Extraordinary rates of transition metal ion-mediated ribozyme catalysis

In pre-steady-state, fast-quench kinetic analysis, the tertiary-stabilized hammerhead ribozyme “RzB” cleaves its substrate RNA with maximal measured k (obs) values of ∼3000 min(−1) in 1 mM Mn(2+) and ∼780 min(−1) in 1 mM Mg(2+) at 37°C (pH 7.4). Apparent pKa for the catalytic general base is ∼7.8–8.5, independent of the corresponding metal hydrate pKa, suggesting potential involvement of a nucleobase as general base as suggested previously from nucleobase substitution studies. The pH-rate profile is bell-shaped for Cd(2+), for which the general catalytic acid has a pKa of 7.3 ± 0.1. Simulations of the pH-rate relation suggest a pKa for the general catalytic acid to be ∼9.5 in Mn(2+) and >9.5 in Mg(2+). The acid pKa's follow the trend in the pKa of the hydrated metal ions but are displaced by ∼1–2 pH units in the presence of Cd(2+) and Mn(2+). One possible explanation for this trend is direct metal ion coordination with a nucleobase, which then acts as general acid

Conformational heterogeneity and the determinants of tertiary stabilization in the hammerhead ribozyme from Dolichopoda cave crickets

Repetitive DNA elements in Dolichopoda cave cricket genomes contain extended hammerhead ribozymes that are functional in adult crickets, but that exhibit very low self-cleavage activity in vitro relative to other extended hammerhead ribozymes. We find that the parental ribozyme tends to misfold into alternate secondary structures in vitro, complicating analysis of contributions by specific nucleotides to activity under biologically relevant magnesium concentrations. However, minor sequence alterations that stabilize the active secondary structure, without altering candidate tertiary interacting nucleotides, boosted observed rates more than 50-fold (4.4 ± 1.7 min−1) and doubled the cleavage extent (>60%) in submillimolar magnesium. Productive alterations included flipping two base pairs in stem I, lengthening stem I and opening stem III to generate a trans-cleaving ribozyme. Specific peripheral nucleotides involved in tertiary stabilization were then identified through kinetic analysis for a series of sequence variants and by correlating plateau cleavage values with band intensity in native gel electrophoresis. These results demonstrate that conformational heterogeneity governs self-cleavage by the wild-type Dolichopoda hammerhead ribozyme in vitro, and they suggest a strategy for improving activity and enhancing the suitability of HHRz for intracellular and biotechnology applications

Distinct reaction pathway promoted by non-divalent-metal cations in a tertiary stabilized hammerhead ribozyme

Divalent ion sensitivity of hammerhead ribozymes is significantly reduced when the RNA structure includes appropriate tertiary stabilization. Therefore, we investigated the activity of the tertiary stabilized “RzB” hammerhead ribozyme in several nondivalent ions. Ribozyme RzB is active in spermidine and Na+ alone, although the cleavage rates are reduced by more than 1,000-fold relative to the rates observed in Mg2+ and in transition metal ions. The trivalent cobalt hexammine (CoHex) ion is often used as an exchange-inert analog of hydrated magnesium ion. Trans-cleavage rates exceeded 8 min−1 in 20 mM CoHex, which promoted cleavage through outersphere interactions. The stimulation of catalysis afforded by the tertiary structural interactions within RzB does not require Mg2+, unlike other extended hammerhead ribozymes. Site-specific interaction with at least one Mg2+ ion is suggested by CoHex competition experiments. In the presence of a constant, low concentration of Mg2+, low concentrations of CoHex decreased the rate by two to three orders of magnitude relative to the rate in Mg2+ alone. Cleavage rates increased as CoHex concentrations were raised further, but the final fraction cleaved was lower than what was observed in CoHex or Mg2+ alone. These observations suggest that Mg2+ and CoHex compete for binding and that they cause misfolded structures when they are together. The results of this study support the existence of an alternate catalytic mechanism used by nondivalent ions (especially CoHex) that is distinct from the one promoted by divalent metal ions, and they imply that divalent metals influence catalysis through a specific nonstructural role

Flavin Recognition by an RNA Aptamer Targeted toward FAD^†

Flavin adenine dinucleotide (FAD) is one of the primary cofactors in biological redox reactions. Designing cofactor-dependent redox ribozymes could benefit from studies of new RNA−cofactor complexes, as would our understanding of ribozyme evolution during an RNA World. We have therefore used the SELEX method to identify RNA aptamers that recognize FAD. Functional analysis of mutant aptamers, S1 nuclease probing, and comparative sequence analysis identified a simple, 45 nt helical structure with several internal bulges as the core-binding element. These aptamers recognize with high specificity the isoalloxazine nucleus of FAD but do not distinguish FAD from FADH2, nor are they removed from an FAD resin with UMP (which shares a pattern of hydrogen bond donors and acceptors along one face). Thus, these aptamers are structurally and functionally distinct from previously identified FMN and riboflavin aptamers. Circular dichroism data suggest a conformational change in the RNA upon FAD binding. These aptamers require magnesium and are active across a wide pH range (4.5−8.9). Since general acid−base catalysis plays a role in some flavin-dependent redox reaction mechanisms, these aptamers may be particularly well-suited to the design of new redox ribozymes

Control of RNA processing by a large non-coding RNA over-expressed in carcinomas

AbstractRNA processing is vital for the high fidelity and diversity of eukaryotic transcriptomes and the encoded proteomes. However, control of RNA processing is not fully established. Σ RNA is a class of conserved large non-coding RNAs (murine Hepcarcin; human MALAT-1) up-regulated in carcinomas. Using antisense technology, we identified that RNA post-transcriptional modification is the most significant global function of Σ RNA. Specifically, processing of the pre-mRNAs of genes including Tissue Factor and Endoglin was altered by hydrolysis of Σ RNA/MALAT-1. These results support the hypothesis that Σ RNA/MALAT-1 is a regulatory molecule exerting roles in RNA post-transcriptional modification

A Novel Tissue-Free Method to Estimate Tumor-Derived Cell-Free DNA Quantity Using Tumor Methylation Patterns

Estimating the abundance of cell-free DNA (cfDNA) fragments shed from a tumor (i.e., circulating tumor DNA (ctDNA)) can approximate tumor burden, which has numerous clinical applications. We derived a novel, broadly applicable statistical method to quantify cancer-indicative methylation patterns within cfDNA to estimate ctDNA abundance, even at low levels. Our algorithm identified differentially methylated regions (DMRs) between a reference database of cancer tissue biopsy samples and cfDNA from individuals without cancer. Then, without utilizing matched tissue biopsy, counts of fragments matching the cancer-indicative hyper/hypo-methylated patterns within DMRs were used to determine a tumor methylated fraction (TMeF; a methylation-based quantification of the circulating tumor allele fraction and estimate of ctDNA abundance) for plasma samples. TMeF and small variant allele fraction (SVAF) estimates of the same cancer plasma samples were correlated (Spearman’s correlation coefficient: 0.73), and synthetic dilutions to expected TMeF of 10−3 and 10−4 had estimated TMeF within two-fold for 95% and 77% of samples, respectively. TMeF increased with cancer stage and tumor size and inversely correlated with survival probability. Therefore, tumor-derived fragments in the cfDNA of patients with cancer can be leveraged to estimate ctDNA abundance without the need for a tumor biopsy, which may provide non-invasive clinical approximations of tumor burden