Screening of nucleic acid agents via particle display

Patent metadata

Field	Value
Publication number	US-2016130575-A1
Application number	US-201314647775-A
Country	US
Kind code	A1
Filing date	Nov 21, 2013
Priority date	Dec 5, 2012
Publication date	May 12, 2016
Grant date	—

Abstract

The present disclosure provides a method for identifying one or more nucleic acid agents, e.g., aptamers, having a desired property from a mixture of candidate nucleic acid agents. The method generally includes immobilizing the mixture of candidate nucleic acid agents onto particles, wherein only a subset of the candidate nucleic acid agents are immobilized on any one of the particles, and wherein the subset is present in multiple copies. The particles are exposed to a target, and particles including candidate nucleic acid agents having the desired property are isolated. In this way, one or more nucleic acid agents having the desired property may be identified. Related compositions and nucleic acid agents identified as having one or more desired properties are also provided.

First claim

1 . A method for identifying one or more nucleic acid agents having one or more desired properties from a mixture of candidate nucleic acid agents, wherein the mixture of candidate nucleic acid agents comprises a plurality of single stranded nucleic acids, the method comprising:
immobilizing the mixture of candidate nucleic acid agents onto a
plurality of particles, wherein only a subset of the candidate nucleic acid agents are immobilized on any one of the plurality of particles, and wherein the subset is present in multiple copies;
exposing the plurality of particles to a target,
isolating a particle or particles from the plurality of particles, wherein the isolated particle or particles comprise one or more candidate nucleic acid agents having the one or more desired properties; and
identifying one or more nucleic acid agents having the one or more desired properties from the isolated particles.

2 . The screening method of claim 1 , wherein the target is a protein target or a small molecule target.

3 .- 6 . (canceled)

7 . The screening method of claim 1 , wherein the single stranded nucleic acids are DNA.

8 . (canceled)

9 . The screening method of claim 1 , wherein one or more of the single stranded nucleic acids comprises a molecule conjugated thereto, wherein the molecule conjugated thereto is a small molecule, a fluorophore, a peptide, or an siRNA.

10 .- 13 . (canceled)

14 . The screening method of claim 1 , wherein the one or more desired properties is a target binding activity or a target-binding induced activity.

15 . The screening method of claim 14 , wherein the target binding activity is affinity, specificity or bi-specificity.

16 . The screening method of claim 15 , wherein the target binding activity is specificity, the screening method comprises exposing the plurality of particles to a first target and a second target, and wherein the nucleic acid agents having the one or more desired properties exhibit a specific binding affinity for either the first target or the second target but not both.

17 .- 20 . (canceled)

21 . The screening method of claim 15 , wherein the target binding activity is bi-specificity, the screening method comprises exposing the plurality of particles to a first target and a second target, and wherein the nucleic acid agents having the one or more desired properties exhibit a specific binding affinity for both the first and second target.

22 .- 46 . (canceled)

47 . The screening method of claim 1 , wherein the plurality of particles comprises from about 1×10 2 to about 1×10 14 particles.

48 . The screening method of claim 1 , wherein the sequence diversity of the mixture is from about 1×10 2 to about 1×10 14 .

49 . The screening method of claim 1 , wherein each of the particles of the plurality of particles comprises from about 1×10 2 to about 1×10 10 candidate nucleic acid agents bound thereto.

50 . The screening method of claim 1 , wherein the sequence diversity of the subset of the candidate nucleic acid agents immobilized on any one of the plurality of particles is from 1 to 10 6 .

51 .- 52 . (canceled)

53 . The screening method of claim 50 , wherein the sequence diversity of the subset of the candidate nucleic acid agents immobilized on any one of the plurality of particles is 1.

54 .- 58 . (canceled)

59 . The screening method of claim 1 , wherein the particle or particles that comprise one or more candidate nucleic acid agents having the one or more desired properties are modified as a result of the one or more desired properties following exposure to the target, which modification allows the particle or particles that comprise the one or more candidate nucleic acid agents having the one or more desired properties to be isolated, wherein the modification results in a physically detectable change, a chemically detectable change, or an optically detectable change.

60 .- 66 . (canceled)

67 . The screening method of claim 1 , wherein the isolating step comprises sorting the plurality of particles using fluorescence activated cell sorting (FACS).

68 .- 71 . (canceled)

72 . The screening method of claim 1 further comprising introducing one or more mutations into the one or more nucleic acid agents having the one or more desired properties.

73 . The screening method of claim 1 , further comprising a step of amplifying the sequences of the one or more nucleic acid agents identified as having the one or more desired properties, and iteratively repeating one or more of the immobilizing, exposing, isolating identifying, and amplifying steps.

74 .- 87 . (canceled)

88 . The aptamer screening method of claim 168 , wherein the method comprises an enriching step, wherein the enriching step comprises preparing an enriched aptamer particle pool from the isolated aptamer particles by amplifying the aptamer sequences of the isolated aptamer particles, wherein the enriched aptamer particle pool comprises aptamer particles, wherein each of the aptamer particles of the enriched aptamer particle pool comprises multiple copies of a single candidate aptamer sequence bound thereto, and wherein the enriched aptamer particle pool has decreased sequence diversity relative to the plurality of aptamer particles from the exposing step.

89 . The aptamer screening method of claim 88 , wherein the exposing, isolating, and enriching steps constitute a first round of screening, and the method comprises one or more additional rounds of screening, wherein the plurality of aptamer particles for each additional round of screening is the enriched aptamer particle pool from the previous round.

90 . The aptamer screening method of claim 89 , wherein following the one or more rounds of screening the identified aptamer sequences are capable of binding the target with a K d of from about 1 pM to about 100 μM.

91 .- 130 . (canceled)

131 . A pool of aptamer particles, the pool comprising aptamer particles comprising DNA aptamer sequences, the pool of aptamer particles comprising:
from about 1×10 2 to about 1×10 14 aptamer particles, wherein each aptamer particle of the pool of aptamer particles comprises from about 1×10 2 to about 1×10 10 copies of one or more DNA aptamer sequences bound thereto, wherein each of the aptamer particles has at least one dimension of from about 50 nm to about 100 μm, and wherein a plurality of the aptamer particles comprise aptamer sequences bound thereto which are capable of binding the target with a K d of from about 1 pM to about 100 μM.

132 .- 167 . (canceled)

168 . An aptamer screening method, the method comprising:
exposing a plurality of aptamer particles to one or more fluorescently labeled targets, wherein each of the aptamer particles comprises two or more copies of one or more candidate DNA aptamer sequences bound thereto; wherein one or more of the one or more fluorescently labeled targets associate to a higher level with particles including one or more candidate DNA aptamer sequences having a first desired property and associate to a lower level with particles which do not include one or more candidate DNA aptamer sequences having the first desired property, or wherein one or more of the one or more fluorescently labeled targets associate

Assignees

• Univ California

Inventors

• Wang Jinpeng
• Soh Hyongsok
• Gong Qiang

Classifications

• C12N2320/11

  for the determination of target sites, i.e. of active nucleic acids · CPC title

• C12N15/1037

  Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display · CPC title

• C12N15/1048 Primary

  SELEX · CPC title

• C12N2320/13

  in a process of directed evolution, e.g. SELEX, acquiring a new function · CPC title

• C12N15/115

  Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith {; Nucleic acids binding to non-nucleic acids, e.g. aptamers} · CPC title