An in vivo selection method to optimize trans-splicing ribozymes

Abstract

Group I intron ribozymes can repair mutated mRNAs by replacing the 3'-terminal portion of the mRNA with their own 3'-exon. This trans-splicing reaction has the potential to treat genetic disorders and to selectively kill cancer cells or virus-infected cells. However, these ribozymes have not yet been used in therapy, partially due to a low in vivo trans-splicing efficiency. Previous strategies to improve the trans-splicing efficiencies focused on designing and testing individual ribozyme constructs. Here we describe a method that selects the most efficient ribozymes from millions of ribozyme variants. This method uses an in vivo rescue assay where the mRNA of an inactivated antibiotic resistance gene is repaired by trans-splicing group I intron ribozymes. Bacterial cells that express efficient trans-splicing ribozymes are able to grow on medium containing the antibiotic chloramphenicol. We randomized a 5'-terminal sequence of the Tetrahymena thermophila group I intron and screened a library with 9 × 10⁶ ribozyme variants for the best trans-splicing activity. The resulting ribozymes showed increased trans-splicing efficiency and help the design of efficient trans-splicing ribozymes for different sequence contexts. This in vivo selection method can now be used to optimize any sequence in trans-splicing ribozymes.

FIGURE 1.

Secondary structure of the trans-splicing ribozyme construct used in this study, and its change during trans-splicing. The ribozyme is shown in gray, with the duplexes labeled. Note that the P4–P6 domain is shown to the right of the catalytic core for clarity. The mRNA is shown in red, with the splice site marked by an arrowhead. The EGS is shown in green. The ribozyme 3′-exon is shown in blue, with the 3′-splice site marked with an arrowhead. The sequence of the EGS used in this figure is that of clone R3C7, which was a result of the in vivo selection. Secondary structures formed by the EGS are labeled as 5′-duplex, internal loop, P1ex (P1 extension), and P1 helix. The reaction from substrate (left) to product (right) is indicated by arrows.

FIGURE 2.

Design of the pool of EGSs for the in vivo selection. Six subpools with six different registers (+6, +3, 0, −3, −6, −9) were generated to represent six different geometries of the internal loop with the mRNA (bottom). The positions of randomized nucleotides are denoted with N. Underlined characters show the position of duplexes between EGS and mRNA. All six subpools form identical P1 interactions at the IGS (GAAGGC) and at the P1 extension, which is constituted by the 3 bp adjacent to the mRNA splice site. The six subpools differ in the position of their 8-bp 5′-duplex on the mRNA, formed with nucleotides 3–10 of the EGS.

FIGURE 3.

Schematic for the work flow of the in vivo selection procedure. SOC_AMP plates refer to agar plates for bacterial growth, supplemented with the antibiotic ampicillin. Selection plates refer to agar plates for bacterial growth, supplemented with IPTG and the antibiotic chloramphenicol. More detailed descriptions are given in the text and in the Materials and Methods.

FIGURE 4.

Enrichment of subpools in the ribozyme library, during five rounds of in vivo selection. Fifty clones were sequenced from the initial pool (round 0), and 20 clones were sequenced from each selected pool (pools 1–5). The relative abundance of each subpool is shown as the size as the corresponding rectangle. Note that subpools −9 and 0 disappeared within two rounds of selection. The corresponding numerical values are given in Supplemental Table S2.

FIGURE 5.

Correlation between the measurements of bacterial growth with two methods. The doubling time in liquid medium containing chloramphenicol and IPTG is shown in the y-axis; the diameter of bacterial colonies on LB agar plates containing IPTG and chloramphenicol is shown on the x-axis. The clone names are indicated. The two clusters are referred to in the text as the class with clone R3C14 (upper) and the class with clone R3C7 (lower).

FIGURE 6.

Predicted secondary structures between (A) the EGS and the mRNA and (B) the EGS and the trans-splicing product. As an example, the secondary structures for clone R3C7 are shown. The two tables show the number of single-stranded nucleotides in the internal loop, on the side on the mRNA substrate or the EGS. The numbers are reported for the most stable secondary structure. For the substrate complex of clone R5C6, two equally stable structures were predicted. The clones are grouped into the two classes observed for the growth behaviors (Fig. 5). Bold letters indicate that these parts of the secondary structure were conserved within each class. The mfold algorithm was used for the prediction of the structures (Zuker 2003).

FIGURE 7.

Correlation between two methods measuring in vivo trans-splicing efficiency. The percentage of repaired cat mRNA was determined by RT-PCR and is shown on the x-axis. The doubling time of E. coli cells expressing the respective ribozyme, in medium containing chloramphenicol, is shown on the y-axis. Error bars, SEM from three quantitations of the doubling time and from six qRT-PCR experiments that determined the fraction of repaired mRNA.