Patent DE-10349441-A1 (DE)
Title: Selecting nucleic acid that binds target molecules, useful potentially as therapeutic agents, by screening short sequences with two fixed regions flanking a random region
Inventor: JAROSCH FLORIAN
IPC: C07H21/00
Filed: 2003-10-23
Abstract: Selecting a nucleic acid (I), especially an aptamer, that binds a target molecule (II), is new. Selecting a nucleic acid (I), especially an aptamer, that binds a target molecule (II) comprises: (a) preparing a heterogeneous population of nucleic acids (NA, preferably D-NA), each of which comprises a randomized sequence (RS), different for each NA, and at least one constant sequence (CS); (b) contacting the population with (II); (c) separating NA that have not interacted with (II); (d) separating, from (II), the NA (NA1) that have interacted; (e) optionally repeating steps (a)-(d); (f) amplifying NA1; (g) optionally repeating steps (a)-(f); and (h) optionally sequencing NA1 isolated from steps (d) or (f). The new feature is that step (f) is performed in two alternative ways: (a) preparing a NA, preferably RNA, to be amplified, that comprises defined 5' and 3' partial sequences, flanking RS; (b) preparing a first adapter molecule (AM1), comprising two strands, the first (S1), preferably RNA, and the second (S2), preferably DNA, where S2 has a 5'-overhang that is at least partly complementary to (part of) the defined 5'-partial sequence; (c) preparing a second adapter molecule (AM2), comprising two strands, one (S11) having a 5'-phosphate group on the 5'-terminal nucleotide, while the other (S12), which is DNA, has a 3'-end at least partly complementary to (part of) the defined 3'-partial sequence, and also includes a cleavage site that generates a first claevage product (CP1), comprising the specified 3'-end of S12; (d) ligating AM1 and AM2 to the NA being amplified to generate a ligation product (LP); (e) reverse transcription of LP to form a product (RTP); (f) optionally performing second strand synthesis to generate a double-stranded product (RTP2); (g) optionally cleaving, in RTP2, the strand that is complementary to the NA being amplified, to form a transcription starting product (TSP); and (h) generating a transcription product from TSP. The second way comprises: (a) preparing the NA, i.e. an RNA, to be amplified, having at the 5'-end a primer sequence (PS1) and first defined partial sequence, at the 3'-end a second defined partial sequence and second primer binding site, and RS between the defined partial sequences; (b) reverse transcription to generate RTP, using S12 of AM2 as primer; (c) performing second strand synthesis, by using S1 of AM1 and S12 of AM2, to generate a double-stranded reaction product; and (d) performing a transcription reaction on the product to produce TP. Independent claims are also included for: (1) two related methods for selecting (I); (2) nucleic acid that comprises, in the 5' to 3' direction, first defined partial sequence, RS and second defined partial sequence, where the two defined sequences are at least partly complementary, particularly sufficiently so that a base-paired stem structure or helix is formed, preferably at 20-40, especially about 37, [deg]C; (3) nucleic acid comprising two defined partial sequences and RS, as in (2), and forming a base-paired stem structure or helix at 20-40, preferably about 37, [deg]C; and (4) population comprising at least two nucleic acid of (2) or (3), with at least two members differing in RS at at least one position.