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.

Figure 1.

Design and validation of the split Spinach aptamer system. (A) Spinach fluorescent aptamer sequence. For the split system, the Spinach aptamer SpFL is divided at the loop structure into two parts resulting in SpA and SpB. (B) Working model of the split Spinach aptamer system. SpA and SpB assemble to a functional Spinach aptamer, which binds to the fluorophore DFHBI-1T and emits green fluorescence. (C) Fluorescence intensities of 500 nM full-length (blue) and 500 nM split (green) Spinach aptamers. SpA and SpB reconstitute a functional Spinach aptamer (green). (D) SpB concentration is kept constant at 2 μM, while SpA is added in different concentrations (0, 1.9, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2000 nM). Background fluorescence levels of buffer containing 2 μM SpB are shown in black. Data are mean ± S.D. of a triplicate experiment.

Figure 2.

Design and validation of the ribozyme performance assay. (A) Nucleotide sequence of the HHR-SpA scaffold. The conserved region is shown as a shaded box and consists of the catalytic core (bold) as well as stem III linked to the 5′-connected split aptamer part SpA (blue). A point mutation A14G (green bold) renders the ribozyme inactive. Variable stem loops can be connected to the HHR-SpA scaffold. (B) Working model for the ribozyme performance assay. Ribozyme cleavage releases SpA, which facilitates the strand-displacement reaction required to reconstitute functional fluorescent Spinach aptamer and results in high-level fluorescence. (C) Nucleotide sequence of the engineered sTRSV stem loops I and II. (D) Design of the stem III variants SpA_L/M/H of the HHR-SpA scaffold with their respective Gibbs free energy values ΔG (italics, calculated using the NUPACK web server (31) and the parameter set of Serra & Turner, 1995). (E–G) Fluorescence intensities of 500 nM active (red) or inactive (blue) sTRSV-SpA_L (E), sTRSV-SpA_M (F) and sTRSV-SpA_H (G) variants. Background fluorescence of buffer containing 500 nM SpB is shown in black. Data are mean ± S.D. of a triplicate experiment.

Figure 3.

Ribozymes with different self-cleavage activities. (A) Nucleotide sequence of the stem loops I/II of Env140, Env140-C3 and Env140-H1 ribozymes. (B) Fluorescence intensities of 500 nM active Env140_ac (red), active Env140-C3_ac (green), active Env140-H1_ac (yellow) or inactive Env140_inac (blue) variants with the HHR-SpA_H scaffold. (C) Nucleotide sequence of the stem loops I/II of sTRSV mutant 1.3, 1.5 and 1.6 ribozymes. (D) Fluorescence intensities of 500 nM sTRSV-1.5_ac (red), sTRSV-1.3_ac (green) or sTRSV-1.6_ac (yellow) variants with the HHR-SpA_H scaffold. Background fluorescence of buffer containing 500 nM SpB is shown in black. Data are mean ± S.D. of three independent experiments performed in duplicates.