Nucleic acid-peptide-nucleic acid conjugate molecules and methods of making the same
View Patent ↗The present disclosure relates to nucleic acid-peptide-nucleic acid conjugate molecules and to methods for synthesizing nucleic acid-peptide-nucleic acid conjugate molecules. In some embodiments, a method for synthesizing a nucleic acid-peptide-nucleic acid conjugate molecule using proximity-enhanced synthesis includes covalently linking a peptide with a first nucleic acid strand via a first reaction, hybridizing the first nucleic acid strand with a second nucleic acid strand to bring the second nucleic acid strand in proximity to the peptide, and covalently linking the peptide with the second nucleic acid strand via a second reaction to provide the nucleic acid-peptide-nucleic acid conjugate molecule. In some embodiments, the peptide of the nucleic acid-peptide-nucleic acid conjugate molecule is a substrate for cleavage by an enzyme, such as matrix metalloproteinase-8 (MMP-8). Exemplary applications of the nucleic acid-peptide-nucleic acid conjugate molecule for drug delivery, molecular assembly of hybrid structures, and constraining the peptide to a biologically active conformation are also disclosed.
1 . A method for synthesizing a nucleic acid-peptide-nucleic acid conjugate molecule, comprising:
covalently linking a peptide, the peptide comprising a terminal azide group and a terminal alkyne group, with a first nucleic acid strand, the first nucleic acid strand comprising an alkyne functional group, via a first reaction, the first reaction comprising an azide-alkyne cycloaddition, to provide a nucleic acid-peptide conjugate;
hybridizing the first nucleic acid strand of the nucleic acid-peptide conjugate with a second nucleic acid strand, the second nucleic acid strand comprising an azide group, to bring the second nucleic acid strand in proximity to the peptide; and
covalently linking the peptide with the second nucleic acid strand via a second reaction, the second reaction comprising an azide-alkyne cycloaddition, to provide the nucleic acid-peptide-nucleic acid conjugate molecule, wherein the first nucleic acid strand and the second nucleic acid strand are partially complementary.
2 . The method of claim 1 , wherein the first nucleic acid strand and the second nucleic acid strand have different nucleic acid sequences and are not fully complementary.
3 . The method of claim 1 , wherein the first reaction and the second reaction are orthogonal azide-alkyne cycloaddition reactions.
4 . The method of claim 1 , wherein the first reaction is a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction, and the second reaction is a copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction.
5 . The method of claim 4 , wherein the peptide is modified with an N-terminal or C-terminal azide group, wherein the first nucleic acid strand is modified with a dibenzocyclooctyne group, and wherein the azide group of the peptide reacts with the dibenzocyclooctyne group of the first nucleic acid strand via the SPAAC reaction.
6 . The method of claim 5 , wherein the peptide is modified with an N-terminal or C-terminal alkyne group, wherein the second nucleic acid strand is modified with an azide group, and wherein the alkyne group of the peptide reacts with the azide group of the second nucleic acid strand via the CuAAC reaction.
7 . The method of claim 1 , wherein the first nucleic acid strand and the second nucleic acid strand are partially hybridized in the nucleic acid-peptide-nucleic acid conjugate molecule.
8 . The method of claim 7 , wherein the nucleic acid-peptide-nucleic acid conjugate molecule includes single-stranded overhangs where the first nucleic acid strand and the second nucleic acid strand are unhybridized.
9 . The method of claim 7 , wherein the nucleic acid-peptide-nucleic acid conjugate molecule is constrained to a hairpin structure by the partial hybridization between the first nucleic acid strand and the second nucleic acid strand.
10 . The method of claim 9 , further comprising displacing the first nucleic acid strand from the second nucleic acid strand using one or more single-stranded nucleic acid strands that are fully complementary to the first nucleic acid strand and/or the second nucleic acid strand.
11 . The method of claim 10 , wherein displacing the first nucleic acid strand from the second nucleic acid strand disrupts the hairpin structure of the nucleic acid-peptide- nucleic acid conjugate molecule.
12 . The method of claim 1 , wherein the peptide is a substrate for enzymatic cleavage, and optionally cleaving the peptide with an enzyme.
13 . The method of claim 1 , wherein
the terminal azide group of the peptide comprises N-terminal azidolysine (azK);
the terminal alkyne group of the peptide comprises C-terminal propargylalanine (prA);
the alkyne group of the first nucleic acid strand comprises dibenzocyclooctyne;
the first reaction comprises strain-promoted azide-alkyne cycloaddition (SPAAC); and
the second reaction comprises copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC).