Methods For Preventing Titration Of Bimolecular Templated Assembly Reactions By Structurally-Determined Differential Hybridizations

Abstract

The present disclosure provides nucleic acid molecules, and kits comprising the same, for producing templated assembly products for a cell.

1 . A nucleic acid molecule comprising:
 a) a first stem portion comprising from about 10 to about 20 nucleotide bases;   b) an anti-target loop portion comprising from about 16 to about 40 nucleotide bases and having a first end to which the first stem portion is linked, wherein the anti-target loop portion is substantially complementary to a target nucleic acid molecule;   c) a second stem portion comprising from about 10 to about 20 nucleotide bases linked to a second end of the anti-target loop portion, wherein the first stem portion is substantially complementary to the second stem portion; and   d) a reactive effector moiety linked to either the first stem portion or the second stem portion;   wherein the T m  of the anti-target loop portion:target nucleic acid molecule is greater than the T m  of the first stem portion:second stem portion; and   wherein the reactive effector moiety is capable of undergoing a chemical reaction with a corresponding reactive effector moiety.   
     
     
         2 . The nucleic acid molecule of  claim 1  wherein:
 the T m  of the first stem portion:second stem portion subtracted from the T m  of the anti-target loop portion:target nucleic acid molecule is from about 10° C. to about 40° C.; and/or 
 the T m  of the first stem portion:second stem portion is from about 40° C. to about 50° C.; and/or 
 the T m  of the anti-target loop portion:target nucleic acid molecule is from about 60° C. to about 80° C.; and/or 
 the T m  of the first stem portion:second stem portion subtracted from the T m  of the anti-target loop portion:target nucleic acid molecule is from about 10° C. to about 20° C. 
 
     
     
         3 . The nucleic acid molecule of  claim 1  wherein:
 the first stem portion comprises from about 12 to about 18 nucleotide bases; and/or 
 the anti-target loop portion comprises from about 18 to about 35 nucleotide bases; and/or 
 the second stem portion comprises from about 12 to about 18 nucleotide bases. 
 
     
     
         4 . The nucleic acid molecule of  claim 1  herein the nucleotide bases of any one or more of the first stem portion, anti-target loop portion, and second stem portion are selected from the group consisting of DNA nucleotides, RNA nucleotides, phosphorothioate-modified nucleotides, 2-O-alkylated RNA nucleotides, halogenated nucleotides, locked nucleic acid nucleotides (LNA), peptide nucleic acids (PNA), morpholino nucleic acid analogues (morpholinos), pseudouridine nucleotides, xanthine nucleotides, hypoxanthine nucleotides, 2-deoxyinosine nucleotides, DNA analogs with L-ribose (L-DNA), Xeno nucleic acid (XNA) analogues, or other nucleic acid analogues capable of base-pair formation, or artificial nucleic acid analogues with altered backbones, or any combination thereof. 
     
     
         5 . The nucleic acid molecule of  claim 1  further comprising a linker between any one or more of the first stem portion and the anti-target loop portion, between the anti-target loop portion and the second stem portion, and between the second stem portion and the reactive effector moiety. 
     
     
         6 . The nucleic acid molecule of  claim 1  wherein the reactive effector moiety is selected from the group consisting of a peptide, a non-active portion of a peptidomimetic structure, a non-active portion of a drug, and a bioactive compound. 
     
     
         7 . The nucleic acid molecule of  claim 1  wherein the reactive effector moiety further comprises a bio-orthogonal reactive molecule. 
     
     
         8 . The nucleic acid molecule of  claim 7  wherein the bio-orthogonal reactive molecule is selected from the group consisting of an azide, an alkyne, a cyclooctyne, a nitrone, a norbornene, an oxanorbornadiene, a phosphine, a dialkyl phosphine, a trialkyl phosphine, a phosphinothiol, a phosphinophenol, a cyclooctene, a nitrile oxide, a thioester, a tetrazine, an isonitrile, a tetrazole, and a quadricyclane, or any derivative thereof. 
     
     
         9 . The nucleic acid molecule of  claim 1  wherein the anti-target loop portion further comprises an internal hinge region, wherein the hinge region comprises one or more nucleotides that are not complementary to the target nucleic acid molecule. 
     
