1, Chee teaches dissociating interacting biomolecules following proximity-dependent barcode formation and sequencing the resulting nucleic acid constructs to determine molecular identity and spatial relationships. Claim 4 further recites that the polypeptide and moiety are dissociated from each other and immobilize on a support prior to determining at least a partial sequence of each.
Immobilization of biomolecules or nucleic acid constructs on solid supports prior to sequencing or amplification was well-known in the art at the time of filling. Solid supports such as beads, planar substrates, or flow-cell surfaces were routinely used to facilitate washing, amplification, spatial segregation, and sequencing reactions. It would have been obvious to a person of ordinary skill in the art to immobilize the dissociated polypeptide and moiety, or their associated nucleic acid tags, on a support prior to sequencing in order to facilitate handling , purification, or sequencing, representing a predictable use of known solid-phase techniques in Chee’s proximity-based tagging systems. Furthermore, Chee teaches a method, wherein the polypeptide (e.g., peptide) and moiety (e.g., binding agent) are dissociated from each other and immobilized on a support (e.g., bead) prior to assessing at least a partial sequence of the polypeptide or the moiety (see Figs. 24, 26, 28-36 , pg. 29 line 25, and throughout).
In regards to claim 5, Chee expressly discloses embodiments wherein polypeptides are fragmented using a protease and analyzing extended tags after fragmentation (see Figs. 1-2, 18-23, 29, 45, pg. 16 lines 27-30, pg. 28 line 1-pg. 29 line 24, and throughout).
In regards to claim 6, Chee expressly discloses that the recording tag anneals to the coding tag via a complementary spacer sequence and that primer extension occurs using the spacer as a priming site (see Fig 5, pg. 30 line 27 – pg. 31 line 17, and throughout). Thus Chee discloses that the two nucleic acid tags comprise complementary sequences and are attached to each other via complementary base pairing prior to extension. This structure reads directly on the claimed “first and second polynucleotides comprise a complementary sequence” and “attached via the complementary sequence.” No further modification of the base combination applied to claim 1 is required to meet this limitation, as such, claim 6 is rejected on the same combination and rationale set forth above with respect to claim 1.
In regards to claim 7, this claim further requires that transferring information between the polypeptide nucleic acid tag and the moiety nucleic acid tag (per claim 1’s step (b)) comprises extending both the first polynucleotide and the second polynucleotide, such that the composite UMI and/or barcode is formed by extension of each.
This limitation is taught by the same combination and rationale applied to claims 1’s limitation (b) above: Chee discloses that the annealed recording tag/coding tag junction (i.e., the hybridizes first and second polynucleotide of claim 6, presents a free 3’-OH end on each strand, positioned to serve as a primer using the complementary strand as template, such that primer extension is inherently available to proceed from either 3’ end absent the affirmative incorporation of a terminator nucleotide blocking one direction (see Chee para. spanning pgs. 131-132). Extension of both the first polynucleotide (using the second as a template) and the second polynucleotide (using the first as a template), i.e., extension of both stands, is accordingly the default unmodified outcome of Chee’s disclosed annealed structure, and results in each of the first and second polynucleotides independently acquiring the composite sequence, as claimed. Peterson is cited to further corroborate that bidirectional polymerase extension of two annealed, complementary end oligonucleotide probes, with each probe’s oligonucleotide being independently extended, was an art-recognized and predictable successful technique, supporting a reasonable expectation of success in arriving at the claimed extension of both polynucleotides. Claim 7 is therefore rejected on the same combination applied to claims 1 and 6, for the reasons discussed above with respect to limitation (b) of claim 1.
In regards to claim 8, the claim requires that determining the sequence of the polypeptide nucleic acid tag and the moiety nucleic acid tag (per claim 1’s step (d)) comprises sequencing the composite UMI and/or barcode. This limitation is taught by the same combination applied to claim 1’s limitation (d) above. Chee discloses sequencing the extended recording tag to obtain the identifying information transferred to it, e.g., UMI and/or barcode, and under the combination applied to claim 1, the extended coding tag is correspondingly sequenced (see Chee 48th-50th embodiments, page 12-13). Because the composite UMI and/or barcode is, under the combination, the sequence information generated by and appended to each tag during the bidirectional extension step of claim 1’s limitation (b), sequencing of either extended tag necessarily comprises sequencing of the composite UMI and/or barcode. The composite sequence is not separable from, but rather constitutes, the newly extended portion of each tag that Chee’s disclosed sequencing step reads out. Peterson is cited to further corroborate that sequencing of the extended, composite portion of a proximity probe following bidirectional extension was an art-recognized readout technique (see Peterson [0004]). Claim 8 is therefore rejected on the same combination and rationale applied to claim 1, for the reasons discussed above with respect to limitation (d). Chee expressly teaches analyzing or sequencing the extended nucleic acid tags.
