A method to select chemically modified aptamers directly.
In vitro selection is a strategy to identify high-affinity ligands of a predetermined target among a large pool of randomized oligonucleotides. Most in vitro selections are performed with unmodified RNA or DNA sequences, leading to ligands of high affinity and specificity (aptamers) but of very short lifetime in the ex vivo and in vivo context. Only a very limited number of modified triphosphate nucleotides conferring nuclease resistance to the oligomer can be incorporated by polymerases. This encourages the development of alternative methods for the identification of nuclease-resistant aptamers. In this paper, we describe such a method. After selection of 2'O-methyl oligonucleotides against the TAR RNA structure of HIV-1, the complementary DNA sequences are fished out of a pool of randomized oligodeoxynucleotides by Watson-Crick hybridization. The DNA-fished sequences are amplified by PCR as double and single strands, the latter being used to fish back the chemically modified candidates from the initial library. This procedure allows an indirect amplification of the selected candidates. This enriched pool of modified sequences is then used for the next selection round against the target.
Boiziau
C
C
Laboratoire de Biophysique Moléculaire, INSERM U 386, Université Victor Segalen Bordeaux 2, France. claudine.boiziau@bordeaux.inserm.fr
Toulmé
J J
JJ
D001483:Base Sequence; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000737:chemistry*; D015195:Drug Design; D015723:Gene Library; D016325:HIV Long Terminal Repeat / Q000187:drug effects; D015497:HIV-1 / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000494:pharmacology; D020319:Oligodeoxyribonucleotides, Antisense / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D013698:Templates, Genetic
D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D020319:Oligodeoxyribonucleotides, Antisense