Methods and systems for identifying candidate nucleic acid agent
Inventors
WANG, Jinpeng • Ferguson, Brian • Gong, Qiang
Assignees
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Publication Number
US-11041849-B2
Publication Date
2021-06-22
Expiration Date
Abstract
The present disclosure provides methods, kits and compositions for identifying nucleic acid agents having a desired property, e.g., a property of specifically binding to a target (such as a protein target) with high affinity. More specifically, the present disclosure provides methods, kits and compositions for identifying candidate nucleic acid agents with both high specificity and affinity for a target.
Core Innovation
The invention provides a method for identifying one or more nucleic acid agents having a desired property from a mixture of candidate nucleic acid agents, where the desired property includes specific binding to a target with high affinity. The mixture comprises a plurality of aptamers, and the method uses a particle-based approach in which candidate nucleic acid agents are immobilized on particles, with each particle containing at most a subset of the candidate nucleic acid agents.
Particles are exposed to a screening composition comprising a target moiety and a reference moiety. An interaction with the target moiety is indicated by a first signal, an interaction with the reference moiety is indicated by a second signal, and the intensities of the first signal and the second signal provide a sorting parameter for a particular particle. The concentration of the target moiety and the concentration of the reference moiety are set to enable the sorting parameter of about 0.05% to about 1% of the particles to be within a predetermined sorting range.
Selected particles having a sorting parameter within the predetermined sorting range are isolated, and the one or more nucleic acid agents having the desired property are identified from the one or more selected particles. In dependent aspects, intensity-based threshold rules define the predetermined sorting range using first-signal and second-signal thresholds, prescreening defines the thresholds using first prescreening and second prescreening compositions, and iterative rounds generate an enriched mixture for repeated screening.
Claims Coverage
The claims cover a particle-based identification method for nucleic acid agents using dual target and reference signals and a sorting parameter defined by those signal intensities. The inventive features include 10 merged features across the independent and dependent claims.
Particle-immobilized candidate aptamers in subsets
providing a plurality of particles with the candidate nucleic acid agents immobilized thereon, wherein each of the plurality of particles comprises at most a subset of the candidate nucleic acid agents within said mixture
Target and reference moiety dual-signal sorting parameter
exposing the plurality of particles to a screening composition comprising a target moiety and a reference moiety, wherein an interaction of said candidate nucleic acid agents with the target moiety is indicated by a first signal, an interaction of said candidate nucleic acid agents with the reference moiety is indicated by a second signal, and an intensity of said first signal together with an intensity of said second signal for a particular particle provide a sorting parameter of the particular particle
Low-population sorting range via set target/reference concentrations
wherein a concentration of the target moiety and a concentration of the reference moiety are respectively set at a value enabling the sorting parameter of about 0.05% to about 1% of the plurality of particles to be within a predetermined sorting range
Isolating selected particles and identifying nucleic acid agents
isolating from said plurality of particles one or more selected particles having a sorting parameter within said predetermined sorting range, wherein the one or more selected particles comprises said one or more nucleic acid agents having the desired property; and identifying the one or more nucleic acid agents having the desired property from the one or more selected particles
First and second signal intensity threshold selection
wherein the predetermined sorting range comprises selecting particles based on the first signal and the second signal being respectively within first and second intensity thresholds
Prescreening-defined first threshold from target-only composition
determining a first threshold using a first prescreening composition that comprises the target moiety and does not comprise the reference moiety, and wherein the first threshold is based on a maximum mean intensity of the first signal
Prescreening-defined second threshold proportional to reference-only maximum
setting a second threshold based on a maximum mean intensity of the second signal obtained from a second prescreening composition that comprises the reference moiety
Reference moiety as a protein or polypeptide
wherein the reference moiety is a protein or a polypeptide moiety
Named protein target moiety embodiments
wherein the target moiety comprises a protein selected from a group including Tumor Necrosis Factor α and Integrins
Iterative rounds using enriched mixture
forming a screening round by performing operations a), b), c), and generating an enriched mixture from selected particles, and repeating for two or more said rounds of screening using the enriched mixture in a next round
Overall, the claims cover selecting high-affinity specific-binding aptamers by immobilizing candidate aptamers on particles, exposing the particles to target/reference dual-moiety screening to produce first and second signals, and sorting via a sorting parameter that captures both signals within a predetermined low-percentage sorting range, with dependent claims refining threshold determination and iterative enrichment across multiple screening rounds.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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