In regards to claim 40, Chee discloses analyzing protein complexes and macromolecular interactions, wherein binding agents associated with coding tags bind to macromolecules and information transfer occurs when the bound molecules are in proximity (see Fig. 1-2). Such embodiments involve a moiety that is at least “in close proximity” to the polypeptide. Accordingly, Chee teaches the limitations recited in claim 40.
In regards to claim 43, Chee expressly discloses binding agents comprising coding nucleic acid tags that bind to macromolecules associated with recording tags (see e.g. Figs. 1 & 2). Chee further discloses that the recording tag anneals to the coding tag and that primer extension or ligation transfers coding information to generate an extended recording tag (see e.g. Figs. 5 & 6). Chee further discloses analyzing the extended recording tag via nucleic acid sequencing (see embodiments 48-53, and 93-96).
In regards to claim 47, Peterson discloses a modified proximity extension assay for protein detection (see Peterson Figs. 2-3, [0006]-[0007], [0029]) employing two antibodies Ab-Oligo A and Ab-Oligo B, each independently conjugated to its own oligonucleotide via art-known conjugation chemistry (see Peterson [0022]-[0026]) prior to use. Oligo A comprises from 5’ to 3’ universal primers, Ab barcode A, and Oligo A/Oligo B complementary sequence; Oligo B comprises from 5’ to 3’ extension product and protein detection probe complementary sequence, Ab barcode B, Oligo A/Oligo B complementary sequence (see Peterson [0006]). Because the mutually complementary annealing region is positioned at the 3’ terminus of both oligonucleotides, and Peterson discloses no terminator nucleotide, dideoxynucleoside, or other blocking modification at either 3’ end, the annealed Oligo A/Oligo B junction is structurally symmetrical and inherently competent to support primer extension from both 3’ ends simultaneously, each oligonucleotide independently serving as primer using the other as a template. Peterson confirms that, upon proximity of the two antibodies, “a signal is produce” and the oligonucleotide “form a DNA template through… extension reaction” (see Peterson [0029]). Figure 2’s illustration of a single extension product (“Oligo A extension product” reflect one arm of this inherently symmetric reaction for ease of illustration, consistent with Peterson’s disclosed goal of apply this chemistry to highly multiplexed panels (see Peterson [0001]-[0003], [0014]) in which the arbitrary A/B labeling has no structural significance.
Peterson’s disclosed assay is designed and optimized for a discovery/screening context, highly multiplexed panels of many antibodies applied broadly across a sample to detect an unknown or unlimited number of proteins (see Peterson [0001]-[0003], [0014]). It was well known in that, however, and had been for decades prior to the priority date of the instant application, to instead perform targeted, hypothesis-driven verification of a specific already suspected pair of interacting proteins, rather than broad blind multiplexed screening, as exemplified by the yeast two-hybrid system1 and its many known variants, which have been standard tools for confirming a specific suspected protein-protein interaction since the late 1980s. A person of ordinary skill in the art seeking to adapt Peterson’s proximity-extension chemistry to this alternative, targeted verification use case, confirming whether two specific, already identified candidate proteins interact, rather than screening broadly across a multiplexed panel, would have had reason to depart from Peterson’s general multiplexed panel workflow and instead pre-form, as a single dedicated reagent, the specific Oligo A/Oligo B pair correspond to the two candidate proteins under investigation. Pre-forming and bidirectionally extending this dedicate pair prior to conjugation and sample exposure, rather than after, as in Peterson’s general multiplexed workflow, would allow the reagent’s proper function to be verified by the practitioner at the bench before that reagent is ever committed to the biological sample, avoiding the risk of consuming a limited, valuable, or difficult to obtain clinical or experimental sample (see Peterson [0001] describing the scarcity and small size of relevant clinical samples) on a reagent whose function has not yet been confirmed. This is a straightforward application of Peterson’s own disclosed, unmodified chemistry to a known, alternative use context (targeted interaction verification, as opposed to blind multiplexed screening) for its known purpose, yielding the predictable results of a pre-validated composite-bearing reagent pair. See KSR International Co. v. Teleflex Inc. (KSR), 550 U.S. 398, 82 USPQ2d 1385 (2007) (“if a technique has been used to improve one device, and a person of ordinary skill in the art would recognize that it would improve similar devices in the same way, using the technique is obvious”; MPEP 2143 (I)(A), (E).
