Structure-function correlations derived from faster variants of a RNA ligase deoxyribozyme

Abstract

We previously reported the in vitro selection of several Mg2+-dependent deoxyribozymes (DNA enzymes) that synthesize a 2'-5' RNA linkage from a 2',3'-cyclic phosphate and a 5'-hydroxyl. Here we subjected the 9A2 deoxyribozyme to re-selection for improved ligation rate. We found two new DNA enzymes (7Z81 and 7Z48) that contain the catalytic core of 7Q10, a previously reported small deoxyribozyme that is unrelated in sequence to 9A2. A third new DNA enzyme (7Z101) is unrelated to either 7Q10 or 9A2. The new 7Z81 and 7Z48 DNA enzymes have ligation rates over an order of magnitude higher than that of 7Q10 itself and they have additional sequence elements that correlate with these faster rates. Our findings provide insight into structure-function relationships of catalytic nucleic acids.

Figure 1

RNA ligation by attack of a 5′-hydroxyl on a 2′,3′-cyclic phosphate. Two isomers are possible: the non-native 2′–5′ linkage (1→2) or the native 3′–5′ junction (1→3). For all reported Mg²⁺-dependent deoxyribozymes that mediate reaction of 1, the product is 2 and not 3.

Figure 2

The selection procedure. (A) Overview of the strategy. RNA is shown as a thick line and DNA as a thin line. The products are separated by denaturing PAGE after each of the three steps A–C of each selection round. The PEG spacer in PCR primer 1 for step C causes the two single-stranded products to have different lengths, thus permitting their separation by PAGE. A detailed description of the overall selection procedure has been published (1). Here, the reaction conditions during the key step B were 50 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM KCl and 40 mM MgCl₂ at 37°C. (B) Progression of ligation activity. For each round, the incubation time for selection step B is indicated; the selection pressure was increased by decreasing the incubation time. The round 7 pool was cloned. Repeating round 5 with a 13 h step B incubation led to 18–21% ligation activity, but we did not further pursue this branch of the selection tree as our principal goal was faster deoxyribozymes.

Figure 3

Sequences of the new 7Z81, 7Z48 and 7Z101 deoxyribozymes. The sequences of the 9A2 and 7Q10 deoxyribozymes are provided for comparison (1,2). Identity between 7Q10 and the new DNA enzymes is marked with vertical lines. The red nucleotides constitute the 7Q10-like catalytic core; other colors are used as shown in Figure 5. Identity between 7Z81 and 7Z48 is marked with bullets.

Figure 4

Representative RNA ligation assays for the new deoxyribozymes. (A) Trimolecular format of the ligation assays. (B and C) Ligation assays for the new DNA enzymes under standard incubation conditions (50 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM KCl, 40 mM MgCl₂, 37°C). Values of k_obs from the curve fits are collected in Table 1, along with similar data obtained in CHES, pH 9.0. Data points for 7Z101 and 7Q10 recorded at 24 h fall on the curve fit lines.

Figure 5

Secondary structure comparison of the previously reported 7Q10 DNA enzyme and the new 7Z81 and 7Z48 DNA enzymes. (A) The 7Q10 DNA enzyme (2,3), with the catalytic core nucleotides in red. (B) The 7Z81 DNA enzyme. The indicated d1–d5 regions on 7Z81 denote the 4 nt deletion mutants (deletants) assayed for ligation activity. The purple nucleotides in the large right-hand loop may together be removed without disrupting activity [the 7Z81d(2+3) deletant in Table 2]. The G nucleotides marked with dashed arrows were additionally tested by deletion in the context of 7Z81d(2+3). As marked, only one of these two G nucleotides may be deleted while retaining full activity. Additional deletions are also not tolerated (Table 22). (C) The 7Z48 DNA enzyme. The purple nucleotides in the large right-hand loop may be deleted without disrupting ligation activity (7Z48d3 deletant in Table 2), which is consistent with the illustrated secondary structure. The parent full-length 7Z48 sequence is shown, along with deletants d1–d9. All secondary structures were predicted by mfold (6,7), which is available online at www.bioinfo.rpi.edu/applications/mfold and also at www.idtdna.com.

Figure 6

Cleavage of 2′–5′ linked RNA by the new deoxyribozymes, shown with ligation assay data from Figure 4C for comparison. The cleavage assays were performed under the standard incubation conditions (50 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM KCl, 40 mM MgCl₂, 37°C) with a 1:5 ratio of (L+R):E. Values of k_obs are collected in Table 1. In parallel assays, a 3′–5′ linked RNA was not cleaved detectably by any of the deoxyribozymes (<5%; data not shown).