Generation of a catalytic module on a self-folding RNA

Abstract

It is theoretically possible to obtain a catalytic site of an artificial ribozyme from a random sequence consisting of a limited numbers of nucleotides. However, this strategy has been inadequately explored. Here, we report an in vitro selection technique that exploits modular construction of a structurally constrained RNA to acquire a catalytic site for RNA ligation from a short random sequence. To practice the selection, a sequence of 30 nucleotides was located close to the putative reaction site in a derivative of a naturally occurring self-folding RNA whose crystal structure is known. RNAs whose activity depended on the starting three-dimensional structure were selected with 3'-5' ligation specificity, indicating that the strategy can be used to acquire a variety of catalytic sites and other functional RNA modules.

FIGURE 1.

(A) Secondary structure of Tetrahymena group I intron (left), the class hc ligase ribozyme (Jaeger et al. 1999) (middle), and P4–P6 ligase ribozyme (right). The P4–P6 domain and the helices containing the reaction site are highlighted in blue and yellow, respectively. Reaction sites are indicated by arrowheads. (B) 3D structure of P4–P6 RNA (Protein Data Bank 1GID) with highlighted L5c (red) and J6/6a (green). (C) Secondary structure of RNA with a pool (red), a reaction site in J6/6a (green), and substituted nucleotides (gray). Long-range interactions are indicated with arrows. Sequences and secondary structures of P5 and P5a regions in the bimolecular system (see last paragraph of Results) are shown in the right box. (D) Nondenaturing PAGE analysis of the P4–P6 RNA (lane 1), its derivative with termini at P6 (lane 2), and another derivative having an additional substitution at internal-loop composed of C217 – A219 and G254 –A256 between P6 and P6a with three consecutive base pairs as follows: 5′-CG, UA and AU-3′ (lane 3), a mutant RNA (this RNA is not a ligase, but a derivative of P4–P6, which has no enzymatic activity) containing L5b consisting of UUCG (L5b UUCG mutant, lane 4), and a derivative of the L5b UUCG mutant with additional substitutions described for lane 3 (lane 5). The conditions were adapted from those reported previously (Ikawa et al. 2002).

FIGURE 2.

Sequences of randomized region in selected clones. Mutations in constant regions are indicated at right; T27C and G45A (italics) were reported previously in the stable mutants of P4–P6 RNA (Juneau and Cech 1999).

FIGURE 3.

Characterization of clone I. (A) Ligation reactions of clone I (lane 1), its derivative containing a modified L5b, which is substituted with GGAA (lane 2), a modified receptor for GGAA (GGAA-receptor) (lane 3), a GGAA at L5b and a GGAA-receptor (lane 4), J5/5a substituted with a stem structure (lane 5), and L5c with original wild-type sequence of UGCAA (lane 6). Conditions: 80 mM of MgCl₂, 50 mM of KCl, 30 mM of Tris-HCl (pH 8.5), 0.5 μM of substrate RNA, and 50 nM of the ribozyme at 37°C for 1 h. (B) Analysis of the ligated phosphodiester bond. Guanylate residues in 3′-substrate was labeled with ³²P. Residues containing ³²P-labeled phosphate at the 3′ side are indicated in red. Substrate RNAs ligated with a modified clone I (gray) were digested with RNase T2. Resulting products were analyzed by two-dimensional denaturing gel electrophoresis.

FIGURE 4.

Ligation reaction of clone I under different conditions. Standard for the assay: 10 mM of MgCl₂, 50 mM of KCl, 30 mM of Tris-HCl (pH 7.5), 1 μM of the substrate RNA, 0.5 μM of the ribozyme at 37°C for 1 h. Reacted fractions of substrate RNA were plotted in the range of pH 7.5–9.5 (A), 37–52°C (B), or 10–80 mM of Mg²⁺ (C). A plot of the observed rate constant of ligation reaction as a function of substrate concentration with 50 mM MgCl₂, 50 mM KCl, and 30 mM Tris-HCl (pH 7.5) at 37°C (D).

FIGURE 5.

Secondary structure analysis and site-directed mutations of the selected sequence in clone I RNA. (A) Autoradiogram of the DMS modification of clone I RNA. Modified bases are indicated at right. (B) Secondary structure model of clone I. Bases modified with DMS are indicated with black circles. The nucleotides derived from the randomized sequence or the constant region, are in black or gray, respectively. Relative activity is indicated for each mutant by using clone I as the standard (1.00). (n.d.) Nondetectable low activity. Conditions: 80 mM of MgCl₂, 50 mM of KCl, 30 mM of Tris-HCl (pH 9.0), 0.5 μM of substrate RNA, and 50 nM of the ribozyme at 37°C for 1 h.

FIGURE 6.

Dissection and reconstruction of clone I RNA. (S) Substrate RNA. (SD and LD) Domains including IGS and random region, respectively (see Fig. 1C) (×1, ×3, ×6) The relative concentration of LD RNA (×1 corresponds to 1 μM). The bands corresponding to ligated products are indicated with an arrow.