In regard to step “a) providing a pre-assembled structure comprising a composite unique molecule identifier (UMI) and/or barcode comprising a middle portion flanked by a polypeptide nucleic acid tag on one side and a moiety nucleic acid tag on the other side, wherein the polypeptide nucleic acid tag comprises a polypeptide UMI and/or barcode, the moiety nucleic acid tag comprises a moiety UMI and/or barcode, and the composite UMI and/or barcode comprises both the polypeptide UMI and/or barcode and the moiety UMI and/or barcode, and wherein the pre- assembled structure is configured such that upon dissociation of the polypeptide nucleic acid tag from the moiety nucleic acid tag, each of the polypeptide nucleic acid tag and the moiety nucleic acid tag independently comprises the composite UMI and/or barcode,” Peterson discloses that Oligo A (polypeptide tag, comprises Ab barcode A) and Oligo B (moiety tag, comprises Ab barcode B), annealed to one another via their complementary sequence, and bidirectionally extended as set forth above such that each oligonucleotide acquires a copy of the others sequence, forming a composite present on both resulting strands, performed as a dedicated, pre-conjugation, pre-validation step for a targeted verification assay directed to a specific candidate pair of suspected interacting proteins, for the reasons discussed above. Each tag comprises its own original barcode plus, from this pre-conjugation extension, a copy of the composite sequence satisfying the requirement that each independently comprise the composite upon later dissociation.
In regards to limitations of step “b) forming a linking structure between a site of a polypeptide in a sample and a site of a moiety in the -staid sample by attaching the said polypeptide nucleic acid tag of the said pre- assembled structure to the site of the -staid polypeptide and attaching the -staid moiety nucleic acid tag of the pre-assembled structure to the site of the moiety,” is taught by the conjugation of the pre-formed, bidirectionally extended, Oligo A and Oligo B to a first antibody (claimed “polypeptide”) and a second antibody (claimed “moiety”), respectively using the antibody-oligonucleotide conjugation chemistry disclosed by Peterson (see Peterson [0022]-[0026]), followed by binding of both antibodies to their target in the sample such that the two antibodies and their conjugated tags are brought into proximity (see Peterson [0029]).
In regards to the limitations of step “c) breaking the linking structure via dissociating the polypeptide from the moiety and dissociating the polypeptide nucleic acid tag from the moiety nucleic acid tag, while maintaining attachment between the -staid polypeptide and the polypeptide nucleic acid tag, and maintaining attachment between the moiety and the moiety nucleic acid tag, wherein the polypeptide nucleic acid tag comprises the composite UMI and/or barcode and the moiety nucleic acid tag comprises the composite UMI and/or barcode”, Peterson teaches denaturation of the annealed Oligo A/Oligo B duplex (a standard step in any PCR-based or sequencing based readout of the extension product (see Peterson [0029]), which separates the two tags from one another. Because Oligo A and Oligo B are never covalently joined to one another or toe each other’s antibody, each oligonucleotide is only conjugated to its own respective antibody, and the two antibodies are held together only transiently via their independent, non-covalent binding to the targets and via the annealed oligo junction, denaturation of that junction results in breaking of the linking structure while each (polypeptide and moiety) tag being associated with their respective covalently bound antibody. Because bidirectional extension occurred prior to conjugation and sample binding, each tag independently retains the composite sequence upon this separation, as claimed.
In regards to limitation of step “d) determining a sequence of the polypeptide nucleic acid tag and at least a partial sequence of the polypeptide, and determining a sequence of the moiety nucleic acid tag and at least a partial sequence of the moiety, wherein the determined sequences of the polypeptide nucleic acid tag and the moiety nucleic acid tag each comprise a sequence of the composite UMI and/or barcode, indicating that the site of the polypeptide and the site of the moiety are in spatial proximity in the sample,” Chee teaches sequencing /readout of the extended recording tag and under the combination above, corresponding sequencing of the extended coding tag, each carrying the composite sequence, with successful paring of the two composite sequences evidencing that the recording tag site and coding tag site were in spatial proximity sufficient for annealing and extension to occur (e.g., “identifying information on the recording tags comprising barcodes can be used to map the extended coding tag or di-tag sequence reads back to the originating macromolecule”) (see Chee pg. 60, last para.; pg. 91, ¶ 1, pg. 145, ¶1).
