An improved method for the in vitro evolution of aptamers and applications in protein detection and purification.
One of the key components of proteomics initiatives is the production of high affinity ligands or probes that specifically recognize protein targets in assays that detect and capture proteins of interest. Particularly versatile probes with tremendous potential for use as affinity molecules are aptamers. Aptamers are short single-stranded DNA or RNA sequences that are selected in vitro based on affinity for a target molecule. Aptamers offer advantages over traditional antibody-based affinity molecules in their ease of production, regeneration and stability, largely due to the chemical properties of nucleic acids versus amino acids. We describe an improved in vitro selection protocol that relies on magnetic separations for DNA aptamer production that is relatively easy and scalable without the need for expensive robotics. We demonstrate the ability of aptamers that recognize thyroid transcription factor 1 (TTF1) to bind their target protein with high affinity and specificity, and detail their uses in a number of assays. The TTF1 aptamers were characterized using surface plasmon resonance, and shown to be useful for enzyme-linked assays, western blots and affinity purification.
Murphy
Michael B
MB
DOE Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, CA 94598, USA.
Fuller
Shirin T
ST
Richardson
Paul M
PM
Doyle
Sharon A
SA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001483:Base Sequence; D001667:Binding, Competitive; D015153:Blotting, Western; D002938:Ciona intestinalis / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000276:immunology; D007700:Kinetics; D009687:Nuclear Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D009842:Oligopeptides / Q000276:immunology; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D020349:Surface Plasmon Resonance; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D000911:Antibodies, Monoclonal; D009687:Nuclear Proteins; D009841:Oligonucleotides; D009842:Oligopeptides; D011506:Proteins; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors; D006639:Histidine