Deconvolution of a complex target using DNA aptamers.
In vitro selection of single-stranded nucleic acid aptamers from large random sequence libraries is now a straightforward process particularly when screening with a single target molecule. These libraries contain considerable shape diversity as evident by the successful isolation of aptamers that bind with high affinity and specificity to chemically diverse targets. We propose that aptamer libraries contain sufficient shape diversity to allow deconvolution of a complex mixture of targets. Using unfractionated human plasma as our experimental model, we aim to develop methods to obtain aptamers against as many proteins as possible. To begin, it is critical that we understand how aptamer populations change with increasing rounds of in vitro selection when using complex mixtures. Our results show that sequence representation in the selected population changes dramatically with increasing rounds of selection. Certain aptamer families were apparent after only three selection rounds. Two additional cycles saw a decline in the relative abundance of these families and the emergence of yet another family that accounted for more than 60% of sequences in the pool. To overcome this population convergence, an aptamer-based target depletion method was developed, and the library screen was repeated. The previous dominant family effectively disappeared from the selected populations but was replaced by other aptamer families. Insights gained from these initial experiments are now being applied in the creation of second generation plasma protein screens and also to the analysis of other complex biological targets.
Fitter
Stephen
S
Department of Gastroenterology and Hepatology, Flinders Medical Centre, Adelaide, South Australia 5042.
James
Robert
R
D052157:Aptamers, Nucleotide / Q000235:genetics*; D001483:Base Sequence; D001798:Blood Proteins; D015723:Gene Library; D014644:Genetic Variation; D006801:Humans; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D010949:Plasma; D052156:SELEX Aptamer Technique; D012641:Selection, Genetic
D052157:Aptamers, Nucleotide; D001798:Blood Proteins