In regards to claim 50, Chee expressly discloses embodiments wherein polypeptides are fragmented using a protease and analyzing extended tags after fragmentation (see Figs. 1-2, 18-23, 29, 45, pg. 16 lines 27-30, pg. 28 line 1-pg. 29 line 24, and throughout).
In regards to claim 51, the claim requires that determining the sequence of the polypeptide nucleic acid tag and the moiety nucleic acid tag (per claim 47’s step (d)) comprises sequencing the composite UMI and/or barcode. This limitation is taught by the same combination applied to claim 1’s limitation (d) above. Chee discloses sequencing the extended recording tag to obtain the identifying information transferred to it, e.g., UMI and/or barcode, and under the combination applied to claim 1, the extended coding tag is correspondingly sequenced (see Chee 48th-50th embodiments, page 12-13). Because the composite UMI and/or barcode is, under the combination, the sequence information generated by and appended to each tag during the bidirectional extension step of claim 1’s limitation (b), sequencing of either extended tag necessarily comprises sequencing of the composite UMI and/or barcode. The composite sequence is not separable from, but rather constitutes, the newly extended portion of each tag that Chee’s disclosed sequencing step reads out. Peterson is cited to further corroborate that sequencing of the extended, composite portion of a proximity probe following bidirectional extension was an art-recognized readout technique (see Peterson [0004]). Claim 8 is therefore rejected on the same combination and rationale applied to claim 47, for the reasons discussed above with respect to limitation (d). Chee expressly teaches analyzing or sequencing the extended nucleic acid tags.
In regards to claim 64, Chee discloses analyzing protein complexes and macromolecular interactions, wherein binding agents associated with coding tags bind to macromolecules and information transfer occurs when the bound molecules are in proximity (see Fig. 1-2). Such embodiments involve a moiety that is at least “in close proximity” to the polypeptide. Accordingly, Chee teaches the limitations recited in claim 64.
In regards to claim 67, Chee expressly discloses binding agents comprising coding nucleic acid tags that bind to macromolecules associated with recording tags (see e.g. Figs. 1 & 2). Chee further discloses that the recording tag anneals to the coding tag and that primer extension or ligation transfers coding information to generate an extended recording tag (see e.g. Figs. 5 & 6). Chee further discloses analyzing the extended recording tag via nucleic acid sequencing (see embodiments 48-53, and 93-96).
In regards to claim 85 and 86, Chee teaches that a DNA coding tag anneals to a DNA recording tag via complementary spacer sequences and that information is transferred between the tags via primer extension or ligation (see e.g. Fig. 5). The spacer sequences that mediate annealing are portions of the coding and recording tags themselves, thus the linking structure formed between the two molecules consist of the respective nucleic acid tags attached to each molecule and hybridized to one another.
In regards to claim 87, Chee teaches embodiments transferring information in each direction (coding tag to recording tag as the primary embodiment (see Chee Fig. 3), and recording tag to coding tag (see Chee pg. 145 1st para.), combined with Chee’s own teaching that the unmodified annealed junction (without the optional terminator nucleotide) supports extension from either 3’ end (see Chee para. spanning pgs. 131-132). It would have been obvious to one of ordinary skill in the art to omit the optional terminator-nucleotide blocking modification, yielding the default bidirectional behavior of Chee’s own disclosed structure, particularity in light of Chee’s own recognition of the value of capturing information via the reverse direction as a hedge against loss of information from the primary direction. Peterson corroborates that bidirectional extension between two annealed complementary-end oligonucleotide probes was a known, predictably successful technique for proximity-based analyte detection generally, supporting a reasonable expectation of success (see Peterson Fig. 2).
In regards to claim 88, Chee discloses a recoding tag and coding tag each provided as pre-formed double-stranded oligonucleotides, each comprising a short single-stranded overhang designed to anneal with a complementary overhang on the other tag upon proximity binding. Chee further discloses that, upon such annealing, the overhang ends of the two double-stranded tags are joined by DNA ligase (e.g., T3, T4, T7 DNA ligase, see Chee pg. 90 lines 18-20, pg. 138 lines 24-26), producing a single, longer double-stranded ligation product spanning from the recording tag derived sequence through to the coding tag derived sequence (see Chee Fig. 6). This constitutes the claimed “ligating the polypeptide nucleic acid tag and the moiety nucleic acid tag to form a composite UMI and/or barcode.” Chee further discloses dissociating a bound binding agent from its analyte following information transfer between tags.
