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 .
Claim Status
Claims 1, 4-9, 16, 40, 43, 47, 50-51, 64, 67 and 85-86 are pending and under examination. Claims 1, 4, 6-9, 16, 43, 47, 51, 67, and 85-86 have been amended since the last office action.
Response to Arguments
Objections Withdrawn
The objection to claims 1, 4, and 47 is withdrawn following the applicants’ amendments.
Rejections Withdrawn
The rejection of claims 1, 4, 40, 47, 64, and 85-86 under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by Chee is withdrawn following the applicants’ amendments.
The rejection of claims 5-9, 16, 43, 50-51, and 67 under 35 U.S.C. 103 as being unpatentable over Chee in view of Chee is withdrawn following the applicants’ amendments.
New Rejection
Claim Rejections - 35 USC § 103
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, 4-9, 16, 40, 43, and 85-86 are rejected under 35 U.S.C. 103 as being unpatentable over Chee et al. (WO 2017/192633 A1, published Nov. 9, 2017, of record).
Regarding claim 1, Chee teaches a multistep method for assessing (e.g., analyzing) the identity and spatial relationship between a polypeptide (e.g., macromolecule, pg. 1, line 12) and a moiety (e.g., binding agent) in a sample (pg. 4, line 17-22), which comprises:
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 (e.g., recording tag) associated with site of the polypeptide and a moiety nucleic acid tag (e.g., coding tag) associated with the site of the moiety, wherein the polypeptide nucleic acid tag and the moiety nucleic acid tag are attached to each other Fig. 3), the polypeptide nucleic acid tag (recording tag) comprises a polypeptide unique molecule identifier (UMI) and/or barcode, and the moiety nucleic acid tag (coding tag) comprises a moiety UMI and/or barcode (see Fig. 3).
Although Chee may not explicitly describe an embodiment in which both tags simultaneously comprise the composite UMI following dissociation, Chee teaches the mechanisms necessary to achieve such reciprocal incorporation (see Figs. 3-5, 8, 11-12, 14, and 27). Chee discloses extending a coding tag to incorporate the UMI of a recording tag (see Figs. 11-12, and 14) and in other embodiments, extending the recording tag to incorporate the information from the coding tag (see Figs. 3-5, 8, 11). Chee further discloses the use of forward and reverse primers capable of amplifying or extending either tag (see Fig. 27), thereby demonstrating bidirectional information transfer between tags. These teachings establish that each tag may be extended to incorporate the identifier of the other.
it would have been obvious to a person of ordinary skill in the art at the time of filing to apply Chee’s disclosed extension strategies in both directions so that each tag retains the composite identifier. Performing reciprocal extension represents a predictable use of Chee’s bidirectional primer and extension system and would have provided the recognized benefit of improved robustness and redundancy in preserving proximity information for downstream sequencing following dissociation of the interacting molecules. Such modification constitutes no more than predictable use of prior art elements according to their established functions and would have been obvious to one of ordinary skill in the art. Thus, Chee teaches “b) transferring information between the associated polypeptide tag and the moiety tag (e.g., transferring information of the first coding tag to the recording tag, Figs. 1-12, pg. 81, line 8-17) or ligating (pg. 19, line 28) the associated polypeptide tag and the moiety tag to form a composite unique molecule identifier (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.
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. Thus Chee demonstrates methods “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.”
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 (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”) (pg. 60, last para.; pg. 91, ¶ 1, pg. 145, ¶1).
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.
In regards to claim 7, as discussed above, Chee teaches the limitations of claim 6 for which claim 7 depends. Claim 7 further recites that transferring information comprises extending both the first polynucleotide of the polypeptide nucleic acid tag and the second polynucleotide of the moiety nucleic acid to form the composite UMI and/or barcode.
Chee expressly discloses extension of the recording tag to incorporate coding tag information (see Fig. 3-5, 8, 11) and also discloses embodiments in which the coding tag is extended to incorporate information from the recording tag (see Figs. 11-12, and 14). Additionally, Chee discloses the use of forward and reverse primers, to generate extended coding and recording tags (see Fig. 27, pg. 166 line 15 - pg. 167 line 7)
Although Chee does not explicitly describe a single embodiment in which both tags are extended in the same reaction, Chee teaches that mechanisms necessary for extension in either direction. It would have been obvious to a person of ordinary skill in the art to perform extension of both polynucleotides so that each tag incorporates information from the other, thereby forming composite identifiers on both tags. Performing reciprocal extension represents a predictable use of Chee’s disclosed primer extension system and would provide the recognized benefit of redundancy and robustness in preserving proximity information following dissociation. Accordingly, claim 7 in unpatentable under 35 USC 103 over Chee.
In regards to claim 8, Chee expressly teaches analyzing or sequencing the extended nucleic acid tags (see 48th-50th embodiments, page 12-13). Because the extended recording tag or coding tag comprises the information from the interacting tag, sequencing the extended tag necessarily sequences the composite identifier corresponding to both interacting molecules.
In regards to claim 9, Chee discloses that the tags may be single or double stranded (see pg. 104, lines 15-23, pg. 132 lines 5-9) . Chee further discloses embodiments involving sticky-end ligation between complementary spacer sequences and illustrates tag information being transferred to an extended recording tag via enzymatic ligation (see Fig. 6, pg. 31 lines 18-26). Because ligation joins the nucleic acid tags to form a construct comprising sequence information from both tags, the ligation forms a composite UMI and/or barcode corresponding to both interacting molecules.
In regards to claim 16, Chee discloses formation of an extended recording tag comprising coding information transferred from a coding tag (see Fig. 5-6). Chee further discloses release, collecting, and sequencing the extended recording tags to determine molecular interactions and spatial relationships (see embodiments 48-53, and 93-96). The extended recording tag constitutes a separate polynucleotide molecule comprising composite coding information derived from both interacting tags.
Because Chee sequences the extended recording tag to determine molecular proximity, Chee teaches forming a sperate record polynucleotide and determining its sequence to establish spatial relationship as recited in claim 16.
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 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.
Claims 1, 4-9, 16, 40, 43, 47, 50-51, 64, 67 and 85-86 are rejected 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).
As discussed above, Chee teaches methods for assessing identity and spatial relationships between biomolecules using nucleic acid tags attached to binding agents such as polypeptides and other moieties.
In regards to claim 47, Chee teaches methods for assessing identity and spatial relationship between a polypeptide and a moiety in a sample, wherein the moiety comprises another polypeptide or a polynucleotide, the method comprises: forming a linking structure between a site of a polypeptide in a sample and a site of a moiety in the sample by attaching the polypeptide nucleic acid tag (reporter tag) to the site of the polypeptide and attaching a moiety nucleic acid tag (coding tag) to the site of the moiety (see Fig. 2, 5-12, pg. 11 lines 31-32, pg. 19 lines 22-23, pg. 25 lines 11-12, pg. 29 1st para., pg. 30 1st para., and throughout).
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. Thus Chee demonstrates methods that include “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.”
Chee further teaches “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 ( see pg. 60 last para., pg. 91 ¶ 1, pg. 145 ¶1, and throughout).
While Chee doesn’t disclose providing a pre-assembled structure, they do to the methods required to generate a linking structure comprising a composite unique molecule identifier comprising a middle portion flanked by a polypeptide nucleic acid tag (reporter tag) on one side and a moiety nucleic acid tag (coding tag) on the other side wherein each tag comprises a UMI and/or barcode (see Figs. 3-5, 8, 11-12, 14, and 27).
Accordingly, Chee teaches the use of polypeptide and moiety nucleic acid tags, 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, the formation of a composite UMI and/or barcode comprising information from both tags, and sequencing of the composite identifier to infer proximity between molecules.
However, Chee does not explicitly teach providing a pre-assembled nucleic acid structure comprising a composite UMI and/or barcode prior to attachment to the polypeptide and moiety. Instead, Chee primarily describes generating the composite identifier during the assay through extension or ligation between tags that are initially separate. Chee therefore does no explicitly disclose a pre-synthesized scaffold comprising a central composite barcode region flanked by two nucleic acid tag regions wherein the composite UMI exists prior to the proximity event.
Like Chee, Landegren is in the field of detecting spatial relationships between molecules, and teaches pre-synthesized splint nucleic acid molecules comprising a barcode or identifying sequence region flanked by regions complementary to two separate nucleic acid templates (see Fig. 13, [0143], [0186]). Landegren discloses that the splint is provided as a pre-assembled nucleic acid structure and that its complementary arms hybridize to distinct nucleic acid strands, enabling ligation and incorporation of the barcode sequence into a resulting product. Structurally, Landegren teaches a pre-assembled scaffold having a central identifying barcode region flanked by two attachment regions designed to associate with separate molecules, when those molecule are in proximity to each other. Thus Landegren teaches a pre-assembled nucleic acid construct comprising an identifying sequence positioned between two regions configured to interact with separate nucleic acid strands.
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to incorporate Landegren’s pre-assembled barcode-containing splint architecture within Chee’s proximity-based tagging system for several technical reasons grounded in routine nucleic acid assay design. Chee teaches generating composite identifiers by enzymatic extension or ligation between separate nucleic acid tags upon proximity of interacting molecules. However, the incorporation of identifying barcode sequences into ligated nucleic acid products was also well-established through pre-synthesized splint architecture as taught by Landegren. A person of ordinary skill in the art would have recognized that embedding barcode sequences within a pre-assembled scaffold provides a predictable and controlled method for incorporating identifying information into ligated products without reliance on extension-mediated sequence copying.
Further, when investigating molecular interaction networks, practitioners routinely design assays to test suspected interaction pairs and hierarchical interaction relationships. I such context, pre-defined barcode scaffolds allow deliberate encoding of candidate interaction combinations, facilitating hypothesis-driven interrogation of molecular proximity relationships. Adapting Landegren’s pre-synthesized splint constructs to Chee’s proximity-based tagging framework would therefore have been an obvious implementation choice to improve assay determinism, allow controlled barcode design, and simplify composite sequence generation.
At the time of filing, splint-mediated ligation and barcode-embedded nucleic acid scaffolds were routine molecular biology techniques, and substituting one known composite-generation strategy (extension/ligation-based formation as in Chee) with another known strategy (pre-assembled barcode-containing splints as in Landegren) represents the predictable use of prior art elements according to their established functions. Such substation falls within the ordinary creativity of a person of ordinary skill in the art and would have been expected to success. Thus the combination of Landegren and Chee would make obvious “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 the combination would read on all limitations of the claim.
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, Chee expressly teaches analyzing or sequencing the extended nucleic acid tags (see 48th-50th embodiments, page 12-13). Because the extended recording tag or coding tag comprises the information from the interacting tag, sequencing the extended tag necessarily sequences the composite identifier corresponding to both interacting molecules.
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).
Conclusion
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/MATTHEW HAROLD RAYMONDA/Examiner, Art Unit 1684 /AARON A PRIEST/Primary Examiner, Art Unit 1681