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 .
Office Action: Notice
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 8/5/2026 has been entered.
Claim Status
Claims 3, 19, 20, 23, 62 and 65 have been amended (8/5/2026). No new matter was added. Thus, claims 3, 4, 7, 8, 11, 19, 20, 23-25, 28-30, 33, and 61-66 are under examination (8/5/2026).
Election/Restrictions
Applicant’s election without traverse of Group I in the reply filed on August 11, 2025 is acknowledged. Claims 36-38, 41-42 and 44 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to nonelected Groups 2 and 3, there being no allowable generic or linking claim. Further, since Group I was elected, species election I of nucleic acids and species II of three-dimensional region were made. Election was made without traverse in the reply filed on 8/11/2025.
Priority
Claims 3, 4, 7, 8, 11, 19, 20, 23-25, 28-20, 33, and 61-66 receive a priority date of 12/12/2019, the effective filing date of GB Provisional Patent GB1918340.9. All priority documents have been received.
Information Disclosure Statement
The listing of references in the specification is not a proper information disclosure
statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information
submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be
incorporated into the specification but must be submitted in a separate paper." Therefore, unless
the references have been cited by the examiner on form PTO-892, they have not been
considered.
Information disclosure statement (IDS) was submitted on 8/5/2026 and is being considered by the examiner.
Objections Withdrawn
Claims:
The minor grammatical objection to claim 62 is withdrawn in view of Applicant’s amendments.
Rejections Withdrawn
Claim Rejections - 35 USC § 102
The rejection of claims 13, 4, 7, 8, 11, 19, 20, 23-25, 28-30 and 33 under 35 U.S.C. 102 (a)(1) and (a)(2) as being anticipated by Vijayan et al., (US PGPub 2019/0093103 A1, published 3/28/2019) is withdrawn in view of Applicant’s amendments of claims 3, 19-20 and 23 and as a result of further clarification set forth in Applicant’s Remarks (8/5/2026). Specifically, the amendment to independent claim 3 requires spatially restricted, illumination-controlled indexing within a defined area of interest in the tissue. Vijayan does not disclose the claimed repeated illumination and index-addition steps wherein indexing is restricted to detection probes within the illuminated defined area to form a spatial barcode unique to that area.
Claim Rejections - 35 USC § 112(b)
The rejection of claim 65 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, is withdrawn in view of the Applicant’s amendment of claim 65 to correct lack of antecedent basis.
New/Amended Rejections
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 3, 4, 7, 8, 11, 19, 20, 23-25, 28-30 and 33 and 61-66 are rejected under 35 U.S.C. 103 as being unpatentable over Vijayan et al., (US PGPub 2019/0093103 A1, published 3/28/2019), in view of in view of Agbavwe et al. (“Efficiency, error and yield in light-directed maskless synthesis of DNA microarrays”, Journal of Nanobiotechnology, published 2011), and Lin et al. (“Intracellular Thiols and Photo-Illumination Sequentially Activate Doubly Locked Molecular Probes for Long-Term Cell Highlighting and Tracking with Precise Spatial Accuracy”, ChemPubSoc Europe, published 6/2014), and Huang et al. (“Photoactivated Specific mRNA Detection in Single Living Cells by Coupling “Signal-on” Fluorescence and “Signal-off” Electrochemical Signals”, NANO Letters, published 2018) and further in view of Nestorova et al. (“Lab-on-a-chip mRNA purification and reverse transcription via a solid-phase gene extraction technique”, Royal Society of Chemistry, published 2017).