Peterson is cited to corroborate that ligation , as an alternative to extension, was independently recognized in the analogous proximity-probe art as a mechanism for forming a proximity-dependent composite DNA product. Peterson discloses that a signal is produced when two antibodies bind and bring their associated oligonucleotides into proximity forming a DNA template “through ligation or extension reaction (see Peterson [0029]). This corroborates that ligation-based composite formation as claimed was a known and interchangeable alternative to extension based composite formation with the same field of antibody directed proximity probes at the time of filling.
In regards to step “a) forming a linking structure between a site of a polypeptide in a sample and a site of a moiety in the sample, the linking structure comprising a polypeptide nucleic acid tag attached to the site of the polypeptide and a moiety nucleic acid tag attached to the site of the moiety, wherein the polypeptide nucleic acid tag and the moiety nucleic acid tag are attached to each other, the polypeptide nucleic acid tag comprises a polypeptide unique molecule identifier (UMI) and/or barcode, and the moiety nucleic acid tag comprises a moiety UMI and/or barcode,” Chee teaches a recording tag (polypeptide nucleic acid tag, attached to the site of the polypeptide, comprising a UMI and/or barcode) and a coding tag (moiety nucleic acid tag, attached to the site of the moiety comprising UMI and/or barcode), which anneal to one another via complementary overhang sequences to form the linking structure, as disclosed in Chee Fig. 6 and corroborated generally by Peterson’s disclosure of paired oligonucleotide conjugated antibody probes.
In regards to step “b) ligating the polypeptide nucleic acid tag and the moiety nucleic acid tag to form a composite UMI and/or barcode, wherein the composite UMI and/or barcode comprises both the polypeptide UMI and/or barcode and the moiety UMI and/or barcode,” Chee discloses double-stranded DNA ligation (see Chee Fig. 6, para. spanning pgs. 70-71) in which DNA ligase joins the annealed overhang ends of the double-stranded recording tag and coding tag producing a single ligated double-stranded product incorporating both the recording tag’s and coding tag’s barcode sequences, the composite UMI and/or barcode. Chee further discloses that this “allows transfer of binding information through a primer extension reaction or ligation to the recording tag, coding tag, or a di-tag construct” (see Chee para. spanning pgs. 70-71). Peterson corroborates that formation of a composite DNA product via ligation, in a proximity dependent antibody directed probe system was a known and art-recognized mechanism (see Peterson [0029]).
In regards to step “c) breaking the linking structure via dissociating the polypeptide from the moiety and dissociating the polypeptide nucleic acid tag from the moiety nucleic acid tag, while maintaining attachment between the polypeptide and the polypeptide nucleic acid tag, and maintaining attachment between the moiety and the moiety nucleic acid tag, wherein the polypeptide nucleic acid tag comprises the composite UMI and/or barcode and the moiety nucleic acid tag comprises the composite UMI and/or barcode,” Chee discloses releasing the bound binding agent from the polypeptide following information transfer (see Chee Figs. 32-33). Although, Chee does not expressly use the phrase “breaking the linking structure,” Chee’s disclosed methods necessarily involves dissociation steps because Chee teaches detecting sequential interactions (see Figs. 5, 8-10, pg. 4 1st embodiment) and downstream amplification and sequencing of nucleic acid products (see pg. 12 48th embodiment and throughout). It is well established in molecular biology that amplification and sequence of nucleic acids products require denaturation of duplex structures and separation of interacting complexes. Thus, practicing Chee’s method would require separation of interacting polypeptide and moiety and separation of hybridized nucleic acid tags for analysis. Such dissociation steps constitute routine processing inherent to ligation and sequencing-based assays and would have been understood by a person of ordinary skill in the art.
In regards to step “d) determining a sequence of the polypeptide nucleic acid tag and at least a partial sequence of the polypeptide, and determining a sequence of the moiety nucleic acid tag and at least a partial sequence of the moiety, wherein the determined sequences of the polypeptide nucleic acid tag and the moiety nucleic acid tag each comprise a sequence of the composite UMI and/or barcode, indicating that the site of the polypeptide and the site of the moiety are in spatial proximity in the sample,” Chee discloses sequencing based readout of ligated tag products which is also generally corroborated by Peterson’s sequencing based readout. Because each determined sequences includes the composite sequence generated by the double-stranded ligation of step b), successful recovery of matching composite sequence from both strands indicates that the polypeptide site and moiety site were in sufficient proximity for annealing and ligation to occur.
Conclusion
No claim is allowed.
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/MATTHEW HAROLD RAYMONDA/Examiner, Art Unit 1684 /AARON A PRIEST/Primary Examiner, Art Unit 1681
1 Fields and Song, “A novel genetic system to detect protein-protein interactions”, Nature, Vol 340, 20 July 1989
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