Polynucleotide-Linked Bioconjugates and Methods of Making and Using

Abstract

Provided for herein is a polynucleotide-modified bioconjugate comprising a substrate such as an antibody or bead linked to a conjugate component via a nucleic acid linker. Also provided are methods of making and using such bioconjugates. Conjugation methods for creating the bioconjugate are stable, not chemically harsh, and efficient enough that post-conjugation purification may not be required. Further this disclosure provides for reducing the logistic overheads related to product lines by eliminating the need for many unique linkers per conjugation pair.

1 - 44 . (canceled) 
     
     
         45 . A fluorescently labeled bioconjugate comprising:
 a substrate linked to an oligonucleotide-based fluorescent label via an at least partially double-stranded nucleic acid linker;   wherein the substrate comprises a first single-stranded nucleic acid linker and the oligonucleotide-based fluorescent label comprises a second single-stranded nucleic acid linker, wherein the first and second single-stranded nucleic acid linkers are at least partially or fully single-stranded;   wherein at least a portion of the first single-stranded nucleic acid linker is hybridized to a portion of the second single-stranded nucleic acid linker thereby forming the at least partially double-stranded nucleic acid linker linking the substrate to the oligonucleotide-based fluorescent label.   
     
     
         46 . The fluorescently labeled bioconjugate of  claim 45 , wherein the substrate is a binding molecule or a bead. 
     
     
         47 . The fluorescently labeled bioconjugate of  claim 46 , wherein the binding molecule is an antibody or an antigen-binding fragment, variant or derivative thereof, a peptide, a recombinant, natural or engineered receptor/ligand protein, an aptamer, a tetramer, a non-antibody protein or an antibody mimetic. 
     
     
         48 . The fluorescently labeled bioconjugate of  claim 45 , wherein the binding molecule is an antibody or an antigen-binding fragment, variant or derivative thereof. 
     
     
         49 . The fluorescently labeled bioconjugate of  claim 45 ,
 wherein the degree-of-labeling (DoL) of the substrate is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, or any range in between inclusive of the endpoints; and   wherein the degree-of-labeling (DoL) of the substrate is controlled stoichiometrically via the availability of the first single-stranded nucleic acid linker.   
     
     
         50 . The fluorescently labeled bioconjugate of  claim 45 , wherein the bioconjugate can be used to specifically label a cell, tissue, or organ for one or more of flow cytometry, antibody screening, ELISA or other sandwich assays, immune monitoring, biomarker assays, lateral flow, point-of-care or rapid diagnostics, imaging, microscopy, molecular diagnostics, next generation sequencing, long read sequencing, in situ sequencing, polymerase chain reaction, microarrays, nucleic acid sequencing, amino acid sequencing, digital pathology, Southern blotting, Northern blotting, or Western blotting. 
     
     
         51 . The fluorescently labeled bioconjugate of  claim 45 , wherein the at least partially double-stranded nucleic acid linker comprises (i) a portion attached to the substrate, (ii) a portion attached to the oligonucleotide-based fluorescent label, and (iii) at least one additional portion that is not (i) or (ii). 
     
     
         52 . The fluorescently labeled bioconjugate of  claim 51  wherein (iii) is a) a second portion attached to or is an extension of the oligonucleotide-based fluorescent label or b) attached to or is an extension of an additional oligonucleotide-based fluorescent label. 
     
     
         53 . The fluorescently labeled bioconjugate of  claim 45 , wherein the bioconjugate is stable in solution. 
     
     
         54 . The fluorescently labeled bioconjugate of  claim 45 , wherein the bioconjugate does not exhibit significant denaturation and re-hybridization of byproducts with other species in solution. 
     
     
         55 . A method of producing the fluorescently labeled bioconjugate of  claim 45 , the method comprising:
 contacting a substrate comprising a first single-stranded nucleic acid linker with an oligonucleotide-based fluorescent label comprising a second single-stranded nucleic acid linker, wherein the first and second single-stranded nucleic acid linkers are at least partially or fully single-stranded and at least a portion of the first at least partially single-stranded linker is complementary to a portion of the second at least partially single-stranded linker; and   wherein when contacted, the first single-stranded nucleic acid linker the second single-stranded nucleic acid linker anneal to form an at least partially double-stranded linker linking the substrate to the oligonucleotide-based fluorescent label.   
     
     
         56 . The method of  claim 55 , wherein prior to contacting the substrate and the oligonucleotide-based fluorescent label, the method further comprises the step of attaching the first single-stranded nucleic acid linker to the substrate. 
     
     
         57 . The method of  claim 55 , wherein prior to contacting the substrate and the oligonucleotide-based fluorescent label, the method further comprises the step of attaching the second single-stranded nucleic acid linker to the oligonucleotide-based fluorescent label. 
     
     
         58 . The method of  claim 55 , wherein the substrate is a binding molecule or a bead. 
     