Regarding claims 3-4, 7-8 and 11, Vijayan teaches a bead or detection probe with a covalently attached chemical compound and a covalently attached DNA barcode and methods for using such beads, where the bead has many substantially identical copies of the chemical compound and many substantially identical copies of the DNA barcode (Abstract). Further, Vijayan teaches that the addition of nucleic acid barcodes to the beads allows the identity of the compound present within the beads to be carried along to the assay volume where very high throughput assays may be performed without needing robotics or spatial indexing of compounds within microtiter plates (Paragraph 7, lines 5-10). Specifically, Vijayan teaches that while incubating cells or tissues under different perturbations within well plates, followed by single-cell analysis and comparisons between transcript profiles can be done, the number of drugs that can be examined is limited by the plate capacity (Paragraph 11, lines 5-10). Vijayan also teaches that the previously described detection probe or bead methodology provides a system for screening chemical compounds, comprising: (a) A picowell array plate comprising a plurality of picowells, wherein each picowell has a top aperture that defines an opening at the top of the picowell, a bottom that is defined by a floor, wherein the top aperture is separated from the floor, and wherein a wall resides in between the top aperture and the floor; (b) A bead or detection probe disposed in a picowell, wherein the bead comprises a plurality of substantially identical bead-bound DNA barcodes, and a plurality of substantially identical bead-bound compounds, (c) Wherein the bead comprises a bead-bound DNA barcode or binding region that takes the form of either a concatenated DNA barcode or an orthogonal DNA barcode, and wherein if the DNA barcode takes the form of a concatenated DNA barcode the concatenated DNA barcode is made by a method that: (i) Uses click chemistry, or (ii) Uses a repeating cycle of steps, wherein the repeating cycle of steps comprises using a splint oligonucleotide (splint oligo) that is capable of hybridizing to a partially made bead-bound DNA barcode, and wherein the hybridizing is mediated by an annealing site on the splint oligo and a corresponding, complementary annealing site in the partially made bead-bound DNA barcode, wherein the annealed splint oligo is used as a template for extending the partially made DNA barcode using DNA polymerase, and wherein the splint oligo contains bases that are complementary to a DNA barcode module that is to be polymerized to the partially made DNA barcode, (d) Wherein each one of the plurality of substantially identical bead-bound compounds comprises one or more chemical library monomers, and wherein each bead-bound DNA barcode module identifies a corresponding chemical library monomer, wherein the term “compound” is used to refer to a completed product that comprises one or more chemical library members, and wherein the completed DNA barcode identifies the compound (Paragraph 12, lines 1-20).
Specifically, Vijayan teaches that the concatenated DNA barcode is coupled to the bead, but is: (i) not coupled to the bead by way of any photocleavable linker or index sequence, (ii) not coupled to the bead by any enzymatically cleavable linker; or (iii ) not coupled to the bead by any kind of cleavable linker (Paragraph 47, lines 1-3). Vijayan also teaches that herein the release-monitor bead comprises a bead, a quencher, a fluorophore, and a photocleavable linker that couples the fluorophore to the bead for illumination, the method comprising, in this order, (i) Providing a resin, (ii) Coupling a lysine linker to the resin, wherein the reagent containing the lysine linker is L-Fmoc-Lys(4-methyltrityl)-OH, (iii) Removing the Fmoc protecting group, (iv) Coupling the quencher using a reagent that is quencher-N-hydroxysuccinimide (quencher-NETS) as the source of quencher, (v) Removing the 4-methyltrityl protecting group using a reagent comprising trifluoroacetic acid, (vi) Coupling a photocleavable linker to the epsilon amino group of lysine, wherein the photocleavable linker is provided by a reagent that is, Fmoc-photocleavable linker-OH, (vii) Coupling the fluorophore (Paragraph 52, lines 1-15).
Vijayan further teaches that the previously described method of spatial barcoding via beads or detection probes can be applied to prepare barcoded mRNA from each sample in isolation and then perform comprehensive RNA profiles for every sample (Paragraph 11, lines 5-10).
Regarding claim 19, Vijayan teaches that the previously described method of spatial barcoding via beads or detection probes includes orthogonal barcoding where each individual module gets covalently bound to its own unique attachment site on the bead and where once a module gets attached to a given site on the bead, no further modules will be connected to the module that is already attached (Paragraph 176, lines 15-20).
