Automated selection of aptamers against protein targets translated in vitro: from gene to aptamer.
Reagents for proteome research must of necessity be generated by high throughput methods. Aptamers are potentially useful as reagents to identify and quantitate individual proteins, yet are currently produced for the most part by manual selection procedures. We have developed automated selection methods, but must still individually purify protein targets. Therefore, we have attempted to select aptamers against protein targets generated by in vitro transcription and translation of individual genes. In order to specifically immobilize the protein targets for selection, they are also biotinylated in vitro. As a proof of this method, we have selected aptamers against translated human U1A, a component of the nuclear spliceosome. Selected sequences demonstrated exquisite mimicry of natural binding sequences and structures. These results not only reveal a potential path to the high throughput generation of aptamers, but also yield insights into the incredible specificity of the U1A protein for its natural RNA ligands.
Cox
J Colin
JC
Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.
Hayhurst
Andrew
A
Hesselberth
Jay
J
Bayer
Travis S
TS
Georgiou
George
G
Ellington
Andrew D
AD
D001331:Automation; D001483:Base Sequence; D019921:Biotinylation; D002460:Cell Line; D005821:Genetic Techniques; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000138:chemical synthesis* / Q000378:metabolism*; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D009841:Oligonucleotides; D011506:Proteins; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C075339:U1A protein