In Vitro Aptamer Selection and Evolution using an Engineered Dual-Ribozyme Complex

Abstract

Small-molecule aptamers are composed of RNA or DNA sequences with intricate secondary and tertiary structure allowing them to perform many functions, including ligand binding and catalytic events. Aptamers are selected from a library of random nucleic acids against a specific target. The binding sequences are amplified and fed back into the process for subsequent rounds of selection and amplification. This is a well-described technology called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). We propose a novel SELEX method to characterize and screen new RNA aptamers using existing aptamers and randomization of a stem-loop sequence in the canonical type-I hammerhead ribozyme. As opposed to other reported techniques, we hypothesize that ours will allow for rapid and efficient selection of small-molecule ligands in solution and extended to an easy model for expression of these aptamers in vivo. This method reduces the number of selection rounds required, potentially selecting viable aptamers within one round. Our method utilizes a binary aptamer binding event in which a randomized sequence is placed in a stem of the hammerhead ribozyme. When the random sequence binds a ligand, it initiates cleavage of the ribozyme and a second “selecting” aptamer folds. The selecting aptamer binds to beads in a column, and the desired aptamers can be eluted after subsequent washes to remove non- and low-specificity binders. Further rounds could increase stringency of selection by means of decreased ligand concentration or increased competing ligands. Advantages of this system are manifested in time saved and resources conserved. Additionally, since our ligands are not immobilized on beads nor is any substrate required for capturing the sequences once selected, more available moieties for binding will be made available and new aptamers more readily discovered.