     
         10 . The nucleic acid molecule of  claim 9  wherein the hinge region comprises from about 1 nucleotide to about 6 nucleotides. 
     
     
         11 . (canceled) 
     
     
         12 . A kit comprising:
 a first nucleic acid molecule according to  claim 1 ; and   a second nucleic acid molecule comprising from about 6 nucleotide bases to about 20 nucleotide bases, which comprises:
 a nucleotide portion that is substantially complementary to the stem portion of the first nucleic acid molecule that is linked to the reactive effector moiety; and 
 a reactive effector moiety which can chemically interact with the reactive effector molecule of the first nucleic acid molecule; 
   wherein the T m  of the second nucleic acid molecule:first or second stem portion linked to the reactive effector moiety is less than or equal to the T m  of the first stem portion:second stem portion.   
     
     
         13 . The kit of  claim 12 , wherein:
 the reactive effector moiety of the second nucleic acid molecule is linked to a bio-orthogonal reactive molecule;   the reactive effector moiety of the first nucleic acid molecule is linked to a bio-orthogonal reactive molecule;   the bio-orthogonal reactive molecule of the second nucleic acid molecule can chemically interact with the bio-orthogonal reactive molecule of the first nucleic acid molecule.   
     
     
         14 . The kit of  claim 12  wherein:
 the T m  of the duplex formed by the second nucleic acid molecule and the first or second stem portion linked to the reactive effector moiety subtracted from the T m  of the first stem portion:second stem portion is from about 0° C. to about 20° C.; and/or 
 the T m  of the duplex formed by the second nucleic acid molecule and the first or second stem portion linked to the reactive effector moiety is from about 30° C. to about 40° C.; and/or 
 the T m  of the duplex formed by the nucleic acid molecule and the first or second stem portion linked to the reactive effector moiety subtracted from the Ina of the first stem portion:second stem portion is from about 5° C. to about 10° C. 
 
     
     
         15 . The kit of  claim 12  wherein the second nucleic acid molecule comprises from about 8 to about 15 nucleotide bases. 
     
     
         16 . The kit of  claim 12  wherein:
 the first nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:13, and the second nucleic acid molecule comprises the nucleotide sequence 5′-AGCTCTCGA GT-3′ (SEQ ID NO:15); or 
 the first nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:14, and the second nucleic acid molecule comprises the nucleotide sequence 5′-GACGTCTCGA GT-3′ (SEQ ID NO:16). 
 
     
     
         17 . The kit of  claim 12  wherein the bio-orthogonal reactive molecule of the first nucleic acid molecule is hexynyl and the bio-orthogonal reactive molecule of the second nucleic acid molecule is azide. 
     
     
         18 . A method of producing a templated assembly product for a cell comprising:
 contacting a target nucleic acid molecule of the cell with a first nucleic acid molecule of  claim 1 ; and   contacting the first nucleic acid molecule with a second nucleic acid molecule, wherein the second nucleic acid molecule comprises:
 a nucleotide portion that is substantially complementary to the stem portion of the first nucleic acid molecule that is linked to the reactive effector moiety; and 
 a reactive effector moiety which can chemically interact with the reactive effector molecule of the first nucleic acid molecule; 
   wherein the T m  of the second nucleic acid molecule:first or second stem portion linked to the reactive effector moiety is less than or equal to the T m  of the first stem portion:second stem portion;   resulting in the combination of the respective reactive effector moieties thereby producing the templated assembly product.   
     
     
         19 . The method of  claim 18  wherein:
 the first nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:13, and the second nucleic acid molecule comprises the nucleotide sequence 5′-AGCTCTCGA GT-3′ (SEQ ID NO:15); or 
 the first nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:14, and the second nucleic acid molecule comprises the nucleotide sequence 5′-GACGTCTCGA GT-3′ (SEQ ID NO:16). 
 
     
     
         20 . The method of  claim 18  wherein the bio-orthogonal reactive molecule of the first nucleic acid molecule is hexynyl and the bio-orthogonal reactive molecule of the second nucleic acid molecule is azide.