Methods for the development of vaccines based on oligosaccharide-oligonucleotide conjugates
Described herein are oligosaccharide-oligonucleotide conjugates useful as vaccines against one or more human or veterinary therapeutic indications, and methods of synthesizing and identifying them. The conjugates may be identified using non-human antibodies as binding targets, thereby expanding the power and scope of the invention. Efficacious conjugates may be identified through an iterative.
1. A method, comprising the steps of:
(a) combining an oligonucleotide, a first DNA polymerase, and a plurality of deoxyribonucleotide triphosphates,
wherein
the oligonucleotide comprises a first primer binding site on the 5′ end, a randomized region, and a stem-loop region;
the randomized region is located between the first primer binding site and the stem-loop region;
the stem-loop region comprises a second primer binding site;
at least one of the deoxyribonucleotide triphosphates comprises a reactive substituent; and
the reactive substituent is ethynyl,
thereby forming an extended oligonucleotide comprising an original strand and an extended strand, wherein the extended strand comprises at least one reactive substituent;
(b) combining a plurality of modifying compounds and the extended oligonucleotide under reaction conditions, wherein the modifying compound is a compound of formula III:
wherein A represents a branched or unbranched oligosaccharide consisting of about 3 to about 15 saccharide moieties,
thereby forming a modified extended oligonucleotide comprising the original strand and a modified extended strand; and
(c) combining a primer complementary to the second primer binding site, a second DNA polymerase, the modified extended oligonucleotide, and a plurality of deoxyribonucleotide triphosphates
thereby creating a duplex with the original strand and displacing the modified extended strand.
2. The method of claim 1 , wherein oligonucleotide is in the form of a partial stem-loop.
3. The method of claim 1 , wherein the deoxyribonucleotide triphosphate comprising a reactive substituent is an unnatural deoxyribonucleotide triphosphate.
4. The method of claim 1 , wherein the deoxyribonucleotide triphosphate comprising a reactive substituent is 5-ethynyl-deoxyuridine triphosphate.
5. The method of claim 1 , wherein the extended oligonucleotide has a hairpin configuration.
6. The method of claim 1 , wherein the modifying compound is represented by formula I or formula II:
7. A method, comprising the steps of
(a) combining a plurality of oligonucleotides, a first DNA polymerase, and a plurality of deoxyribonucleotide triphosphates,
wherein
the oligonucleotides comprise a first primer binding site on the 5′ end, a randomized region, and a stem-loop region;
the randomized region is located between the first primer binding site and the stem-loop region;
the stem-loop region comprises a second primer binding site;
at least one of the deoxyribonucleotide triphosphates comprises a reactive substituent; and
the reactive substituent is ethynyl,
thereby forming a plurality of extended oligonucleotides comprising an original strand and an extended strand, wherein the extended strand comprises at least one reactive substituent;
(b) combining a plurality of modifying compounds and the plurality of extended oligonucleotides under reaction conditions, wherein each modifying compound is a compound of formula III:
wherein A represents a branched or unbranched oligosaccharide consisting of about 3 to about 15 saccharide moieties,
thereby forming a plurality of modified extended oligonucleotides comprising the original strand and a modified extended strand;
(c) combining a plurality of primers complementary to the second primer binding site, a second DNA polymerase, the plurality of modified extended oligonucleotides, and a plurality of deoxyribonucleotide triphosphates thereby creating duplexes with the original strands, displacing the modified extended strands, and forming a plurality of modified single-stranded oligonucleotides;
(d) combining the plurality of modified single-stranded oligonucleotides and a target protein;
(e) isolating the modified single-stranded oligonucleotides that bind to the target protein, thereby identifying a plurality of selected oligonucleotides;
(f) amplifying the plurality of selected oligonucleotides, thereby forming a plurality of complementary oligonucleotides; and
(g) preparing a plurality of regenerated selected oligonucleotides from the plurality of complementary oligonucleotides.
8. The method of claim 7 , wherein the oligonucleotide has the form of a partial stem-loop.
9. The method of claim 7 , wherein the deoxyribonucleotide triphosphate comprising a reactive substituent is an unnatural deoxyribonucleotide triphosphate.
10. The method of claim 7 , wherein the deoxyribonucleotide triphosphate comprising a reactive substituent is 5-ethynyl-deoxyuridine triphosphate.
11. The method of claim 7 , wherein the extended oligonucleotide has a hairpin configuration.
12. The method of claim 7 , wherein the modifying compound is represented by formula I or formula II:
13. The method of claim 7 , wherein the target protein is an antibody.
14. An oligonucleotide, wherein the oligonucleotide comprises at least one non-natural deoxynucleoside of formula IV:
wherein A represents a branched or unbranched oligosaccharide consisting of about 3 to about 15 saccharide moieties.