Evolution of aptamers with a new specificity and new secondary structures from an ATP aptamer

Abstract

Small changes in target specificity can sometimes be achieved, without changing aptamer structure, through mutation of a few bases. Larger changes in target geometry or chemistry may require more radical changes in an aptamer. In the latter case, it is unknown whether structural and functional solutions can still be found in the region of sequence space close to the original aptamer. To investigate these questions, we designed an in vitro selection experiment aimed at evolving specificity of an ATP aptamer. The ATP aptamer makes contacts with both the nucleobase and the sugar. We used an affinity matrix in which GTP was immobilized through the sugar, thus requiring extensive changes in or loss of sugar contact, as well as changes in recognition of the nucleobase. After just five rounds of selection, the pool was dominated by new aptamers falling into three major classes, each with secondary structures distinct from that of the ATP aptamer. The average sequence identity between the original aptamer and new aptamers is 76%. Most of the mutations appear to play roles either in disrupting the original secondary structure or in forming the new secondary structure or the new recognition loops. Our results show that there are novel structures that recognize a significantly different ligand in the region of sequence space close to the ATP aptamer. These examples of the emergence of novel functions and structures from an RNA molecule with a defined specificity and fold provide a new perspective on the evolutionary flexibility and adaptability of RNA.

Comprehensive experimental fitness landscape and evolutionary network for small RNA

“…Indeed, the RNA landscape itself is likely to be highly dependent on conditions such as ionic composition (e.g., the concentration of Mg 2+ and other divalent cations), presence of cofactors, temperature, and water activity, and therefore may be quite dynamic in response to changing conditions (53,54). Fitness peaks for one functional activity can be near in sequence space to peaks for another activity, potentially leading to adoption of new function (55)(56)(57)(58), suggesting that changing selection conditions could have an important role. Further studies are required to evaluate the generality of these findings, their robustness to environmental conditions, and possible mechanisms for optimization within the fitness landscape.…”

Gene Knockdown by EpCAM Aptamer–siRNA Chimeras Suppresses Epithelial Breast Cancers and Their Tumor-Initiating Cells

“…Spleens, harvested at sacrifice 16 hours after treatment, were stored in RNAlater (Qiagen) before extracting RNA by adding TRIzol (Invitrogen) to single-cell suspensions generated using the gentleMACS Dissociator (MACS Miltenyi Biotec). cDNA was synthesized using Superscript III and random hexamers (Invitrogen) and PCR was performed using SsoFast EvaGreen Supermix and a Bio-Rad CFX96 Real-Time PCR System (Bio-Rad Laboratories) using the following primers: (42,43). A handful of EpCAM-AsiCs that linked either the sense or antisense strand of the siRNA to the 3 0 -end of the aptamer by several linkers were designed and synthesized with 2 0 -fluoropyrimidine substitutions and 3 0 -dTdT overhangs to enhance in vivo stability, avoid off-target knockdown of partially complementary genes, and limit innate immune receptor stimulation.…”

In Vitro Selection of Functional Oligonucleotides and the Origins of Biochemical Activity

“…Structures with completely new secondary (and presumably) tertiary configurations were obtained upon evolving the specificity of three aptamers from flavin-adenine diphosphate (FAD) to guanosine monophosphate (GMP), even though the closest overlapping solutions differ by only three sequence mutations (Held et al, 2003). Similarly, selection for GTP binders from a pool of mutagenized ATP aptamers produced molecules with highly diverged sequences and secondary structures (Huang and Szostak, 2003).…”