Regarding claims 20 and 23, Vijayan teaches that the previously described method of spatial barcoding via beads or detection probes includes a stepped picowell, a three dimensional structure or physical structure with three compartments and two steps where the top compartment is widest and is configured for accepting cap where most of the top compartment is occupied by the cap in the situation where the picowell is capped and the middle compartment is configured for being occupied mainly by, or solely by, reagents (Figure 29). Further, Vijayan teaches that the reagents can include buffer, enzyme substrates, one or more salts, and a preservative or stabilizer such as dithiothreitol, RNAse inhibitor, glycerol, or DMSO and the lowest compartment is configured for being occupied by bead, that is, a bead with coupled both a DNA library and with releasable compounds (Figure 29; Paragraph 381, lines 1-15).
Regarding claim 24, Vijayan teaches that the previously described method of spatial barcoding via beads or detection probes includes cleaving the DNA barcode from the bead before sequencing and can encompass a method where bead-bound DNA barcode is cleaved from the bead, thereby releasing the DNA barcode in a soluble form, prior to amplification, or prior to sequencing, or prior to any type of sequence identification technique such as hybridizing with a nucleic acid probe (Paragraph 401, lines 1-5).
Regarding claim 25, Vijayan teaches that the previously described method of spatial barcoding via beads or detection probes includes bead-bound compositions, systems, and methods, that uses at least one splint oligo or split detection probes, at least two splint oligos, at least three splint oligos, at least four splint oligos, at least five splint oligos, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at last 13, at least 14, at least 20 splint oligos, or less than 20, less than 15, less than 10, less than 8, less than 6, less than 4, less than 3, less than 2 splint oligos (Paragraph 291, lines 1-5).
Regarding claim 28, Vijayan teaches that the previously described method of spatial barcoding via beads or detection probes can be applied to amplified DNA while attached to a bead and DNA in amplified form is easier to sequence that non-amplified DNA and once single stranded, a splint oligo is added to bridge the ends of the tag DNA, and this is followed by extension and ligation of the splint oligo (Paragraph 407, lines 1-5).
Further, Vijayan teaches that the distance from primer annealing site to a DNA barcode module comprises a polynucleotide including a first nucleic acid that is an annealing site for a sequencing primer, and a second nucleic acid that is a DNA barcode module, the first nucleic acid can be immediately upstream of the second nucleic acid. where, the first nucleic acid can be upstream of the second nucleic acid, where the first and second nucleic acids are separated from each other by one, two, three, four, five, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleotides, or by about one, about two, about three, about four, about five, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 nucleotides (Paragraph 221, lines 1-10).
Regarding claims 29-30, Vijayan teaches that the previously described method of spatial barcoding via beads or detection probes includes the option of creating a DNA barcode that includes a terminal nucleic acid that encodes DNA hairpin which includes, at the 3-prime end, a nucleic acid that possesses an annealing site for a sequencing primer, a bend taking the form of about four bases that are not base-paired, and a sequencing primer that is capable of bending around and forming base pairs with the sequencing primer annealing site (Paragraph 242, lines 1-5). Specifically, Vijayan teaches that these optional encoding regions can include: (1) Linkers to attach chemical library member to a substrate, such as a bead; (2) Linkers to attach nucleic acid barcode to a substrate, such as a bead; (3) Cleavable linkers, for example, cleavable by UV light, cleavable by an enzyme such as a protease; (4) Non-cleavable linkers; (5) Bifunctional linkers; (6) Multi-functional linkers; and (7) Plurality of beads used for linking (Paragraph 314, lines 1-5).
Regarding claim 33, Vijayan teaches that the previously described method of spatial barcoding via beads or detection probes includes beads or probes where DNA barcodes are mainly attached on the exterior surface; however one reason to NOT make and use beads with internal DNA barcodes, is the low permeation of DNA oligomers to the interior spaces, and low permeation of DNA ligases to interior spaces (ligases for connecting DNA modules to each other to create the finished DNA barcode) (Paragraph 355, lines 1-5). Specifically, Vijayan teaches that the previously described method of spatial barcoding via beads or detection probes can be applied to amplified DNA while attached to a bead and DNA in amplified form is easier to sequence that non-amplified DNA and once single stranded, a splint oligo is added to bridge the ends of the tag DNA, and this is followed by extension and ligation of the splint oligo (Paragraph 407, lines 1-5).
