Comparison of the Structures and Mechanisms of the Pistol and Hammerhead Ribozymes

Comparison of the secondary and three-dimensional structures of the hammerhead and pistol ribozymes reveals many close similarities, so in this work we have asked if they are mechanistically identical. We have determined a new crystal structure of the pistol ribozyme and have shown that G40 acts as general base in the cleavage reaction. The conformation in the active site ensures an in-line attack of the O2′ nucleophile, and the conformation at the scissile phosphate and the position of the general base are closely similar to those in the hammerhead ribozyme. However, the two ribozymes differ in the nature of the general acid. 2′-Amino substitution experiments indicate that the general acid of the hammerhead ribozyme is the O2′ of G8, while that of the pistol ribozyme is a hydrated metal ion. The two ribozymes are related but mechanistically distinct

General Acid–Base Catalysis Mediated by Nucleobases in the Hairpin Ribozyme

The catalytic mechanism by which the hairpin ribozyme accelerates cleavage or ligation of the phosphodiester backbone of RNA has been incompletely understood. There is experimental evidence for an important role for an adenine (A38) and a guanine (G8), and it has been proposed that these act in general acid-base catalysis. In this work we show that a large reduction in cleavage rate on substitution of A38 by purine (A38P) can be reversed by replacement of the 5′-oxygen atom at the scissile phosphate by sulfur (5′-PS), which is a much better leaving group. This is consistent with A38 acting as the general acid in the unmodified ribozyme. The rate of cleavage of the 5′-PS substrate by the A38P ribozyme increases with pH log-linearly, indicative of a requirement for a deprotonated base with a relatively high pK(a). On substitution of G8 by diaminopurine, the 5′-PS substrate cleavage rate at first increases with pH and then remains at a plateau, exhibiting an apparent pK(a) consistent with this nucleotide acting in general base catalysis. Alternative explanations for the pH dependence of hairpin ribozyme reactivity are discussed, from which we conclude that general acid-base catalysis by A38 and G8 is the simplest and most probable explanation consistent with all the experimental data

Hachimoji DNA and RNA: A genetic system with eight building blocks

Reported here are DNA and RNA-like systems built from eight (hachi-) nucleotide letters (-moji) that form four orthogonal pairs. This synthetic genetic biopolymer meets the structural requirements needed to support Darwinism, including a polyelectrolyte backbone, predictable thermodynamic stability, and stereoregular building blocks that fit a Schrödinger aperiodic crystal. Measured thermodynamic parameters predict the stability of hachimoji duplexes, allowing hachimoji DNA to double the information density of natural terran DNA. Three crystal structures show that the synthetic building blocks do not perturb the aperiodic crystal seen in the DNA double helix. Hachimoji DNA was then transcribed to give hachimoji RNA in the form of a functioning fluorescent hachimoji aptamer. These results expand the scope of molecular structures that might support life, including life throughout the cosmos

Recognition of an expanded genetic alphabet by type-II restriction endonucleases and their application to analyze polymerase fidelity

To explore the possibility of using restriction enzymes in a synthetic biology based on artificially expanded genetic information systems (AEGIS), 24 type-II restriction endonucleases (REases) were challenged to digest DNA duplexes containing recognition sites where individual Cs and Gs were replaced by the AEGIS nucleotides Z and P [respectively, 6-amino-5-nitro-3-(1′-β-d-2′-deoxyribofuranosyl)-2(1H)-pyridone and 2-amino-8-(1′-β-d-2′-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one]. These AEGIS nucleotides implement complementary hydrogen bond donor–donor–acceptor and acceptor–acceptor–donor patterns. Results allowed us to classify type-II REases into five groups based on their performance, and to infer some specifics of their interactions with functional groups in the major and minor grooves of the target DNA. For three enzymes among these 24 where crystal structures are available (BcnI, EcoO109I and NotI), these interactions were modeled. Further, we applied a type-II REase to quantitate the fidelity polymerases challenged to maintain in a DNA duplex C:G, T:A and Z:P pairs through repetitive PCR cycles. This work thus adds tools that are able to manipulate this expanded genetic alphabet in vitro, provides some structural insights into the working of restriction enzymes, and offers some preliminary data needed to take the next step in synthetic biology to use an artificial genetic system inside of living bacterial cells

Revisiting Molossus (Mammalia: Chiroptera: Molossidae) diversity: Exploring southern limits and revealing a novel species in Argentina

Abstract Understanding species diversity and delineating their boundaries are crucial for effective management and conservation efforts. In the case of bats, species identification holds particular importance from an epidemiological standpoint. The genus Molossus (Chiroptera: Molossidae) encompasses 15 species distributed across the Neotropics, ranging from the southeastern United States to Argentina. This genus exhibits two contrasting patterns of variation: some species are cryptic, while others are morphologically distinct yet genetically similar. This study explores the diversity of Molossus in Argentina through a molecular phylogenetic approach. We analyzed sequences from three molecular markers (cyt b, COI, and FGB) along with morphology data obtained from a sample of 64 individuals. Uni- and multivariate analyses of external and cranial measurements were conducted, alongside comparisons of external and cranial characteristics among species. Based on molecular and morphological differences, we describe a new species within the Molossus genus. This newly discovered species exhibits a broad distribution spanning the Paraná River basin across three distinct ecoregions. It is noteworthy that this species is pseudo-cryptic with respect to similar-sized species such as M. molossus and M. melini. Additionally, it is important to mention that all species in Argentina have overlapping distribution ranges. In summary, this study provides valuable insights into the diversity and distribution of Molossus bats in Argentina, employing molecular and morphological analyses. The discovery of a new species underscores the ongoing importance of comprehensive research efforts in understanding and conserving bat populations in the Neotropics

Reprogramming the assembly of unmodified DNA with a small molecule

The ability of DNA to store and encode information arises from base pairing of the four-letter nucleobase code to form a double helix. Expanding this DNA ‘alphabet’ by synthetic incorporation of new bases can introduce new functionalities and enable the formation of novel nucleic acid structures. However, reprogramming the self-assembly of existing nucleobases presents an alternative route to expand the structural space and functionality of nucleic acids. Here we report the discovery that a small molecule, cyanuric acid, with three thymine-like faces reprogrammes the assembly of unmodified poly(adenine) (poly(A)) into stable, long and abundant fibres with a unique internal structure. Poly(A) DNA, RNA and peptide nucleic acid all form these assemblies. Our studies are consistent with the association of adenine and cyanuric acid units into a hexameric rosette, which brings together poly(A) triplexes with a subsequent cooperative polymerization. Fundamentally, this study shows that small hydrogen-bonding molecules can be used to induce the assembly of nucleic acids in water, which leads to new structures from inexpensive and readily available materials

A portable RNA sequence whose recognition by a synthetic antibody facilitates structural determination

RNA crystallization and phasing represent major bottlenecks in RNA structure determination. Seeking to exploit antibody fragments as RNA crystallization chaperones, we have used an arginine-enriched synthetic Fab library displayed on phage to obtain Fabs against the class I ligase ribozyme. We solved the structure of a Fab–ligase complex at 3.1-Å resolution using molecular replacement with Fab coordinates, confirming the ribozyme architecture and revealing the chaperone's role in RNA recognition and crystal contacts. The epitope resides in the GAAACAC sequence that caps the P5 helix, and this sequence retains high-affinity Fab binding within the context of other structured RNAs. This portable epitope provides a new RNA crystallization chaperone system that easily can be screened in parallel to the U1A RNA-binding protein, with the advantages of a smaller loop and Fabs′ high molecular weight, large surface area and phasing power.National Institutes of Health (U.S.) (GM61835