Engineering a ribozyme cleavage-induced split fluorescent aptamer complementation assay

Abstract

Hammerhead ribozymes are self-cleaving RNA molecules capable of regulating gene expression in living cells. Their cleavage performance is strongly influenced by intra-molecular loop–loop interactions, a feature not readily accessible through modern prediction algorithms. Ribozyme engineering and efficient implementation of ribozyme-based genetic switches requires detailed knowledge of individual self-cleavage performances. By rational design, we devised fluorescent aptamer-ribozyme RNA architectures that allow for the real-time measurement of ribozyme self-cleavage activity in vitro. The engineered nucleic acid molecules implement a split Spinach aptamer sequence that is made accessible for strand displacement upon ribozyme self-cleavage, thereby complementing the fluorescent Spinach aptamer. This fully RNA-based ribozyme performance assay correlates ribozyme cleavage activity with Spinach fluorescence to provide a rapid and straightforward technology for the validation of loop–loop interactions in hammerhead ribozymes.

Measurement of Kinetics of Hammerhead Ribozyme Cleavage Reactions using Toehold Mediated Strand Displacement

“…It is important that the experimenters understand that and hence, utilizes the method most appropriate to the particular needs of their own projects. (Singh et al 1999;Li et al 2005;Porecha and Herschlag 2013;Auslander et al 2016).…”

Synthetic Biology—The Synthesis of Biology

“…[165] Importantly,a ll the components can be freeze-dried and applied on paper disks for easy handling and storage at ambient temperature. [168] Similar to cell-based gene circuits,ligand-responsive gene switches are important building blocks for the design of programmable circuits by providing control over transcription output. Recently, RNA-based fluorogenic aptamers have been developed and used as an alternative output module in cell-free systems.…”

“…Based on this aptamer,several biosensors have been designed that are capable of measuring c-di-GMP, [166] RNAs equences, [167] and ribozyme cleavage performances. [168] Similar to cell-based gene circuits,ligand-responsive gene switches are important building blocks for the design of programmable circuits by providing control over transcription output. [16d, 169] Fore xample,acell-based oscillatory gene circuit, the repressilator, [170] was reconstructed to run in acellfree system.…”

RNA Fluorescence with Light-Up Aptamers

“…Extra sequences are added to the hammerhead ribozyme to bind one half of the sequence of Spinach. Once the ribozyme self-cleaves, the half-Spinach sequence is released and binds its cognate Spinach second half to reconstitute a bi-molecular Spinach aptamer to induce fluorescence activation upon DFHBI binding (Ausländer et al, 2016 ). The hammerhead ribozyme cleavage activity can therefore be monitored in real-time in a fluorimeter, rather than quenching radioactive aliquots that are resolved on a gel and then imaged with expensive instrumentation.…”