Regarding claims 61-63, Vijayan teaches that the previously described detection probe or bead methodology provides a system for screening chemical compounds, comprising: (a) A picowell array plate comprising a plurality of picowells, wherein each picowell has a top aperture that defines an opening at the top of the picowell, a bottom that is defined by a floor, wherein the top aperture is separated from the floor, and wherein a wall resides in between the top aperture and the floor; (b) A bead or detection probe disposed in a picowell, wherein the bead comprises a plurality of substantially identical bead-bound DNA barcodes, and a plurality of substantially identical bead-bound compounds, (c) Wherein the bead comprises a bead-bound DNA barcode or binding region that takes the form of either a concatenated DNA barcode or an orthogonal DNA barcode, and wherein if the DNA barcode takes the form of a concatenated DNA barcode the concatenated DNA barcode is made by a method that: (i) Uses click chemistry, or (ii) Uses a repeating cycle of steps, wherein the repeating cycle of steps comprises using a splint oligonucleotide (splint oligo) that is capable of hybridizing to a partially made bead-bound DNA barcode, and wherein the hybridizing is mediated by an annealing site on the splint oligo and a corresponding, complementary annealing site in the partially made bead-bound DNA barcode, wherein the annealed splint oligo is used as a template for extending the partially made DNA barcode using DNA polymerase, and wherein the splint oligo contains bases that are complementary to a DNA barcode module that is to be polymerized to the partially made DNA barcode, (d) Wherein each one of the plurality of substantially identical bead-bound compounds comprises one or more chemical library monomers, and wherein each bead-bound DNA barcode module identifies a corresponding chemical library monomer, wherein the term “compound” is used to refer to a completed product that comprises one or more chemical library members, and wherein the completed DNA barcode identifies the compound (Paragraph 12, lines 1-20).
Specifically, Vijayan teaches that the concatenated DNA barcode is coupled to the bead, but is: (i) not coupled to the bead by way of any photocleavable linker or index sequence, (ii) not coupled to the bead by any enzymatically cleavable linker; or (iii ) not coupled to the bead by any kind of cleavable linker (Paragraph 47, lines 1-3). Vijayan also teaches that herein the release-monitor bead comprises a bead, a quencher, a fluorophore, and a photocleavable linker that couples the fluorophore to the bead for illumination, the method comprising, in this order, (i) Providing a resin, (ii) Coupling a lysine linker to the resin, wherein the reagent containing the lysine linker is L-Fmoc-Lys(4-methyltrityl)-OH, (iii) Removing the Fmoc protecting group, (iv) Coupling the quencher using a reagent that is quencher-N-hydroxysuccinimide (quencher-NETS) as the source of quencher, (v) Removing the 4-methyltrityl protecting group using a reagent comprising trifluoroacetic acid, (vi) Coupling a photocleavable linker to the epsilon amino group of lysine, wherein the photocleavable linker is provided by a reagent that is, Fmoc-photocleavable linker-OH, (vii) Coupling the fluorophore (Paragraph 52, lines 1-15).
Vijayan further teaches that the previously described method of spatial barcoding via beads or detection probes can be applied to prepare barcoded mRNA from each sample in isolation and then perform comprehensive RNA profiles for every sample (Paragraph 11, lines 5-10).
Regarding claims 64-66, Vijayan teaches that reducing damage via coupling DNA to beads via their 3 ' – end, where certain chemical transformation may damage exposed 3 ' - hydroxyl groups of nucleic acids and for instance Mitsunobu reactions allow the conversion of primary and secondary alcohols to esters, phenyl ethers, thioethers and various other compounds, which might render exposed 3 ' - ends unreactive to subsequent processing steps, or cause the now modified 3 ' - end to participate in further chemical reactions (Paragraph 310, lines 1-5). Further, Vijayan teaches that in some embodiments , the DNA tags may be attached to beads via their 3 ' - end , so only the 5 ' - end is exposed to solution (Paragraph 310, lines 5-10).
