A Mechanistic Explanation for the Regioselectivity of Nonenzymatic RNA Primer Extension

A working model of nonenzymatic RNA primer extension could illuminate how prebiotic chemistry transitioned to biology. All currently known experimental reconstructions of nonenzymatic RNA primer extension yield a mixture of 2′-5′ and 3′-5′ internucleotide linkages. Although long seen as a major problem, the causes of the poor regioselectivity of the reaction are unknown. We used a combination of different leaving groups, nucleobases, and templating sequences to uncover the factors that yield selective formation of 3′-5′ internucleotide linkages. We found that fast and high yielding reactions selectively form 3′-5′ linkages. Additionally, in all cases with high 3′-5′ regioselectivity, Watson–Crick base pairing between the RNA monomers and the template is observed at the extension site and at the adjacent downstream position. Mismatched base-pairs and other factors that would perturb the geometry of the imidazolium bridged intermediate lower both the rate and regioselectivity of the reaction

Synthesis of a Nonhydrolyzable Nucleotide Phosphoroimidazolide Analogue That Catalyzes Nonenzymatic RNA Primer Extension

We report the synthesis of guanosine 5′-(4-methylimidazolyl)­phosphonate (ICG), the third member of a series of nonhydrolyzable nucleoside 5′-phosphoro-2-methylimidazolide (2-MeImpN) analogues designed for mechanistic studies of nonenzymatic RNA primer extension. The addition of a 2-MeImpN monomer to a primer is catalyzed by the presence of a downstream activated monomer, yet the three nonhydrolyzable analogues do not show catalytic effects under standard mildly basic primer extension conditions. Surprisingly, ICG, which has a p<i>K</i>_a similar to that of 2-MeImpG, is a modest catalyst of nonenzymatic primer extension at acidic pH. Here we show that ICG reacts with 2-MeImpC to form a stable 5′–5′-imidazole-bridged guanosine-cytosine dinucleotide, with both a labile nitrogen–phosphorus and a stable carbon–phosphorus linkage flanking the central imidazole bridge. Cognate RNA primer–template complexes react with this GC-dinucleotide by attack of the primer 3′-hydroxyl on the activated N–P side of the 5′-5′-imidazole bridge. These observations support the hypothesis that 5′–5′-imidazole-bridged dinucleotides can bind to cognate RNA primer–template duplexes and adopt appropriate conformations for subsequent phosphodiester bond formation, consistent with our recent mechanistic proposal that the formation of activated 5′–5′-imidazolium-bridged dinucleotides is responsible for 2-MeImpN-driven primer extension

Downstream Oligonucleotides Strongly Enhance the Affinity of GMP to RNA Primer–Template Complexes

Origins of life hypotheses often invoke a transitional phase of nonenzymatic template-directed RNA replication prior to the emergence of ribozyme-catalyzed copying of genetic information. Here, using NMR and ITC, we interrogate the binding affinity of guanosine 5′-monophosphate (GMP) for primer–template complexes when either another GMP, or a helper oligonucleotide, can bind downstream. Binding of GMP to a primer–template complex cannot be significantly enhanced by the possibility of downstream monomer binding, because the affinity of the downstream monomer is weaker than that of the first monomer. Strikingly, GMP binding affinity can be enhanced by ca. 2 orders of magnitude when a helper oligonucleotide is stably bound downstream of the monomer binding site. We compare these thermodynamic parameters to those previously reported for T7 RNA polymerase-mediated replication to help address questions of binding affinity in related nonenzymatic processes

Selective Access to Trisubstituted Macrocyclic <i>E</i>- and <i>Z</i>‑Alkenes from the Ring-Closing Metathesis of Vinylsiloxanes

Macrocyclic (<i>E</i>)-alkenylsiloxanes, obtained from <i>E</i>-selective ring-closing metathesis reactions, can be converted to the corresponding (<i>Z</i>)-alkenyl bromides and (<i>E</i>)-alkenyl iodides allowing access to both <i>E-</i> and <i>Z</i>-trisubstituted macrocyclic alkenes. The reaction conditions and substrate scope of these stereoselective transformations are explored

Common and Potentially Prebiotic Origin for Precursors of Nucleotide Synthesis and Activation

We have recently shown that 2-aminoimidazole is a superior nucleotide activating group for nonenzymatic RNA copying. Here we describe a prebiotic synthesis of 2-aminoimidazole that shares a common mechanistic pathway with that of 2-aminooxazole, a previously described key intermediate in prebiotic nucleotide synthesis. In the presence of glycolaldehyde, cyanamide, phosphate and ammonium ion, both 2-aminoimidazole and 2-aminooxazole are produced, with higher concentrations of ammonium ion and acidic pH favoring the former. Given a 1:1 mixture of 2-aminoimidazole and 2-aminooxazole, glyceraldehyde preferentially reacts and cyclizes with the latter, forming a mixture of pentose aminooxazolines, and leaving free 2-aminoimidazole available for nucleotide activation. The common synthetic origin of 2-aminoimidazole and 2-aminooxazole and their distinct reactivities are suggestive of a reaction network that could lead to both the synthesis of RNA monomers and to their subsequent chemical activation

Bidirectional Direct Sequencing of Noncanonical RNA by Two-Dimensional Analysis of Mass Chromatograms

Mass spectrometry (MS) is a powerful technique for characterizing noncanonical nucleobases and other chemical modifications in small RNAs, yielding rich chemical information that is complementary to high-throughput indirect sequencing. However, mass spectra are often prohibitively complex when fragment ions are analyzed following either solution phase hydrolysis or gas phase fragmentation. For all but the simplest cases, ions arising from multiple fragmentation events, alternative fragmentation pathways, and diverse salt adducts frequently obscure desired single-cut fragment ions. Here we show that it is possible to take advantage of predictable regularities in liquid chromatographic (LC) separation of optimized RNA digests to greatly simplify the interpretation of complex MS data. A two-dimensional analysis of extracted compound chromatograms permits straightforward and robust de novo sequencing, using a novel Monte Carlo algorithm that automatically generates bidirectional paired-end reads, pinpointing the position of modified nucleotides in a sequence. We demonstrate that these advances permit routine LC–MS sequencing of RNAs containing noncanonical nucleotides, and we furthermore examine the applicability of this approach to the study of oligonucleotides containing artificial modifications as well as those commonly observed in post-transcriptionally modified RNAs

Enhanced Nonenzymatic RNA Copying with 2‑Aminoimidazole Activated Nucleotides

Achieving efficient nonenzymatic replication of RNA is an important step toward the synthesis of self-replicating protocells that may mimic early forms of life. Despite recent progress, the nonenzymatic copying of templates containing mixed sequences remains slow and inefficient. Here we demonstrate that activating nucleotides with 2-aminoimidazole results in superior reaction kinetics and improved yields of primer extension reaction products. This new leaving group significantly accelerates monomer addition as well as trimer-assisted RNA primer extension, allowing efficient copying of a variety of short RNA templates with mixed sequences