Single stranded oligonucleotide ligands, known as aptamers, can be raised to theoretically any target and have rapid development times of up to 12 weeks. These advantages over the standard antibody affinity reagent have meant that aptamers are well placed to serve as antibody alternatives to the research, diagnostic and therapeutic markets.
Aptamers are generated by SELEX, which is an iterative in vitro process entailing three main steps: 1) binding, where the target molecule is incubated with a random library of oligonucleotide aptamers 2) separation of the target bound aptamers from unbound ones and 3) amplification, where the enriched pool of aptamers are amplified to be used in the next round of selection. Finally, the enriched aptamer pool is analysed by high-throughput sequencing and bioinformatics analysis to identify candidate aptamers. Further examination of these candidates is then necessary to determine they possess the required binding affinity, specificity and any desired properties such as target inhibition and stability.
Despite the multiple rounds of panning involved in SELEX aimed to increase the stringency in aptamer selection, all too often this process fails to deliver an aptamer with the required characteristics to be used as an affinity reagent.
Earlier this month a paper in Biotechnology and Bioengineering showed that researchers at the University of British Columbia have identified a number of issues that limit the performance of standard SELEX. These include limitations in the aptamer library being screened, the retention of non-specific aptamers during the screening rounds, the accumulation of amplification artefacts that reduce yields and impede the enrichment of a high-affinity aptamer pool and the unintended isolation of candidate aptamers lacking sufficient specificity for the target due, in part, to the use of ion-exchange binding mechanisms that do not reflect therapeutic activity. Together these limitations may ultimately prevent the development of aptamers to therapeutically relevant targets.
With these issu
es in mind the researchers developed a new platform, termed high-fidelity SELEX or Hi-Fi SELEX, that they hope will accelerate and improve theselection of DNA aptamers by overcoming some of the limitations of the current SELEX methodology. This platform is designed to enhance the diversity of the initial aptamer library and reduce the number of selection rounds required to identify therapeutically relevant aptamers. To achieve this blocking elements in the form of complimentary oligonucleotides were bound to the constant regions of the aptamer to prevent the aptamer folding upon itself, thus preserving the functional diversity of the library. The chemistry of the target-display surface and composition of the equilibration solvent were also engineered to inhibit the non-specific retention of aptamers improving partition efficiencies. This technology was showcased by selecting aptamers to ?-thrombin and human factors IXa, X and D, with the identified aptamers offering nM order dissociation constants (Kd) achieved within three selection rounds.
Though these improvements in SELEX seem to offer the long-promised realisation of the potential to deliver specific aptamers to any molecular target, the difficulties of the incompatibility of hydrophobic, negatively charged oligonucleotides to bind hydrophobic proteins and the challenge this presents to identifying high-affinity binders remain, in addition to the narrowly confined chemistry of oligonucleotides compared to proteins, which ultimately restricts the potential interactions between oligonucleotide aptamers and protein targets.
Affimer technology overcomes these difficulties because the Affimer peptide structure presents none of the chemical limitations associated with aptamers, allowing high-affinity, specific Affimer binders to truly be developed to any target. Additionally, the Affimer custom service can be delivered in just seven weeks preventing any stall in your research and allowing you to get on with producing publication quality data. While the need for antibody alternatives is increasing the problems inherent in aptamers makes Affimer technology the optimal affinity reagent for researchers.