# US 2023/0053360 A1 · Nucleic acid-peptide-nucleic acid conjugate molecules and methods of making the same > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2023/0053360 A1 - Document ID: US20230053360A1 - Status: Published application - Kind: A1 - Application number: 17/818,856 - Filed: 2022-08-10 - Published: 2023-02-23 - Earliest claimed: 2021-08-11 - Assignees: Arizona Board of Regents on behalf of Arizona State University - Inventors: Nicholas Stephanopoulos; Tara MacCulloch - Web page: https://patentgrep.com/patent/US20230053360A1 ## CPC - A61K 47/6925: {the form being a microcapsule, nanocapsule, microbubble or nanobubble} - C07K 1/1077: {by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids} - A61K 47/549: {Sugars, nucleosides, nucleotides or nucleic acids} - A61K 47/64: Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent (peptidic linkers A61K47/65) - C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} - C12N 2310/18: acting by a non-sequence specific mechanism - C12N 2310/3513: Protein; Peptide - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract 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. ## Claims ### Claim 1 A method for synthesizing a nucleic acid-peptide-nucleic acid conjugate molecule, comprising: covalently linking a peptide with a first nucleic acid strand via a first reaction 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 to bring a 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, wherein the first reaction and the second reaction are orthogonal chemical reactions. ### Claim 2 (depends on 1) 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. ### Claim 3 (depends on 1) The method of claim 1, wherein the first reaction and the second reaction are orthogonal azide-alkyne cycloaddition reactions. ### Claim 4 (depends on 1) 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. ### Claim 5 (depends on 4) 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. ### Claim 6 (depends on 5) 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. ### Claim 7 (depends on 1) 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. ### Claim 8 (depends on 7) 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. ### Claim 9 (depends on 7) 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. ### Claim 10 (depends on 9) 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. ### Claim 11 (depends on 10) 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. ### Claim 12 (depends on 1) The method of claim 1, wherein the peptide is a substrate for enzymatic cleavage, and optionally cleaving the peptide with the enzyme. ### Claim 13 A drug delivery vehicle, comprising: a nucleic acid-based nano-container; a therapeutic agent within the nucleic acid-based nano-container; and a nucleic acid-peptide-nucleic acid conjugate including a peptide modified with a first nucleic acid strand and a second nucleic acid strand, the first nucleic acid strand and the second nucleic acid strand being hybridized with complementary nucleic acid strands of the nucleic acid-based nano-container to secure the therapeutic agent within the nucleic acid-based nano-container. ### Claim 14 (depends on 13) The drug delivery vehicle of claim 13, wherein the first nucleic acid strand is coupled at an N-terminal end of the peptide, and wherein the second nucleic acid strand is coupled at a C-terminal end of the peptide. ### Claim 15 (depends on 13) The drug delivery vehicle of claim 13, wherein the first nucleic acid strand and the second nucleic acid strand have different sequences. ### Claim 16 (depends on 13) The drug delivery vehicle of claim 13, wherein the peptide is a substrate for enzymatic cleavage. ### Claim 17 (depends on 16) The drug delivery vehicle of claim 16, wherein cleavage of the peptide with an enzyme triggers release of the therapeutic agent from the nucleic acid-based nano-container. ### Claim 18 A method of drug delivery, comprising: encapsulating a therapeutic agent inside of a nucleic acid-based nano-container; securing the therapeutic agent inside of the nucleic acid-based nano-container with a nucleic acid-peptide-nucleic acid conjugate molecule having at least a first nucleic acid strand and a second nucleic acid strand that hybridize with complementary nucleic acid strands of the nucleic acid-based nano-container; and degrading the peptide to release of the therapeutic agent from the nucleic acid-based nano-container. ### Claim 19 (depends on 18) The method of claim 18, wherein the peptide is a substrate for enzymatic cleavage, and wherein degrading the peptide comprises cleaving the peptide with the enzyme. ### Claim 20 (depends on 19) The method of drug delivery of claim 19, wherein the peptide is a substrate for matrix metalloproteinase-8 (MMP8), and wherein degrading the peptide comprises cleaving the peptide with MMP8.