Additionally, Vijayan teaches that the reagents , systems , and methods of the present disclosure encompass bead - bound nucleic acids , such as a bead - bound DNA or a bead - bound DNA tags , where coupling to the bead involves the 3 ' - terminus (or the 3 ' - end) of
the DNA, where ssDNA that comprises a DNA barcode is coupled by way of the 3 ' - end , of the ssDNA, sequencing can be initiated by hybridizing only one sequencing primer, where this sequencing primer hybridizes upstream of the entire DNA barcode , and where this hybridizing is at or near the bead - bound end of the coupled ssDNA (Paragraph 311, lines 1-10). Vijayan also teaches that as an alternative to using only one sequencing primer , a plurality of sequencing primers can be used, where each sequencing primer hybridizes upstream to a particular DNA barcode module (Paragraph 311, lines 1-10). Vijayan further teaches that for example, if a given DNA barcode contains five DNA barcode modules, and where the DNA is coupled to a bead by way of its 3 ' - end, the DNA barcode can include five different primer annealing sites, where each primer anneals given DNA barcode modulating site is located just upstream of a given DNA barcode molecule (Paragraph 311, lines 10-15).
However, Vijayan does not teach or suggest a specific binding region of the detection probe comprised of RNA, specifically mRNA, that is elongated via reverse transcription. Further, Vijayan does not teach or suggest light-directed synthesis in which illumination selectively removes a photolabile protecting group to permit addition of a nucleic acid component at an illuminated location, or repeated cycles of such light-directed deprotection and coupling. Further, Vijayan does not teach the claimed use of photocleavable or light-responsive probe architecture in a cellular environment for interaction with intracellular target mRNA.
Nestorova teaches extraction and purification of high-quality RNA is a crucial initial step required for a variety of genomic assays, using a solid phase gene extraction (SPGE) method for automated extraction, purification and reverse transcription of mRNA in a microfluidic device (Abstract). Nestorova further teaches that the specificity and RNA loading capacity of the probes were evaluated using conventional qPCR, where this procedure was successfully used to extract, purify, and transcribe mRNA from rat glioblastoma cell spheroids in less than seven minutes and analysis of the product confirmed that the SPGE technique selectively captures and inherently purifies high-quality mRNA directly from biological material with no need for additional pre-processing steps (Abstract). Further, Nestorova teaches that nucleic acid-based assays require high quality RNA from cells and tissues that is free of proteins or inhibitors that interfere with downstream reverse transcription (RT) and PCR amplification and traditional methods for RNA extraction involve multiple steps and rely on tissue homogenization to release the genetic material and eliminate inhibitors (Introduction: Paragraph 1). Nestorova also teaches that this method simplifies the isolation of RNA from tissue or cells to a single pin prick and specifically, solid phase gene extraction (SPGE) was performed with miniature needles functionalized with oligo(-dT) or gene-specific sequences via simply inserting the needle into the specimen, the target RNA is collected on its tip (Introduction: Paragraphs 2-3).
More so, Nestorova teaches that the reported method describes the integrated collection, purification and reverse transcription of mRNA, while eliminating the need for cell lysis and multi-step mRNA purification and the resulting heterogeneous sample of whole mRNA can be analyzed by shotgun sequencing (RNA-Seq) or RT-PCR quantification of one or more gene-specific mRNA sequences where mRNA extraction is achieved using needles with amino-modified dT(15) oligonucleotides immobilized to their surface (Materials and Methods: Paragraph 1; Fig. 1). Further, Nestorova teaches that the specificity and loading capacity of the SPGE needles were evaluated using total rat RNA (20 ng μL−1) as a source of biological material, where the specificity was assessed using electrophoretic sizing of the RNA; the loading capacity was quantified using a RT-qPCR LightScanner®32 via functionalized steel probes (SPGE of mRNA from total RNA sample: specificity and concentration studies: Paragraph 1).
