Deconvolution of a Complex Target Using DNA Aptamers

Abstract

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.

Single-Step Selection of Bivalent Aptamers Validated by Comparison with SELEX Using High-Throughput Sequencing

“…In the single-step method most enrichment occurred in the last 4 cycles, but no peak was reached suggesting that further enrichment could be achieved by additional cycles. The initial increase of motifs 4 and 5 followed by a plateau is typical of SELEX,[25] and shows that enrichment was complete after three or four rounds. Linker 6 is different; it was first detected at low (0.01%) abundance in Round 4 and then increased more rapidly than any other motif in Round 5.…”

Gene therapy progress and prospects: RNA aptamers

“…In particular, the concept of 'complex-target' selection, where targets are not pure proteins but may include whole organisms, intact cells or human plasma, may lead to the identification of aptamers against proteins that can only be targeted in their physiological milieu. [65][66][67][68] In some biological systems, agonists may be of more therapeutic importance than antagonists and we envision that agonistic aptamers will soon be realized. Furthermore, the increasing focus on the use of aptamers as tools for diagnostics, target validation and drug discovery will maximize the potential of aptamers in therapeutics.…”

The Selection of DNA Aptamers for Two Different Epitopes of Thrombin Was Not Due to Different Partitioning Methods

“…This leaves unanswered the ultimate question of why there are 2 aptamers for thrombin. We suspect that the solution to this mystery is related to the small library used by Bock, which may have had a diversity of less than 10 12 , and the tendency of aptamers to converge on a single epitope after many rounds of selection (Fitter and James 2005), such as the 11 rounds applied by Tasset. We are currently investigating these factors using next generation sequencing and hope to show how they contributed to the discovery of the thrombin aptamers in the near future.…”

“…Electrophoresis of thrombin solutions stored at room temperature showed that the intensity of the 27-kDa band increased with time. b-thrombin does not have an intact exosite 1 (Fitter and James 2005), and therefore APT-15 should have a lower affinity for it. Blots interrogated with fluorescent APT-15 and APT-29 (Fig.…”