SELF-ASSEMBLING OLIGONUCLEOTIDES AND METHODS

• US 20090299046A1
• Filed: 08/29/2007
• Published: 12/03/2009
• Est. Priority Date: 08/29/2006
• Status: Active Grant

• Current Assignee: Creatrone, Inc.
• Original Assignee: Creatrone, Inc.
• Inventors: Crea, Roberto, Cappuccilli, Guido
• Granted Patent: US 9,315,536 B2
• Time in Patent Office: Days
• US Class Current: 536/25.3
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical

Abstract

A composition comprising individual single-stranded oligonucleotides capable of self assembling to form a pair of complementary, substantially contiguous single strands of a double-stranded nucleic acid filament, and a method for forming such filaments are disclosed.

Claims

1. A composition comprising individual single-stranded oligonucleotides, each of which is composed of a first, 5′

-end segment and a second, 3′

-end segment, said oligonucleotides being capable of forming, by self assembly under oligonucleotide hybridization conditions, a pair of complementary, substantially contiguous single strands of a double-stranded nucleic acid filament in which the first and second segments of an oligonucleotide in one strand of the filament are hybridized with complementary first and second segments, respectively, of two adjacent oligonucleotides in the second strand of the filament, and each strand is composed of a plurality of said oligonucleotides.

2. The composition of claim 1, wherein the segments of the oligonucleotides are palindromic and the molecules are symmetric with respect to the lengths of their first and second segments.

3. The composition of claim 1, wherein the segments of molecules are palindromic and the molecules are asymmetric with respect to the lengths of their first and second segments.

4. The composition of claim 1, wherein the oligonucleotides each have a 5′

-end phosphate group, permitting ligation of the 5′

-end of one oligonucleotide in each strand to the 3′

-end of an adjacent oligonucleotide in the same strand.

5. The composition of claim 1, wherein the individual oligonucleotides each contain between 1-3 nucleotide bases between said first and second segments, and each substantially contiguous strand in the double stranded filament contains periodic gaps that are 1-3 bases in length and offset from gaps in the other strand in the filament.

6. The composition of claim 1, wherein the first segment of the oligonucleotides is composed substantially of A and T bases only, and the second segment of the oligonucleotides is composed of substantially of G and C bases only, and the double stranded filament has regions composed substantially of paired A and T bases only alternating with regions of paired G and C bases only.

7. The composition of claim 1, which includes a plurality of N sets of different-sequence, but same-length oligonucleotides, where the 5′

-end sequence in each set of oligonucleotides is complementary to the 5′

-end sequence in only one other set of oligonucleotides, and where the 3′

-end sequence in each set of oligonucleotides is complementary to 3′

-end sequence in only one other set of oligonucleotides, wherein the pair of complementary, substantially contiguous single strands of a double-stranded nucleic acid filament are composed of only one oligonucleotide from each of the N sets of oligonucleotides.

8. The composition of claim 1, which includes a plurality of N sets of different-sequence, different-length oligonucleotides, where the 5′

-end sequence in each set of oligonucleotides is the same length as and complementary to the 5′

-end sequence in only one other set of oligonucleotides, and where the 3′

-end sequence in each set of oligonucleotides is the same length as and complementary to 3′

-end sequence in only one other set of oligonucleotides, wherein the pair of complementary, substantially contiguous single strands of a double-stranded nucleic acid filament are composed of only one oligonucleotide from each of the N sets of oligonucleotides.

9. A method of forming a double-stranded duplex nucleic acid filament by self-assembly, comprising

reacting in solution, under nucleic acid-acid hybridization conditions, individual single-stranded oligonucleotides, each of which is composed of a first, 5′

-end segment and a second, 3′

-end segment, wherein the oligonucleotides self-assemble to form a pair of complementary, substantially contiguous single strands of a double-stranded nucleic acid filament in which the first and second segments of an oligonucleotide in one strand of the filament are hybridized with complementary first and second segments, respectively, of two adjacent oligonucleotides in the second strand of the filament, and each strand is composed of a plurality of said oligonucleotides.

10. The method of claim 9, wherein the segments of the oligonucleotides are palindromic and the molecules are symmetric with respect to the lengths of their first and second segments.

11. The method of claim 9, wherein the segments of molecules are palindromic and the molecules are asymmetric with respect to the lengths of their first and second segments.

12. The method of claim 9, wherein the oligonucleotides each have a 5′

-end phosphate group, permitting ligation of the 5′

-end of one oligonucleotide in each strand to the 3′

-end of an adjacent oligonucleotide in the same strand.

13. The method of claim 9, wherein the individual oligonucleotides each contain between 1-3 nucleotide bases between said first and second segments, and each substantially contiguous strand in the double stranded filament contains periodic gaps that are 1-3 bases in length and offset from gaps in the other strand in the filament.

14. The method of claim 9, wherein the first segment of the oligonucleotides is composed substantially of A and T bases only, and the second segment of the oligonucleotides is composed of substantially of G and C bases only, and the double stranded filament has regions composed substantially of paired A and T bases only alternating with regions of paired G and C bases only.

