Emergence of a dual-catalytic RNA with metal-specific cleavage and ligase activities: the spandrels of RNA evolution

Abstract

In vitro selection, or directed molecular evolution, allows the isolation and amplification of rare sequences that satisfy a functional-selection criterion. This technique can be used to isolate novel ribozymes (RNA enzymes) from large pools of random sequences. We used in vitro evolution to select a ribozyme that catalyzes a novel template-directed RNA ligation that requires surprisingly few nucleotides for catalytic activity. With the exception of two nucleotides, most of the ribozyme contributes to a template, suggesting that it is a general prebiotic ligase. More surprisingly, the catalytic core built from randomized sequences actually contains a 7-nt manganese-dependent self-cleavage motif originally discovered in the Tetrahymena group I intron. Further experiments revealed that we have selected a dual-catalytic RNA from random sequences: the RNA promotes both cleavage at one site and ligation at another site, suggesting two conformations surrounding at least one divalent metal ion-binding site. Together, these results imply that similar catalytic RNA motifs can arise under fairly simple conditions and that multiple catalytic structures, including bifunctional ligases, can evolve from very small preexisting parts. By breaking apart and joining different RNA strands, such ribozymes could have led to the production of longer and more complex RNA polymers in prebiotic evolution.

Figure 1

(a) Possible secondary structure of one of the ligase ribozymes selected from round 13, shown ligated to the substrate guide RNA (gRNA). The 5′ and 3′ constant regions of the pool are shaded (and sequences derived from the 5′ constant region are shown in blue throughout Fig. 1). The 3′-U₆ tail (underlined and in green) and the 5′ biotin of the guide RNA substrate are marked. (CAT)₄, a DNA sequence tag, and other sequences in red are not needed. The core region of the ribozyme is boxed in the center. The ∗ arrow indicates the 3′-terminal base needed for efficient ligation. Dashed lines and arrows indicate sites of allowed breakage for trans-ligation or truncation experiments. U–A pairs in magenta are weakly supported by sequence covariation. Substitutions in black increase activity, and mutations in light gray abolish activity, following a grayscale. The small arrow indicates attack of the 2′-hyrdoxyl of the 3′-U₆ tail on the 5′-triphosphate of the G at the end of the RNA. (b) Very small cis-ligation with the same substrate as in a, k_obs = 3.4 × 10⁻⁴ min⁻¹. (c) Very small trans-ligation with a 10-nt 3′ substrate and a 29-nt ribozyme (boxed), k_obs = 5.4 × 10⁻⁴ min⁻¹ at 25°C and 30 × 10⁻⁴ min⁻¹ at 45°C. (d and e) Two very simple catalytic motifs still ligate with up to 10% (d) and 4% (e) the efficiency of the ligation in b or c, demonstrating remarkable versatility of the small ligase motif. Randomized nucleotides (N) are not intended to be base paired.

Figure 2

Alignment of 12 representative functional ribozyme clones from a total of 51 such clones. A dash indicates identity with the top sequence. A . indicates a deletion. The most conserved region is boxed. The constant regions are shaded in the reference sequence.

Figure 3

(Left) Time course of representative trans-ligation shown in Fig. 1c with 10-nt 3′ substrate; Lower band, labeled 22-nt guide RNA band; upper band, 32-nt ligated product band. k_obs = 2.7 × 10⁻⁴ min⁻¹. (Right) Time course of randomized cis-ligation shown in Fig. 1e. M, marker lane; lower band, labeled 41-nt partially randomized RNA; upper band, ligated 63-nt product.

Figure 4

Ribonuclease T2 digestion to determine regiospecificity of bond formation. Lane 2 is the RNase T2 digestion product of the ligation in Fig. 1c (see Methods). Lane 1 is the RNase T2 digestion product of a ligated RNA of identical sequence, but with a labeled 3′,5′ linkage generated by T4 DNA ligase and a complementary DNA splint (42). Digestion products were analyzed on a 20% denaturing acrylamide gel. Lanes 3 and 4 are a time course of alkali hydrolysis at 5 and 10 min, respectively, of the ligated product digested in lane 1.

Figure 5

Biochemistry of ligation reaction. (Left) pH dependence and (Center and Right) effect of replacing Mg²⁺ with a series of divalent cations using ribozyme/substrate complex in Fig. 1c. (Center) k_obs as a function of Mg²⁺ (⋄) and Mn²⁺ (□) at pH 7.0. (Right) k_obs as a function of Mg²⁺ (□), Ca²⁺ (⋄), Cd²⁺ (○), Sr²⁺ (▵), and Ba²⁺ (crossed squares) at pH 9.0.

Figure 6

Ligation of 5′,3′-(UUUUUU) (lower band) to a 10-nt substrate. Upper band is the 16-nt product. Lane 1 was incubated without substrate and lane M is a marker; lane 2, 3-day incubation; lane 3, 6-day incubation.

Figure 7

Mn²⁺-dependent RNA cleavage. The complex of UUU and UGAAA undergoes site specific Mn-dependent cleavage at the arrowhead (15). The U is not required in the substrate but was present in the original sequence (19) and, surprisingly, the entire 8-nt motif shown here appears in our selected ligase, just opposite the ligation site (Figs. 1 a–c and e). The lines represent optional RNA sequences, and the circle represents at least one binding site for a divalent ion—Mn²⁺ or Cd²⁺ to promote cleavage—and presumably Mg²⁺ or other metals to promote ligation. Lanes M and 1 contain standards; lane 2 is unreacted 3′-end labeled 22-nt positive control synthetic RNA5 (15), and lane 3 is RNA5 digested to an 18-nt product. Lanes 4–6 represent Mn²⁺ cleavage of the 29-nt ribozyme strand shown in Fig. 1c to a 9-nt shorter product in the presence of Mn²⁺ and 0.05, 0.5, and 5 ng/μl poly(U).