METHOD TO GENERATE BIOCOMPATIBLE DENDRITIC POLYMERS FOR ANALYTE DETECTION WITH MULTIMODAL LABELING Ahttps://dav.uspto.gov/prex/index.html#domesticContinuityND SIGNAL AMPLIFICATION

Patent metadata

Field	Value
Publication number	US-2023193363-A1
Application number	US-202218047215-A
Country	US
Kind code	A1
Filing date	Oct 17, 2022
Priority date	Apr 17, 2017
Publication date	Jun 22, 2023
Grant date	—

Abstract

Described herein is a method to create dendritic biocompatible polymers from pairs of complementary dendritic nucleic acid monomers in a controlled manner, using polymerization triggers. The dendritic monomers are constituted of nucleic acids and an organic polymer capable of self-assembly. Each polymer contains approximately 200 dendrites that can be used to attach labels and constitute a biologically compatible signal amplification technology. Depending on the context this technology could be used to reveal the presence of a large variety of analytes such as specific nucleic acid molecules, small molecules, proteins, and peptides.

First claim

1 . An assembly, comprising:
at least two molecules, wherein each molecule comprises:
a) a nucleic acid hairpin,
b) a nucleic acid stem,
c) nucleic acid dendrites comprising a binding dendrite and extension dendrite, and
d) an organic polymer, wherein the organic polymer is about 16-20 carbon lengths, about 2 nm in length, or about 2-8 base pairs in length, and further wherein the nucleic acid hairpin sequence of at least one first molecule is complementary to the nucleic acid binding dendrite sequence of at least one second molecule, and also wherein the nucleic acid hairpin loop sequence of the at least one second molecule is complementary to the nucleic acid binding dendrite sequence of the at least one first molecule; and
at least one nucleic acid trigger coupled to an analyte binding agent, wherein the nucleic acid trigger is complementary to the nucleic acid stem and the binding dendrite of one of the at least two molecules.

2 . The assembly of claim 1 , wherein the hairpin loop sequence and binding dendrite sequence are each about 6-10 nucleotides.

3 . The assembly of claim 1 , wherein the hairpin loop sequence and binding dendrite sequence are each about 11-13 nucleotides.

4 . The assembly of claim 1 , wherein the extension dendrite comprises about 13-16 nucleotides.

5 . The assembly of claim 1 , wherein the extension dendrite comprises about 10-25 nucleotides.

6 . The assembly of claim 1 , wherein the nucleic acid trigger comprises about 12-48 nucleotides.

7 . The assembly of claim 1 , wherein the nucleic acid trigger comprises about 34-38 nucleotides.

8 . The assembly of claim 1 , wherein the nucleic acid stem comprises about 6-15 nucleotides.

9 . The assembly of claim 1 , wherein the nucleic acid stem comprises about 22-26 nucleotides.

10 . (canceled)

11 . (canceled)

12 . The assembly of claim 1 , wherein the analyte binding agent comprises a polynucleotide.

13 . The assembly of claim 1 , wherein the analyte binding agent comprises a peptide or protein.

14 . The assembly of claim 1 , wherein the analyte binding agent comprises an antibody.

15 . The assembly of claim 1 , further comprising a labeling polynucleotide complementary to the extension dendrite of one of the at least two molecules.

16 . The assembly of claim 15 , wherein the labeling polynucleotide further comprises fluorophores, chromophores, chromogens, quantum dots, fluorescent microspheres, nanoparticles, elemental labels, metal chelating polymers, barcodes and/or sequential barcodes.

17 . The assembly of claim 1 , wherein the assembly further comprises at least two additional molecules, wherein each of the at least two additional molecules comprises:
a) a nucleic acid hairpin loop, b) a nucleic acid stem, c) nucleic acid dendrites comprising a binding dendrite and extension dendrite, and d) an organic polymer, wherein the organic polymer is about 16-20 carbon lengths, about 2 nm in length, or about 2-8 base pairs in length, and further wherein the nucleic acid hairpin loop sequence of a first one of the at least two additional molecules is complementary to the nucleic acid binding dendrite sequence of a second one of the at least two additional molecules, and also wherein the nucleic acid hairpin loop sequence of the second one of the at least two additional molecules is complementary to the nucleic acid binding dendrite sequence of the first one of the at least two additional molecules; and a linker comprising a nucleic acid address complementary to an extension dendrite of the at least two molecules, and a second nucleic acid trigger complementary to a nucleic acid stem and a binding dendrite of the at least two additional molecules.

18 . The assembly of claim 17 , further comprising a labeling polynucleotide complementary to an extension dendrite of the at least two additional molecules.

19 . The assembly of claim 18 , wherein the labeling polynucleotide further comprises fluorophores, chromophores, chromogens, quantum dots, fluorescent microspheres, nanoparticles, elemental labels, metal chelating polymers, barcodes and/or sequential barcodes.

20 . A method of polymerization to form an assembly, comprising:
adding at least two molecules, each comprising:
a nucleic acid hairpin loop,
a nucleic acid stem,
nucleic acid dendrites comprising a binding dendrite and an extension dendrite, and
an organic polymer, wherein the organic polymer is about 16-20 carbon lengths, about 2 nm in length, or about 2-8 base pairs in length,
wherein the nucleic acid hairpin loop sequence of at least one first molecule is complementary to the nucleic acid binding dendrite sequence of at least one second molecule, and wherein the nucleic acid hairpin loop sequence of the at least one second molecule is complementary to the nucleic acid binding dendrite sequence of the at least one first molecule;
further adding a trigger molecule comprising a nucleic acid, wherein the trigger molecule is coupled to an analyte binding agent, and wherein the nucleic acid of the trigger molecule is complementary to the nucleic acid stem and the binding dendrite of one of the at least two molecules; and triggering self-assembled polymerization of the at least two molecules, thereby forming the assembly.

21 . (canceled)

22 . (canceled)

23 . The method of claim 20 , comprising generating a detectable signal by binding a labeling polynucleotide complementary to the extension dendrite of one of the at least two molecules, wherein the labeling polynucleotide comprises a labeling agent.

24 . (canceled)

25 . (canceled)

26 . The method of claim 20 , further comprising adding: at least two additional molecules, wherein each of the at least two additional molecules comprises:
a) a nucleic acid hairpin loop, b) a nucleic acid stem, c) nucleic acid dendrites comprising a binding dendrite and an extension dendrite, and d) an organic polymer, wherein the organic polymer is about 16-20 carbon lengths, about 2 nm in length, or about 2-8 base pairs in length, and further wherein the nucleic acid hairpin loop sequence of a first one of the at least two additional molecules is complementary to the nucleic acid binding dendrite sequence of a second one of the at least two additional molecules, and also wherein the nucleic acid hairpin loop sequence of the second one of the at least two additional molecules is complementary to the nucleic acid binding dendrite sequence of the first one of the at least two additional molecules; and
a linker comprising a nucleic acid address complementary to an extension dendrite of the at least two molecules, and
a second nucleic acid trigger complementary to a nucleic acid stem and a binding dendrite of the at least two additional molecules.

Assignees

• Univ Southern California

Inventors

• Fraser Scott E
• Restrepo Simon
• Dunham Joseph P

Classifications

• G01N2021/6439

  with indicators, stains, dyes, tags, labels, marks · CPC title

• C12Q1/682 Primary

  Signal amplification · CPC title

• C12N15/11

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title

• C12Q1/6841

  In situ hybridisation · CPC title

• C12N2310/531

  Stem-loop; Hairpin · CPC title