Stability of split-aptamers
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Abstract
Oligonucleotide aptamers are widely used in fundamental research, diagnostics and technical applications, because of their property to bind analytes with high affinity and specificity. Analyte-binding can cause a change of the flanking nucleotides and thus of the aptamer structure. By combining two binding-sites with a so called communication module, the structural change of one binding-site can be transduced to the other binding-site. However, for such a construct, at least one aptamer has to be split into two strands. The aim is, to develop split-aptamers, which regained their analyte-binding property upon hybridization of the two strands. A significant stabilization of the aptamer upon analyte-binding is desired, which can be determined by monitoring the dissociation of the oligonucleotide-strands.
A force-induced dissociation of a split-aptamer, which binds streptomycin, was investigated by means of single-molecule force spectroscopy measurements. Rate dependent measurements revealed a decrease in the off-rate for the aptamer-streptomycin complex (koff-COMPLEX = 0.22 ± 0.16 s-1) compared to the aptamer, having an empty binding pocket (koff-APTAMER = 0.49 ± 0.11 s-1). In the context of the Bell-Evans potential, a decrease in the Gibbs free energy of ≈ 3.4 kJ mol-1 emerged. The results led to the conclusion that the hydrogen-bonds between both RNA strands mainly contribute to the stability of the aptamer system studied.
In a different approach, the dissociation of oligonucleotides was investigated by means of temperature dependent UV/Vis-absorbance measurements. Here, an increase in the melting temperature upon analyte-binding indicated the functionality of the binding-site. A suitable splitting position was identified for the neomycin-aptamer between the nucleotides A14 and G15. The split neomycin-aptamer showed the same shift in the melting temperature as the original neomycin-aptamer. Thus, the functionality of the binding-site was verified. The binding-sites were connected with a communication module of three nucleotides length. The truncation of the communication module to a length of one nucleotide revealed a decreased functionality of the Hg2 -binding DNA. The simultaneous presence of neomycin molecules compensated the destabilization and the affinity of Hg2 -ions to the binding-site was restored. Thus, an oligonucleotide with a positive allostery was developed, which exhibits a ≈ 13 °C larger melting temperature upon binding of both analytes, compared to the unbound aptamer. This system might be the basis for molecular switches.