Therapeutic nucleic acid-3′-conjugates
View Patent ↗Methods are described for improvement of the serum half life of therapeutic nucleic acids by 3′ conjugation to useful target proteins, or other large molecules with useful function. In one embodiment, a 3′ A, C or G overhang is added to ds-DNA and the primary amines conjugated using biocompatible bifunctional linkers to proteins. The resulting nucleic acid-3′ conjugates are serum nuclease-resistant and retained in vivo for long periods without rapid kidney clearance. Further, the choice of conjugate imparts additional functionality to the nucleic acid-3′ conjugate.
1. A synthetic aptamer comprising:
a first polynucleotide;
wherein the first polynucleotide comprises at least 60 nucleotides;
wherein a 3′ terminal oligonucleotide of the first polynucleotide is selected from the group consisting of adenine, cytosine and guanine; and
a protein moiety that is operatively coupled to a primary amine group of the 3′ terminal oligonucleotide to form a single-stranded polynucleotide-3′ protein conjugate.
2. The synthetic aptamer of claim 1 , further comprising:
a second polynucleotide hybridized to the first polynucleotide to form a double-stranded polynucleotide-3′ protein conjugate;
wherein the double-stranded polynucleotide-3′ protein conjugate comprises an overhang consisting of the 3′ terminal oligonucleotide of the first polynucleotide and the protein moiety.
3. The synthetic aptamer of claim 2 , wherein the second polynucleotide has at least about 70% complementarity to the first polynucleotide.
4. The synthetic aptamer of claim 2 , wherein the second polynucleotide has at least about 95% complementarity to the first polynucleotide.
5. The synthetic aptamer of claim 2 , wherein the first polynucleotide is selected from at least one of SEQ ID NO: 107.
6. The synthetic aptamer of claim 1 , wherein the first polynucleotide is selected from at least one of SEQ ID NO: 107.
7. The synthetic aptamer of claim 1 , wherein the protein moiety is selected from the group consisting of a biocidal protein, a phage lysis protein, a protein that recruits the cells of the immune system, a protein that activates the immune system, and a serum stable protein.
8. The synthetic aptamer of claim 1 , wherein the protein moiety is selected from the group consisting of Clqrs, Fc, C3b, C4b, C5a, C567, alpha-globulins, beta-globulins, gamma-globulins, serum albumin, and hemoglobin.
9. The synthetic aptamer of claim 1 , wherein the aptamer specifically binds to a toxin, and the protein moiety prevents degradation and clearance of said aptamer when administered in vivo.
10. The synthetic aptamer of claim 9 , wherein the toxin is selected from the group consisting of bacterial biotoxins, botulinum toxins, cholera toxin, ricin, staphylococcal enterotoxins, plant toxins, insect toxins, arachnid toxins, or reptilian venoms.
11. The synthetic aptamer of claim 1 , further comprising:
a biocompatible bifunctional linker operatively coupling the first polynucleotide to the protein moiety.
12. The synthetic aptamer of claim 1 , wherein the protein moiety prevents degradation and clearance of said aptamer when administered in vivo.
13. The synthetic aptamer of claim 1 , wherein operative coupling of the protein moiety to the primary amine group comprises covalent bonding of the protein moiety to the primary amine group.
14. A nucleic acid-protein conjugate comprising:
a first polynucleotide;
wherein a 3′ terminal oligonucleotide of the first polynucleotide is selected from the group consisting of adenine, cytosine and guanine; and
a protein moiety that is operatively coupled to a primary amine group of the 3′ terminal oligonucleotide to form a single-stranded nucleic acid-protein conjugate; and
wherein said single-stranded nucleic acid-protein conjugate has a greater in vivo stability relative to the first polynucleotide alone.
15. The nucleic acid-protein conjugate of claim 14 , further comprising:
a second polynucleotide hybridized to the first polynucleotide to form a double-stranded nucleic acid-protein conjugate;
wherein the double-stranded nucleic acid-protein conjugate comprises an overhang consisting of the 3′ terminal oligonucleotide of the first polynucleotide and the protein moiety.
16. The nucleic acid-protein conjugate of claim 15 , wherein the second polynucleotide has at least about 70% complementarity to the first polynucleotide.
17. The nucleic acid-protein conjugate of claim 15 , wherein the second polynucleotide has at least about 95% complementarity to the first polynucleotide.
18. The nucleic acid-protein conjugate of claim 14 , wherein the protein moiety is selected from the group consisting of a biocidal protein, a phage lysis protein, a protein that recruits the cells of the immune system, a protein that activates the immune system, and a serum stable protein.
19. The nucleic acid-protein conjugate of claim 14 , wherein the protein moiety is selected from the group consisting of Clqrs, Fc, C3b, C4b, C5a, C567, alpha-globulins, beta-globulins, gamma-globulins, serum albumin, and hemoglobin.
20. The nucleic acid-protein conjugate of claim 14 , wherein operative coupling of the protein moiety to the primary amine group comprises covalent bonding of the protein moiety to the primary amine group.