202563724
Dynamical ensemble of the active state and transition state mimic for the RNA-cleaving 8–17 DNAzyme in solution
Abstract We perform molecular dynamics simulations, based on recent crystallographic data, on the 8–17 DNAzyme at four states along the reaction pathway to determine the dynamical ensemble for the active state and transition state mimic in solution. A striking finding is the diverse roles played by Na+ and Pb2+ ions in the electrostatically strained active site that impact all four fundamental catalytic strategies, and share commonality with some features recently inferred for naturally occurring hammerhead and pistol ribozymes. The active site Pb2+ ion helps to stabilize in-line nucleophilic attack, provides direct electrostatic transition state stabilization, and facilitates leaving group departure. A conserved guanine residue is positioned to act as the general base, and is assisted by a bridging Na+ ion that tunes the pKa and facilitates in-line fitness. The present work provides insight into how DNA molecules are able to solve the RNA-cleavage problem, and establishes functional relationships between the mechanism of these engineered DNA enzymes with their naturally evolved RNA counterparts. This adds valuable information to our growing body of knowledge on general mechanisms of phosphoryl transfer reactions catalyzed by RNA, proteins and DNA.
9351450
DNA Catalysis: The Chemical Repertoire of DNAzymes
Deoxyribozymes or DNAzymes are single-stranded catalytic DNA molecules that are obtained by combinatorial in vitro selection methods. Initially conceived to function as gene silencing agents, the scope of DNAzymes has rapidly expanded into diverse fields, including biosensing, diagnostics, logic gate operations, and the development of novel synthetic and biological tools. In this review, an overview of all the different chemical reactions catalyzed by DNAzymes is given with an emphasis on RNA cleavage and the use of non-nucleosidic substrates. The use of modified nucleoside triphosphates (dN*TPs) to expand the chemical space to be explored in selection experiments and ultimately to generate DNAzymes with an expanded chemical repertoire is also highlighted.
5138957
Allergen-induced asthmatic responses modified by a GATA3-specific DNAzyme.
BACKGROUND
The most prevalent phenotype of asthma is characterized by eosinophil-dominated inflammation that is driven by a type 2 helper T cell (Th2). Therapeutic targeting of GATA3, an important transcription factor of the Th2 pathway, may be beneficial. We evaluated the safety and efficacy of SB010, a novel DNA enzyme (DNAzyme) that is able to cleave and inactivate GATA3 messenger RNA (mRNA).


METHODS
We conducted a randomized, double-blind, placebo-controlled, multicenter clinical trial of SB010 involving patients who had allergic asthma with sputum eosinophilia and who also had biphasic early and late asthmatic responses after laboratory-based allergen provocation. A total of 40 patients could be evaluated; 21 were assigned to receive 10 mg of SB010, and 19 were assigned to receive placebo, with each study drug administered by means of inhalation once daily for 28 days. An allergen challenge was performed before and after the 28-day period. The primary end point was the late asthmatic response as quantified by the change in the area under the curve (AUC) for forced expiratory volume in 1 second (FEV1).


RESULTS
After 28 days, SB010 attenuated the mean late asthmatic response by 34%, as compared with the baseline response, according to the AUC for FEV1, whereas placebo was associated with a 1% increase in the AUC for FEV1 (P=0.02). The early asthmatic response with SB010 was attenuated by 11% as measured by the AUC for FEV1, whereas the early response with placebo was increased by 10% (P=0.03). Inhibition of the late asthmatic response by SB010 was associated with attenuation of allergen-induced sputum eosinophilia and with lower levels of tryptase in sputum and lower plasma levels of interleukin-5. Allergen-induced levels of fractional exhaled nitric oxide and airway hyperresponsiveness to methacholine were not affected by either SB010 or placebo.


