Polynucleotide backbones for complexing proteins
AssigneeAffomix Corporation
InventorsMichael P. Weiner, Michael I. Sherman
Life of this patent
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Abstract
We use the Tus-Ter interaction to enable the utilization of nucleic acid analytical methodologies for proteins. We also use the Tus-Ter interaction to make polymers and oligomers that have a nucleic acid backbone with protein functionalities. These methods are useful for molecular modeling, for efficiently running enzymatic pathway reactions, and for analyzing presence and/or amount of particular proteins.
Claims21
A polymer comprising a plurality of monomers, each monomer comprising a non-covalent complex of:
- a fusion protein and
- a nucleic acid molecule,
- wherein the fusion protein comprises a Tus protein according to SEQ ID NO:
- 5 and a polypeptide wherein the nucleic acid molecule comprises a Ter site according to SEQ ID NO:
- 7.
-16. (canceled)
(canceled)
A method of assembling a polymer comprising a plurality of monomers, comprising the steps of:
- ligating a plurality of monomers to each other using a DNA ligase enzyme, each monomer comprising a non-covalent complex of:
- a fusion protein, and
- a nucleic acid molecule,
- wherein the fusion protein comprises a Tus protein according to SEQ ID NO:
- 5 and a polypeptide, and wherein the nucleic acid molecule comprises a Ter site according to SEQ ID NO:
- 7.
-24. (canceled)
A protein-DNA complex which comprises:
- a fusion protein;
- and
- a nucleic acid molecule;
- wherein a first portion of the nucleic acid molecule is double stranded and a second portion of the nucleic acid molecule is single stranded;
- wherein the first portion comprises a Ter sequence according to SEQ ID NO:
- 7 and the second portion comprises an addressing sequence of at least 6 nucleotides;
- wherein the fusion protein comprises a Tus protein according to SEQ ID NO:
- 5 and a binding polypeptide.
-33. (canceled)
A method to measure a target molecule which can be bound by two distinct binding polypeptides, comprising:
- mixing a first and a second binding polypeptides with a target molecule to form a mixture;
- wherein each binding polypeptide is part of a fusion protein with a Tus protein according to SEQ ID NO:
- 5, wherein the Tus protein is bound to a DNA molecule which comprises a double-stranded portion and a single-stranded portion, wherein the double-stranded portion comprises a Ter sequence according to SEQ ID NO:
- 7, wherein the single stranded portion comprises a tag sequence, wherein the tag sequence uniquely corresponds to the binding polypeptide;
- adding a bridging oligonucleotide to said mixture under conditions in which complementary DNA single strands will form double strands;
- wherein the bridging oligonucleotide comprises a first and a second portion, wherein the first portion is complementary to the tag sequence of the first binding polypeptide and the second portion is complementary to the tag sequence of the second binding polypeptide, wherein the first and the second portion of the bridging oligonucleotide are separated by 0 to 6 nucleotides;
- adding DNA ligase to said mixture, wherein said ligase joins 5′ and 3′ ends of nicked double-stranded DNA molecules to form an analyte DNA strand comprising a ligated junction between said first tag sequence and said second tag sequence;
- amplifying the first tag sequence, the ligated junction, and the second tag sequence to form an amplified analyte DNA strand;
- assaying to determine amount in the mixture of the amplified analyte DNA strand, wherein the amount of the amplified analyte DNA strand is related to the amount of the target molecule.
-40. (canceled)
A method for attaching an enzyme to a substratum, comprising:
- attaching a nucleic acid molecule to a substratum by means of covalent or non-covalent coupling, wherein the nucleic acid molecule comprises a Ter sequence according to SEQ ID NO:
- 7;
- forming a complex between the nucleic acid molecule and a fusion protein, wherein the fusion protein comprises a Tus protein according to SEQ ID NO:
- 5 and an enzyme.
-49. (canceled)
A method of forming an arrayed library of diverse protein-DNA complexes, comprising the step of:
- mixing together one or more substrata comprising arrayed single stranded probes and a library of diverse protein-DNA complexes in which each complex comprises:
- a fusion protein;
- and
- a nucleic acid molecule;
- wherein a first portion of the nucleic acid molecule is double stranded and a second portion of the nucleic acid molecule is single stranded;
- wherein the first portion comprises a Ter sequence according to SEQ ID NO:
- 7 and the second portion comprises an addressing sequence;
- wherein the fusion protein comprises a Tus protein according to SEQ ID NO:
- 5 and a binding polypeptide;
- wherein each addressing sequence is complexed with a fusion protein comprising a unique binding polypeptide;
- wherein the single stranded probes each comprise a sequence of at least 6 nucleotides which is complementary to an addressing sequence in the nucleic acid molecules;
- whereby upon mixing, the protein-DNA complexes bind to single stranded probes having complementary sequences.
-56. (canceled)
Dependencies are inferred from the claim text.