Imaging Tetrahymena ribozyme splicing activity in single live mammalian cells

Abstract

Tetrahymena ribozymes hold promise for repairing genetic disorders but are largely limited by their modest splicing efficiency and low production of final therapeutic proteins. Ribozyme evolution in intact living mammalian cells would greatly facilitate the discovery of new ribozyme variants with high in vivo activity, but no such strategies have been reported. Here we present a study using a new reporter enzyme, beta-lactamase, to report splicing activity in single living cells and perform high-throughput screening with flow cytometry. The reporter ribozyme constructs consist of the self-splicing Tetrahymena thermophila group I intron ribozyme that is inserted into the ORF of the mRNA of beta-lactamase. The splicing activity in single living cells can be readily detected quantitatively and visualized. Individual cells have shown considerable heterogeneity in ribozyme activity. Screening of Tetrahymena ribozymes with insertions in the middle of the L1 loop led to identification of better variants with at least 4-fold more final in vivo activity than the native sequence. Our work has provided a new reporter system that allows high-throughput screening with flow cytometry of single living mammalian cells for a direct and facile in vivo selection of desired ribozyme variants.

Fig. 1.

Splicing-dependent Bla reporter gene in vitro and in vivo assays. (A) Schematic presentation of splicing-dependent Bla reporter gene strategy. The ribozyme reporter Rz156 consists of the Tetrahymena intron and a broken Bla ORF (BlaI and BlaII). Ribozyme self-splicing produces uninterrupted mRNA of Bla, which is translated into the reporter enzyme Bla. (B) Hydrolysis of nonfluorescent CC1 by Bla generates a blue fluorescent product that emits at 465 nm when excited at 360 nm. (C) The in vivo assay uses CCF2/AM (adapted from ref. 10). Membrane-permeable CCF2/AM is converted to CCF2 by intracellular esterases. When no Bla is present, CCF2 fluoresces green (at 520 nm) because of fluorescence resonance energy transfer (FRET) from the coumarin donor to the fluorescein acceptor. Bla hydrolysis splits off the fluorescein, disrupts FRET, and shifts the emission to blue (at 447 nm).

Fig. 2.

In vitro and in vivo assays of Rz156. (A) RT-PCR analyses of RNA extracts from COS-1 cells transiently transfected with CMV, CMV-bla, and Rz156. The expected splicing product Bla was as indicated. The top bright band was unspliced Rz156, and the faint middle band might be mRNA spliced at the cryptic splice site (19). Samples in Upper contained reverse transcriptase, and, as a control, samples in Lower did not. Shown in the first lane of each panel are molecular mass markers. (B) In vitro Bla assay using CC1. Shown is the hydrolysis rate, determined from lysates of COS-1 cells transiently transfected with CMV, CMV-bla, or Rz156 in three independent transfections and normalized against total lysates protein content in a fluorescence units (FU) per s per μg of proteins. Note that hydrolysis rates are plotted in a log scale. (C–E) Fluorescence microscopy images of the COS-1 cells transfected with an empty vector (CMV) (C) or transiently transfected with CMV-bla (D) or with Rz156 (E). (C–E Left) An overlay of frames captured at 530 nm (green emission) and 460 nm (blue emission). (C–E Right) DsRed emission at 605 nm with excitation at 560 nm. Nontransfected cells show no Bla activity (green in Left and black in Right).

Fig. 3.

Ribozyme variants with insertion of nucleotides at the indicated site of the L1 loop of the Tetrahymena ribozyme. Some have spontaneous deletions or mutations around the insertion site. The nucleotides displayed for each variant replace those in Rz156 starting from the indicated insertion site to the beginning of IGS (shown in italics). For RzL + 46 and RzL + 84, the third nucleotide after the splice site is G instead of A, shown in parentheses.

Fig. 4.

In vitro and in vivo assays of ribozyme variants with insertion at the L1 loop. (A) RT-PCR analyses of RNA extracts from COS-1 cells transiently transfected with RzL + 4, RzL + 23, RzL + 24, RzL + 44, RzL + 46, or RzL + 84. Reverse transcriptase was present in samples in Upper but not in Lower. The expected splicing product Bla was as indicated. (B) In vitro Bla assay using CC1 of lysates of COS-1 cells transiently transfected with CMV, Rz156, RzL + 4, RzL + 23, RzL + 24, RzL + 44, RzL + 46, or RzL + 84. Shown is the hydrolysis rate, determined from three independent transfections and normalized against total lysate protein contents in fluorescence units (FU) per s per μg of proteins. (C) The apparent dependence of the Bla activity of mutants on the number of inserted nucleotides. The relative Bla activity of each mutant is the ratio of its hydrolysis rate to that of Rz156. (D–F) Fluorescence microscopy of Cos-1 cells transiently transfected with ribozyme variant RzL + 4 (D), RzL + 23 (E), or RzL + 24 (F). (D–F Left) An overlay of frames captured at 530 nm and 460 nm with excitation at 405 nm after a 1-h loading of CCF2/AM. (D–F Right) Red emission at 605 nm of DsRed.

Fig. 5.

Fluorescence-activated cell sorter analysis of COS-7 cells transiently transfected with construct CMV-bla (positive control), RzL + 4, RzL + 24, RzL + 23, Rz156, RzL + 44, RzL + 46, RzL + 84, or a ribozyme dead mutant (RzDead, a negative control). DsRed cDNA was cotransfected as a transfection marker. Cells were incubated with CCF2/AM for 1 h before fluorescence-activated cell sorter analysis. The populations depicted consist of cells that were healthy (as judged by forward and side scatter) and DsRed-positive. For easy comparison of different constructs, the percentages of Bla-positive (dark area) and Bla-negative (light gray area) cells (defined as blue/green ratios >1 and <1, respectively) are indicated for each construct.