     
         59 . A method of producing the fluorescently labeled bioconjugate of  claim 45 , the method comprising:
 contacting   (i) a substrate comprising a first single-stranded nucleic acid linker,   (ii) an oligonucleotide-based fluorescent label comprising a second single-stranded nucleic acid linker, and   (iii) a third single-stranded nucleic acid linker that is not the single-stranded nucleic acid linker of (i) or (ii),   wherein the first, second and third single-stranded nucleic acid linkers are each at least partially or fully single-stranded,   wherein at least a portion of the first single-stranded nucleic acid linker is complementary to a portion of the third single-stranded nucleic acid linker, and/or wherein at least a portion of the second single-stranded linker is complementary to a portion of the third single-stranded nucleic acid linker, and   wherein when contacted, the first single-stranded nucleic acid linker, the second single-stranded nucleic acid linker, and the third single-stranded nucleic acid linker anneal to form an at least partially double-stranded linker linking the substrate to the oligonucleotide-based fluorescent label.   
     
     
         60 . The method of  claim 59 , wherein the substrate is a binding molecule or a bead. 
     
     
         61 . A composition comprising:
 (i)(a) a substrate comprising a first single-stranded nucleic acid linker, and   (i)(b) an oligonucleotide-based fluorescent label comprising a second single-stranded nucleic acid linker,   wherein the first and second single-stranded nucleic acid linkers are at least partially or fully single-stranded,   wherein at least a portion of the first single-stranded nucleic acid linker is hybridized to a portion of the second single-stranded nucleic acid linker to form an at least partially double-stranded linker thereby linking the substrate to the oligonucleotide-based fluorescent label; or   (ii)(a) a substrate comprising a nucleic acid linker, and   (ii)(b) an oligonucleotide-based fluorescent label capable of attachment to the nucleic acid linker to link the substrate to the oligonucleotide-based fluorescent label,   wherein the nucleic acid linker is at least partially or fully single-stranded, comprises a double-stranded segment, or is entirely double-stranded; or   (iii)(a) an oligonucleotide-based fluorescent label comprising a nucleic acid linker,   (iii)(b) a substrate capable of attachment to the nucleic acid linker to link the oligonucleotide-based fluorescent label to the substrate,   wherein the nucleic acid linker is at least partially or fully single-stranded, comprises a double-stranded segment, or is entirely double-stranded; or   (iv)(a) a nucleic acid linker, wherein the nucleic acid linker is single-stranded, comprises a double-stranded segment, or wherein the nucleic acid linker is entirely double stranded, and   (iv)(b) a substrate capable of attachment to the nucleic acid linker, and an oligonucleotide-based fluorescent label capable of attachment to the nucleic acid linker; or   (v)(a) a substrate comprising a first at least partially single-stranded nucleic acid linker,   (v)(b) an oligonucleotide-based fluorescent label comprising a second at least partially single-stranded nucleic acid linker; and   (v)(c) a third at least partially single-stranded nucleic acid linker,   wherein (v)(a), (v)(b), and (v)(c) can anneal to form an at least partially double-stranded nucleic acid linker comprising (v)(a), (v)(b), and (v)(c) thereby linking (v)(a) to (v)(b).   
     
     
         62 . The composition of  claim 61 , wherein the substrate is a binding molecule or a bead. 
     
     
         63 . A method of labeling a cell, tissue, or organ, the method comprising:
 contacting the cell, tissue, or organ with the fluorescently labeled bioconjugate of  claim 45 , wherein the substrate portion of the bioconjugate binds to an antigen, epitope, or hapten of the cell, tissue, or organ.   
     
     
         64 . The method of  claim 63 , wherein the labeled cell can be used for one or more of flow cytometry, antibody screening, ELISA or other sandwich assays, immune monitoring, biomarker assays, lateral flow, point-of-care or rapid diagnostics, imaging, microscopy, molecular diagnostics, next generation sequencing, long read sequencing, in situ sequencing, polymerase chain reaction, microarrays, nucleic acid sequencing, amino acid sequencing, digital pathology, Southern blotting, Northern blotting, Western blotting, as a reference sample such as a control or for calibration of an instrument, assay, or system. 
     
     
         65 . A method of quantitatively measuring a signal from a labeled cell, tissue, and/or organ, the method comprising:
 (a) contacting the cell, tissue, and/or organ with the fluorescently labeled bioconjugate of  claim 45 , wherein the substrate portion of the bioconjugate binds to an antigen, epitope, membrane, genomic material, and/or hapten of the cell, tissue, or organ; and   (b) measuring the presence of the fluorescently labeled bioconjugate wherein the degree-of-labeling (DoL) of the substrate is known, thus allowing a quantitative measurement to be made.   
     
     
         66 . The method of  claim 65 , wherein the degree-of-labeling (DoL) of the substrate is controlled stoichiometrically via the availability of the first single-stranded nucleic acid linker; and
 wherein the degree-of-labeling (DoL) of the substrate is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or any range in between inclusive of the endpoints.   
     
     
         67 . A kit comprising:
 (a) the fluorescently labeled bioconjugate of  claim 45 ; and   (b) instructions for use.   
     
     
         68 . The kit of  claim 67 , wherein the kit further comprises one or more of the following:
 instructions either printed or on an electronic storage medium, buffers, additional reagents, or packaging materials.