USE OF RNA POLYMERASE AS AN INFORMATION-DEPENDENT MOLECULAR MOTOR

• US 20070077575A1
• Filed: 09/05/2006
• Published: 04/05/2007
• Est. Priority Date: 03/05/2004
• Status: Abandoned Application

• Current Assignee: The Research Foundation for The State University of New York (State University of New York)
• Original Assignee: The Research Foundation for The State University of New York (State University of New York)
• Inventors: McAllister, William, Anikin, Michael, Pomerantz, Richard
• Application Number:
  US11/469,957
• Publication Number:
  US 20070077575A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C07K 2319/20 containing a tag with affinity for a non-protein ligand
• CPC Class Codes: C07K 2319/80 containing a DNA binding domain, e.g. Lacl or Tet-repressor
• CPC Class Codes: C12N 15/1068 Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis

Abstract

Materials and methods are described in which the information dependence of RNA polymerase is employed to enable its use as a molecular motor adaptable for movement within DNA grid arrays and to actuate, move, position or alter cargo such as physical structures and normally inanimate substances and objects.

Claims

1. A molecular motor for actuating cargo in a controlled and information-dependent manner comprising a nucleotide polymerase enzyme (NP) having a high-affinity binding domain capable of binding to the cargo and being able to move along a DNA template.

2. The motor according to claim 1 wherein the high-affinity binding domain is reversibly or irreversibly bound by attachment or fusion to the NP.

3. The motor according to claim 1 wherein the NP is a single-subunit NP.

4. The motor according to claim 3 wherein the high-affinity binding domain is bound at or near the N-terminus of the NP.

5. The motor according to claim 4 wherein the binding domain is capable of binding to the cargo and to a solid surface.

6. The motor according to claim 4 wherein the high-affinity binding domain is an amino acid sequence.

7. The motor according to claim 6 wherein the amino acid sequence is selected from a yeast GAL4 polypeptide sequence, a Zif268 zinc-finger polypeptide sequence, a streptavidin polypeptide sequence, a metallotheionein polypeptide sequence, a transcription factor Sp1³⁰ polypeptide sequence, or a histidine sequence.

8. The motor according to claim 4 wherein the high-affinity binding domain is a DNA or RNA sequence.

9. The motor according to claim 8 wherein the DNA or RNA sequence is an aptamer.

10. A linear array of molecular motors comprising of a plurality of nucleotide polymerases as described and claimed in claim 3.

11. The array according to claim 10 wherein each motor is fused to a different high-affinity binding domain.

12. The array according to claim 10 wherein each motor comprises a different, phage NP.

13. A plurality of linear arrays according to claim 12, arranged and positioned in a two dimensional grid.

14. A method of actuating cargo in an information dependent manner comprising the steps of (a) creating a start-up complex by adding to a solution of a DNA template having a RNA polymerase promoter and the molecular motor according to claim 1 and sufficient nucleotide triphosphates complementary to the nucleic acids of the DNA template to cause formation of a stable EC, (b) washing the solution to remove excess substrate, (c) adding substrate to the solution, the substrate being composed of one or more nucleotide triphosphates complementary to the nucleic acid(s) of the DNA template downstream from the location of the stable EC, (c) incubating the solution under suitable transcription conditions.

15. The method of actuating cargo according to claim 14 wherein the start-up complex is formed by the addition of at least 14 nucleotide triphosphates complementary to the DNA template downstream from the polymerase binding site.

16. The method of actuating cargo according to claim 15 further comprising the steps of adding in a sequential manner a substrate composed of nucleotide triphosphate complementary to each successive nucleic acid in the DNA template sequence and washing the solution after each addition to remove excess substrate.

17. A method of making a molecular motor comprising binding a NP enzyme to a high affinity binding domain capable of binding to a surface support or a ligand sequence.

18. The method of claim 17 wherein the NP enzyme is a single subunit NP enzyme.

19. The method of claim 18 wherein the high affinity binding domain is a DNA sequence, a RNA sequence or an amino acid sequence selected from a yeast GAL4 polypeptide sequence, a Zif268 zinc-finger polypeptide sequence, a streptavidin polypeptide sequence, a metallotheionein polypeptide sequence, a transcription factor Sp1³⁰ polypeptide sequence, a β

-galactosidase polypeptide sequence or a histidine sequence.

20. The method of claim 19 wherein the high affinity binding domain is fused to the NP at or near its N-terminus.