Method for Obtaining Aptamers
The invention relates to a new method for obtaining aptamers directed against protein targets comprising a histidine-containing surface domain, and aptamers obtaining by said method.
1 .- 15 . (canceled)
16 . A method for obtaining an aptamer against a protein target comprising a histidine-containing surface domain, said method comprising:
a) contacting the protein target with a candidate mixture of nucleic acids at a pH promoting the formation of positive charge on the histidine-containing surface domain of said protein target;
b) recovering nucleic acids that bind to the protein target, while removing unbound nucleic acids, wherein step b) comprises the steps of:
(i) separating the complex formed in step (a) from unbound nucleic acids, and
(ii) releasing the nucleic acids from the complex by dissociation, wherein the dissociation of the complex between the bound nucleic acids and the protein target is performed by increasing the pH at a value higher than the pH used in step a);
c) amplifying the nucleic acids obtained in step (b) to yield a candidate mixture of nucleic acids with increased affinity to the protein target; and
d) repeating steps (a), (b), (c) until obtaining one or several aptamers against the protein target of interest.
17 . The method of claim 16 , wherein in step b), sub-step (ii), dissociation of the complex between the bound nucleic acids and the protein target is performed by increasing the pH of a ΔpH of at least 0.8
18 . The method of claim 16 wherein the pH in step a) is less than 7.0.
19 . The method of claim 16 , wherein the pH in step a) is from 5.0 to 6.9.
20 . The method of claim 16 , wherein the pH in step a) is selected so that the electrostatic surface potential of at least one of the histidine-containing surface domain of the protein target is positive.
21 . The method of claim 16 , wherein:
the protein target has an isoelectric point (pI) of less than 7.5, and/or
the protein target is devoid of any surface domain with positive electrostatic potential at a pH of more than 7.0.
22 . The method of claim 16 , wherein the protein target is selected from the group consisting of fibrinogen, immunoglobulin, Fc fragment, and variants thereof.
23 . The method of claim 16 , wherein the pH of step (a) is determined by obtaining surface electrostatic potential maps at different pH and selecting a pH that yields a positive surface potential on at least one histidine-containing surface domain of the protein target and which is included in the pH stability range of the protein target.
24 . The method of claim 16 , which further comprises the step of
(i) sequencing an aptamer obtained in step (c);
(ii) optionally optimizing the sequence of said aptamer; and
(iii) producing the aptamer.
25 . An aptamer obtainable or obtained by the method as defined in claim 16 .
26 . The aptamer of claim 25 , wherein the aptamer binds to a protein target comprising a histidine-containing surface domain in a pH dependent-manner.
27 . The aptamer of claim 26 , wherein the aptamer binds to the protein target at an acidic pH but which does not bind to the protein target at a pH of more than 7.0.
28 . The aptamer of claim 26 , wherein the aptamer binds to the protein target at an acidic pH selected from 5.0 to 6.5, but does not bind to the protein target at a pH of more than 7.0.
29 . An affinity ligand comprising an aptamer moiety as defined in claim 25 and at least one moiety for immobilization onto a support.
30 . An affinity support comprising thereon a plurality of aptamers as defined in claim 25 .
31 . A method for obtaining an aptamer against a protein target, said method comprising:
(i) determining a pH value promoting positive charges in at least one surface domain of the protein target,
(a) contacting the protein target with a candidate mixture of nucleic acids at the pH determined in step (i) in conditions favorable for binding of the protein target with nucleic acids having affinity for said targets,
(b) recovering nucleic acids which bind to the protein target, while removing unbound nucleic acids,
(c) amplifying the nucleic acids obtained in step (b) to yield to a candidate mixture of nucleic acids with increased affinity to the protein target, and
(d) repeating steps (a), (b), (c) until obtaining one or several aptamers against the protein target of interest.
32 . The method of claim 31 , further comprising the steps of:
determining the presence of a histidine-containing surface domain in the protein target, and
if said domain is present, determining a pH value promoting positive charges in said histidine-containing surface domain.
33 . The method of claim 32 , wherein the pH value promoting positive charges is a pH value resulting in a local positive surface electrostatic potential in said histidine-containing surface domain.