Methods and systems for identifying candidate nucleic acid agent
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.
1. A method for identifying one or more nucleic acid agents having a desired property from a mixture of candidate nucleic acid agents, said desired property is specific binding to a target with high affinity, wherein the mixture of candidate nucleic acid agents comprises a plurality of aptamers, the method comprising:
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;
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, 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, wherein the reference moiety is a protein or a polypeptide moiety;
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.
2. The method according to claim 1 , wherein said sorting range is determined with a first threshold and a second threshold, and the sorting parameter of a particular particle is within said sorting range when the intensity of the first signal of the particular particle is above said first threshold and the intensity of the second signal of the particular particle is below said second threshold.
3. The method according to claim 2 , wherein said first threshold is determined by a process comprising:
exposing the plurality of particles with the mixture of candidate nucleic acid agents immobilized thereon to a first prescreening composition comprising a saturating concentration of the target moiety, and
determining a maximum mean intensity of a signal indicating an interaction of said candidate nucleic acid agents with the target moiety in said first prescreening composition,
wherein said first prescreening composition does not comprise the reference moiety.
4. The method according to claim 3 , wherein said first threshold is set to be at least one half of said maximum mean intensity of the signal indicating an interaction of said candidate nucleic acid agents with the target moiety in said first prescreening composition.
5. The method according to claim 2 , wherein said second threshold is determined by a process comprising:
exposing the plurality of particles with the mixture of candidate nucleic acid agents immobilized thereon to a second prescreening composition comprising a saturating concentration of the reference moiety, and
determining a maximum mean intensity of a signal indicating an interaction of said candidate nucleic acid agents with the reference moiety in said second prescreening composition,
wherein said second prescreening composition does not comprise the target moiety.
6. The method according to claim 5 , wherein said second threshold is set to be at most one tenth of the maximum mean intensity of the signal indicating an interaction of said candidate nucleic acid agents with the reference moiety in said second prescreening composition.
7. The method according to claim 1 , wherein a ratio between the concentration of the target moiety and the concentration of the reference moiety in the screening composition is from about 1:10 9 to about 1:1.
8. The method according to claim 1 , wherein the target moiety is a protein or a polypeptide moiety.
9. The method according to claim 1 , wherein the reference moiety comprises serum proteins.
10. The method according to claim 1 , further comprising c2) generating an enriched mixture of candidate nucleic acid agents from the selected particle prior to the operation d).
11. The method according to claim 10 , wherein operations a), b), c), and c2) constitute one round of screening, and the method comprises two or more said rounds of screening, wherein the enriched mixture of candidate nucleic acid agents obtained from operation c2) of one round of screening is used as the mixture of candidate nucleic acid agents to be immobilized onto the plurality of particles in operation a) of the next round of screening.
12. The method according to claim 1 , wherein the target moiety comprises a protein or a part thereof selected from the group consisting of Tumor Necrosis Factor α, Neutrophil Gelatinase-Associated Lipocalin, Histidine-Rich Protein 2, Platelet-Derived Growth Factors, Vascular Endothelial Growth Factors, Angiopoietins, Complement proteins and Integrins.