Agbavwe teaches light-directed in situ synthesis of oligonucleotides on a solid surface using maskless array synthesis (MAS), wherein a digital micromirror device delivers patterned ultraviolet light to selected locations on a synthesis surface (Background). Agbavwe teaches that the patterned illumination selectively removes photocleavable 5’-hydroxyl protecting groups from terminal phosphonamidites at the illuminated locations, thereby exposing terminal hydroxyl groups for coupling of a subsequent phosphonamidite (Background; Figure 1; Chemical Synthesis). Additionally, Agbavwe teaches an iterative phosphoramidite synthesis cycle comprising coupling, capping, and photo-deprotection, whereby selected features are exposed to ultraviolet light to remove the 5’-NIPPOC protecting group and permit coupling of a new nucleotide during a subsequent synthesis cycle (Figure 1; Chemical Synthesis). Thus, Agbavwe teaches that illumination controls the spatially selective addition of nucleic acid components during iterative oligonucleotide synthesis by selectively deprotecting illuminated locations and permitting subsequent nucleotide coupling at those locations.
Lin teaches a photoconvertible fluorescent molecular probe configured for controlled activation within living cells (Abstract). Specifically, Lin teaches a probe comprising a 7-aminocoumarin phototrigger, a thiol-removable energy acceptor, and a caged fluorescein florescent reporter, wherein intracellular thiols remove the energy acceptor and thereby enable subsequent light-induced photocleavage and photoactivation of the caged fluorescein (Introduction; Scheme 1). Further, Lin teaches that upon illumination the fluorescein is liberated to fluorescently label the selected cell, such that activation is spatially restricted to the particular region subjected to illumination (Scheme 1; Results and Discussion: Designs). Thus, Lin teaches that a photolabile probe component can remain in a caged or inactive state until exposure to light and that spatially controlled illumination can selectively photocleave and activate the probe at a desired location within a cellular environment.
Huang teaches photoactivated detection of a target mRNA within individual living cells using photocleavable nucleic acid hairpin probes (Abstract). Further, Huang teaches a fluorescent hairpin probe comprising an ortho-nitrophenylphosphate ester photocleavable group and a FAM fluorophore maintained in a caged, quenched configuration, wherein the probe is modified with an AS1411 aptamer to facilitate targeting and internalization into MCF-7 cells (Introduction, Paragraphs 1-4; Scheme 1). Further, Huang teaches that upon UV irradiation, the hairpin is photocleaved, thereby permitting intracellular target mRNA to initiate toehold-mediated strand displacement and generate a fluorescent signal indicative of the target mRNA (Abstract). Specifically, Huang teaches an alternative electrochemical configuration in which a nanoelectrode functionalized with a redox-active photocleavable hairpin probe is introduced into the cytoplasm of a single cell, wherein photocleavage permits target mRNA-mediated strand displacement of probe-associated strand (Abstract). Thus, Huang demonstrates the use of light to control activation of a nucleic acid probe within a cellular environment, such that irradiation renders the probe available for subsequent interaction with intracellular target mRNA (Introduction, Paragraphs 3-4).
It would have been obvious to one of ordinary skill in the art at the time of the invention to modify the system of Vijayan to incorporate mRNA-binding probes and reverse transcription as taught by the SPGE reference in order to enable gene expression analysis and transcript profiling of biological samples. Both references are directed to nucleic acid analysis of biological samples and share the common objective of identifying and characterizing molecular content within cells or tissues. Vijayan expressly contemplates RNA analysis (Paragraph 11), and Nestorova provides a known and established technique for selectively capturing and reverse transcribing mRNA for such analysis. Therefore, a person of ordinary skill in the art would have been motivated to combine these teachings to enhance Vijayan’s system with known mRNA capture and reverse transcription techniques.
Furthermore, a person of ordinary skill in the art would have had a reasonable expectation of success in making this combination because both references operate within the same technical field of nucleic acid hybridization, amplification, and sequencing, and because reverse transcription of mRNA using oligo(dT) or sequence-specific probes was a well-understood and routine technique at the time of the invention. Vijayan already teaches nucleic acid constructs capable of hybridization and enzymatic manipulation (Paragraph 311), which are directly compatible with reverse transcription workflows, and the SPGE reference demonstrates that mRNA can be captured on probe surfaces and successfully reverse transcribed in situ. Thus, the combination would have predictably resulted in a system in which mRNA bound to a detection probe is elongated via reverse transcription for downstream analysis. Accordingly, it would have been obvious to modify Vijayan in view of Nestorova to arrive at the claimed invention, including a detection probe comprising an RNA binding region that is elongated via reverse transcription.
