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 À1 in 1 mM Mn 2+ and ; ;780 min À1 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 6 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.

Tertiary stabilizing motifs (TSMs) between terminal loops or internal bulges facilitate folding of natural hammerhead ribozymes (hRz) under physiological conditions. However, both substrate and enzyme strands contribute nucleotides to the TSMs of trans-cleaving hRz, complicating the design of hRz that exploit TSMs to target specific mRNA. To overcome this limitation, we used SELEX to identify new, artificial TSMs that are less sensitive to sequence context. Nucleotides in loop II or in a bulge within the ribozyme strand of stem I were randomized, while the interaction partner was held constant. All nucleotides of the substrate pair with the ribozyme, minimizing their possible recruitment into the TSM, as such recruitment could constrain choice of candidate target sequences. Six cycles of selection identified cis-acting ribozymes that were active in 100 µM MgCl 2 . The selected motifs partially recapitulate TSMs found in natural hRz, suggesting that the natural motifs are close to optimal for their respective contexts. Ribozyme "RzB" showed enhanced thermal stability by retaining trans-cleavage activity at 80°C in 10 mM MgCl 2 and at 70°C in 2 mM MgCl 2 . A variant of ribozyme "RzB" with a continuously paired stem 1 rapidly lost activity as temperature was increased. The selected motifs are modular, in that they permit trans-cleavage of several substrates in submillimolar MgCl 2 , including two substrates derived from the U5 genomic region of HIV-1. The new, artificial tertiary stabilized hRz are thus nearly independent of sequence context and enable for the first time the use of highly active hRz targeting almost any mRNA at physiologically relevant magnesium concentrations.

Edited by Tamas DalmayKeywords: Hepcarcin R RNA/MALAT-1 RNA processing Alternative splicing a b s t r a c t RNA 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. R 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 R RNA. Specifically, processing of the pre-mRNAs of genes including Tissue Factor and Endoglin was altered by hydrolysis of R RNA/MALAT-1. These results support the hypothesis that R RNA/MALAT-1 is a regulatory molecule exerting roles in RNA post-transcriptional modification.

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 Mg 2+ 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 À1 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 Mg 2+ , unlike other extended hammerhead ribozymes. Site-specific interaction with at least one Mg 2+ ion is suggested by CoHex competition experiments. In the presence of a constant, low concentration of Mg 2+ , low concentrations of CoHex decreased the rate by two to three orders of magnitude relative to the rate in Mg 2+ alone. Cleavage rates increased as CoHex concentrations were raised further, but the final fraction cleaved was lower than what was observed in CoHex or Mg 2+ alone. These observations suggest that Mg 2+ 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 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 FADH(2), 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.

The discovery of ribozymes has inspired exploration of RNA’s potential to serve as primordial catalysts in a hypothesized RNA world. Modern oxidoreductase enzymes employ differential binding between reduced and oxidized forms of redox cofactors to alter cofactor reduction potential and enhance the enzyme’s catalytic capabilities. The utility of differential affinity has been underexplored as a chemical strategy for RNA. Here we show an RNA aptamer that preferentially binds oxidized forms of flavin over reduced forms and markedly shifts flavin reduction potential by −40 mV, similar to shifts for oxidoreductases. Nuclear magnetic resonance structural analysis revealed π–π and donor atom–π interactions between the aptamer and flavin that cause unfavorable contacts with the electron-rich reduced form, suggesting a mechanism by which the local environment of the RNA-binding pocket drives the observed shift in cofactor reduction potential. It seems likely that primordial RNAs could have used similar strategies in RNA world metabolisms.

Introduction: Cancer detection blood tests have shown clinical utility after cancer diagnosis, but many evaluate only single cancers and require tumor tissue, thereby limiting their utility. We developed a versatile, tissue-free (ie, no tumor tissue required), multi-cancer detection test (“Post-Diagnosis Cancer Research Solution”) based on methylation sequencing of cfDNA from blood. This technology solution can be used to evaluate cfDNA applications in cancer research, including treatment evaluation, recurrence monitoring, and prognostic guidance. We report the analytical validation of this Post-Diagnosis Cancer Research Solution, characterizing sensitivity, specificity, precision, and input range. Methods: cfDNA samples from cancer and non-cancer donors were analyzed. Analytical sensitivity (LoD95, limit of detection with ≥95% probability) was determined as a function of methyl variant allele fraction (MVAF), a measure of circulating tumor allele fraction. A total of 6 ng of cfDNA was used (cancer cfDNA titrated into a background of non-cancer cfDNA). LoD95 was defined as the lowest observed MVAF with ≥95% cancer signal detection across tested replicates or was estimated using probit regression for eligible samples. Analytical specificity was the rate of non-cancer classification among samples from 128 non-cancer donors. Precision (defined as concordance with the expected cancer/non-cancer result) was evaluated for 15 cancer donors tested near sample LoD95 and 8 non-cancer donors, with ≥18 replicates/donor. Reliability of classification was evaluated as a function of cfDNA input mass (0.25-100 ng total cfDNA, 14 cancer and 2 non-cancer donors). Results: A total of 12 different solid cancer types from 22 individuals with cancer were assessed in the LoD95 analysis. Median LoD95 at 6 ng total cfDNA input was 0.023% (10th percentile 0.0037%; 90th percentile 0.04%) MVAF. LoD95 estimates from in silico titration analyses of >200 clinical samples across a subset of the 12 cancer types were consistent with experimental LoD95 values. Analytical specificity was determined to be 98.47% (95% CI: 94.60-99.58%). Median precision across individuals was 100% (10th percentile 87%; 90th percentile 100%). Classification performance was accurate and consistent across a wide range of cfDNA input mass (100% correct cancer/non-cancer classification from 1.5-100 ng). Conclusions: Results demonstrate that this multi-cancer Post-Diagnosis Cancer Research Solution has high analytical sensitivity, specificity, and precision, with reliable performance across a broad cfDNA input range. This technology does not require a tumor sample and provides a cancer signal estimate in terms of MVAF. These features may enable understanding of cfDNA dynamics in a wide variety of cancers for research studies. Citation Format: Mohini Desai, Svetlana Rakhmanova Shchegrov, Shoujie Chai, Yifan Zhou, Tracy Nguyen, Yirang Cho, Collin Melton, Eric Scott, Manami Roychowdhury-Saha, Pei-Yun Chang, Rita Shaknovich, Byoungsok Jung. Analytical validation of a tissue-free, multi-cancer, post-diagnosis cancer research test that uses cell-free DNA methylation profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB297.

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.