15. The method of claim 9, for forming a double-stranded duplex nucleic acid filament of a defined length, wherein the individual single-stranded oligonucleotides include a plurality of N sets of different-sequence, but same-length oligonucleotides, where the 5′

-end sequence in each set of oligonucleotides is complementary to the 5′

-end sequence in only one other set of oligonucleotides, and where the 3′

-end sequence in each set of oligonucleotides is complementary to 3′

-end sequence in only one other set of oligonucleotides, wherein the pair of complementary, substantially contiguous single strands of a double-stranded nucleic acid filament formed are composed of only one oligonucleotide from each of the N sets of oligonucleotides.

16. The method of claim 9, for forming a double-stranded duplex nucleic acid filament of a defined length, wherein the individual single-stranded oligonucleotides include a plurality of N sets of different-sequence, different-length oligonucleotides, where the 5′

-end sequence in each set of oligonucleotides is the same length as and complementary to the 5′

-end sequence in only one other set of oligonucleotides, and where the 3′

-end sequence in each set of oligonucleotides is the same length as and complementary to 3′

-end sequence in only one other set of oligonucleotides, wherein the pair of complementary, substantially contiguous single strands of a double-stranded nucleic acid filament are composed of only one oligonucleotide from each of the N sets of oligonucleotides.

17. The method of claim 9, for use in forming a bundle of sequence-aligned double-stranded nucleic acid filaments, wherein said reacting step is carried out in the presence of a selected-length double-stranded template filament having a sequence formed by self-assembly of such oligonucleotides, wherein the template filament directs the assembly of the oligonucleotides to form a bundle of sequence-aligned double-stranded nucleic acid filaments.

18. The method of claim 9, for producing a double-stranded duplex nucleic acid filament bridging a pair of selected regions on a substrate, comprising

(a) anchoring at each region on the substrate, a single-stranded nucleic acid fragment whose sequence is complementary to one segment of an oligonucleotide composed of a first, 5′

-end segment and a second, 3′

-end segment, where said oligonucleotides is capable of forming, under oligonucleotide hybridization conditions, a pair of complementary, substantially contiguous single strands of a double-stranded nucleic acid filament in which the first and second segments of an oligonucleotide in one strand of the filament are hybridized with complementary first and second segments, respectively, of two adjacent oligonucleotides in the second strand of the filament, and each strand is composed of a plurality of said oligonucleotides,

(b) exposing the substrate and anchored single-stranded nucleic acid fragments to a solution of said oligonucleotides, under nucleic acid hybridization conditions, and

(c) by said exposing, forming between the two regions, and connected therewith, a double-stranded nucleic acid filament composed of overlapping oligonucleotides.

19. The method of claim 18, wherein each region of the solid support is a gold film electrode, and the fragments are attached to the film through a disulfide linkage between a surface-bound thiol reagent and a phosphorothio group at the 3′

end the fragment.

20. The method of claim 18, for use in creating an electrically conductive filament between the first and second regions, wherein the double-stranded nucleic acid filament formed in step (c) is coated with an electrically conductive film.

21. The method of claim 18, for use in creating an electrically conductive filament between the first and second regions, wherein the double-stranded nucleic acid filament formed in step (c) is treated with a solution of a Zn, Co, or Ni salt, under conditions effective to incorporate Zn, Co, or Ni atoms between the filament'"'"'s nucleic acid bases.

22. The method of claim 18, for forming a double-stranded duplex nucleic acid filament of a defined length spanning the two substrate regions, wherein the individual single-stranded oligonucleotide include a plurality of N sets of different-sequence, but same-length oligonucleotides, where the 5′

-end sequence in each set of oligonucleotides is complementary to the 5′

-end sequence in only one other set of oligonucleotides, and where the 3′

-end sequence in each set of oligonucleotides is complementary to 3′

-end sequence in only one other set of oligonucleotides, wherein the pair of complementary, substantially contiguous single strands of a double-stranded nucleic acid filament formed are composed on only one oligonucleotide from each of the N sets of oligonucleotides.

23. The method of claim 18, for forming a double-stranded duplex nucleic acid filament of a defined length spanning the two regions, wherein the individual single-stranded oligonucleotide include a plurality of N sets of different-sequence, different-length oligonucleotides, where the 5′

-end sequence in each set of oligonucleotides is the same length as and complementary to the 5′

-end sequence in only one other set of oligonucleotides, and where the 3′

-end sequence in each set of oligonucleotides is the same length as and complementary to 3′

-end sequence in only one other set of oligonucleotides, wherein the pair of complementary, substantially contiguous single strands of a double-stranded nucleic acid filament are composed on only one oligonucleotide from each of the N sets of oligonucleotides.

24. The method of claim 18, for use in forming a bundle of sequence-aligned double-stranded nucleic acid filaments between the first and second regions, which further includes forming between the two regions, a desired-length double-stranded template filament having a sequence formed by self-assembly of such oligonucleotides, wherein the template filament directs the assembly of the oligonucleotides to form a bundle of sequence-aligned double-stranded nucleic acid filaments.