It would further have been obvious to one of ordinary skill in the art at the time of the invention to modify the system of Vijayan, as modified by Nestorova, to incorporate light-directed nucleic acid synthesis and spatially controlled photoactivation as taught by Agbavwe, Lin and Huang. Agbavwe teaches an established technique for light-directed synthesis of oligonucleotides in which patterned ultraviolet illumination selectively removes photocleavable protecting groups from the 5’e ends of growing oligonucleotides, thereby exposing terminal hydroxyl groups for subsequent phosphoramidite coupling. Agbavwe further teaches repeating such photo deprotection and coupling steps to synthesize predetermined nucleic acid sequences at selected locations.
A person of ordinary skill in the art would have been motivated to incorporate the light-directed synthesis technique of Agbavwe into Vijayan’s nucleic-acid probe and barcode system because Agbavwe provides a known technique for controlling the location at which nucleic acid synthesis occurs through selective illumination. Such a modification would have predictably provided spatial control over the synthesis or extension of nucleic acid components associated with Vijayan’s detection probes while retaining Vijayan’s use of nucleic acid barcodes for identifying and analyzing molecular content.
Lin further demonstrates that photolabile probe components may be selectively activated by illumination within a cellular environment, thereby proving spatial control over probe activation. Huang likewise teaches photocleavable nucleic acid probes introduced into living cells, wherein irradiation activates the probe and permits subsequent interaction with intracellular target mRNA. Accordingly, Lin and Huang demonstrate that the photochemical control taught by Agbavwe was compatible with biological and cellular probe applications and provide additional motivation to employ light-responsive probe chemistry for spatially controlled interaction with intracellular nucleic acids.
Therefore, a person of ordinary skill in the art would have had a reason to combine these teachings to provide Vijayan’s detection-probe system, as modified to capture and reverse transcribe mRNA according to Nestorova, with known light-directed synthesis and photoactivation techniques, thereby permitting illumination to selectively control nucleic acid synthesis and/or probe activation at desired locations. The combination would have involved the application of known light-responsive nucleic acid and probe techniques to Vijayan’s known detection probe system to obtain the predictable benefit of spatially controlled probe synthesis and activation.
Applicant’s Response: The Applicant argues that Vijayan does not teach the newly claimed light-directed synthesis/activation limitations, including selective illumination to control addition or activation of nucleic acid components associated with the detection probe. Applicant further contends that the cited art fails to teach or suggest the claimed combination of these light-controlled features with the cellular mRNA-binding and reverse-transcription aspects of the detection-probe system.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully considered and are found to be partially persuasive, as discussed below.
As of note, the new rejection does not rely on Vijayan alone to teach the newly claimed light-controlled limitations. Rather, Vijayan provides the underlying detection-probe and barcode system, as modified by Nestorova for mRNA capture and reverse transcription, while Agbavwe teaches iterative light-directed oligonucleotide synthesis through selective photodeprotection followed by nucleotide coupling; Lin further teaches spatially controlled photoactivation of photolabile probes in a cellular environment, and Huang teaches photocleavable nucleic acid probes activated by irradiation for subsequent interaction with intracellular target mRNA.
Thus, the cited references collectively teach the claimed features and provide a reason to incorporate known light-directed synthesis and photoactivation techniques into Vijiyan’s probe system to obtain spatial control over nucleic acid synthesis and probe activation. Such modification represents the application of known techniques to a related nucleic acid probe system for their established functions and predictable results. See MOE 2143.
Conclusions
No claim is allowed.
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/ELIZABETH ROSE LAFAVE/
Examiner, Art Unit 1684
/HEATHER CALAMITA/Supervisory Patent Examiner, Art Unit 1684
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