Incorporation of Polymerizable Linkers into Aptamers for High-affinity Nanoscale Molecularly Imprinted Polymer Hybrids: Analysis of Positional Selectivity

Aptamers are short single strand nucleic acid sequences that exhibit high-affinity molecular recognition towards non nucleic acid targets. They offer many benefits over antibodies, but still suffer from variable affinities and stability issues. Recently, aptamers have been incorporated as functional recognition agents into molecularly imprinted polymers, a competing recognition technology, to create hybrid materials, AptaMIPs, that exhibit the benefits of both classes. Specifically, this process can increase target affinity while preventing aptamer degradation. For the first time, using a lysozyme aptamer as an exemplar, we have undertaken a systematic and fundamental study to identify the optimal number and location of polymer connection points on an aptameric sequence for boosting AptaMIP target affinity and selectivity creating high affinity recognition elements. Clear patterns have emerged showing "fixing" throughout the molecule is required, but only in particular regions of the sequence. The results suggest that conformationally flexible regions within the polymer-bound aptameric sequence are detrimental to strong target binding, supporting the hypothesis that a successful imprinting process must lock the aptamer into its ideal binding conformation to achieve observable marked improvement in recognition. Conversely, too much flexibility in the embedded oligo (demonstrated through limited binding points) leads to poor performance. These findings offer a clear direction for development of aptamer-polymer hybrids. We also demonstrate the effectiveness of the developed materials in sensitive detection of the template using surface plasmon resonance, through improved quality of the recognition element.