CONCLUSIONS
Treatment with SB010 significantly attenuated both late and early asthmatic responses after allergen provocation in patients with allergic asthma. Biomarker analysis showed an attenuation of Th2-regulated inflammatory responses. (Funded by Sterna Biologicals and the German Federal Ministry of Education and Research; ClinicalTrials.gov number, NCT01743768.).
9534016
Potent inhibition of HIV-1 gene expression and TAT-mediated apoptosis in human T cells by novel mono- and multitarget anti-TAT/Rev/Env ribozymes and a general purpose RNA-cleaving DNA-enzyme.
One of the hallmarks of progression of HIV-1/AIDS is the rapid depletion of CD4+T cells that is known to occur at the late stages of the disease when usually X4 tropic HIV-1 predominates. Besides direct killing of T lymphocytes, HIV-1 infection leads to extensive apoptosis of naïve/uninfected bystander T cells, which is predominantly mediated by HIV-1 TAT protein. Therefore, reduction of HIV-1 TAT protein is likely to reduce substantially the pathogenesis associated with HIV-1 infection. We designed two non-GUX hammerhead ribozymes (Rzs) and a Di-Rz by placing them in direct tandem. These were targeted against the most conserved second exon of HIV-1 TAT/Rev/Env region. Although very impressive in vitro cleavage of the target RNA by the two hammerhead Rzs was obtained under standard conditions of cleavage, only one of them was active under simulated physiological conditions. Sequence-specific cleavage by the Di-Rz was most efficient under standard conditions. Cleavage reactions carried out under simulated physiological conditions by the Di-Rz, however, indicated that both mono-Rzs were functional. We also assembled a 10-23 catalytic motif containing general purpose RNA-cleaving DNA-enzyme (Dz) against the same region, which cleaved the target RNA very efficiently. Both Rzs and Dz showed not only potent inhibition of HIV-1 gene expression but also showed remarkable protection against HIV-1 TAT protein-mediated apoptosis in Jurkat T cells. Possible implications of these findings are discussed.
102482892
DNAzyme-based biosensor for detection of lead ion: A review
Abstract Lead ion (Pb2 +) is a kind of toxic metal pollutant that widely spread in the environment, which is of serious concern to human health. So it is of great significance to develop simple, fast method for the detection of Pb2 +. Up to now, a number of DNA sensors have been designed to recognize Pb2 +, especially DNAzyme-based sensors have attracted much attention due to its advantages, such as high selectivity and sensitivity. In this review, we summarize the recent progress of two types of DNAzyme-based sensors for the detection of toxic Pb2 +, including Pb2 +-specific DNAzyme and G-quadruplex DNAzyme sensors. On the basis of the sensor transducers and their signal types, development and applications of fluorescent DNAzyme sensors, colorimetric DNAzyme sensors and electrochemical DNAzyme sensors are to be discussed in this paper. These DNAzyme-based sensors are highly sensitive and selective toward Pb2 +, and has proved to be a useful tool in the potential application for on-site and real-time real sample (such as environmental and biological samples) monitoring in the future.
20886222
In vitro selection of RNA-cleaving DNAzymes for bacterial detection.
DNAzymes refer to single-stranded DNA molecules with catalytic activity and can be isolated from synthetic random-sequence DNA pools using the technique of in vitro selection. DNAzymes that cleave RNA, known as "RNA-cleaving DNAzymes", represent one of the best-studied classes of DNAzymes and have been widely used for the development of biosensors and bioassays for various analytes. We have been interested in developing RNA-cleaving DNAzymes as bacterial sensors and these DNAzymes are engineered to perform three linked functions: recognition of a bacterial biomarker, RNA cleavage, and fluorescence generation. These fluorogenic DNAzymes emit fluorescence automatically in the presence of a bacterium of interest and can be used to set up a simple "mix-and-read" assay to detect this bacterium. In this article, we will discuss this DNAzyme system and present a proven strategy for isolating highly specific bacteria-responding DNAzyme probes from random-sequence DNA pools. We will also provide an in vitro selection protocol successfully used to derive RNA-cleaving fluorogenic DNAzyme probes that are capable of recognizing a targeted strain of Clostridium difficile.
157058140
An Ontology for Facilitating Discussion of Catalytic Strategies of RNA-Cleaving Enzymes.
A predictive understanding of the mechanisms of RNA cleavage is important for the design of emerging technology built from biological and synthetic molecules that have promise for new biochemical and medicinal applications. Over the past 15 years, RNA cleavage reactions involving 2'-O-transphosphorylation have been discussed using a simplified framework introduced by Breaker that consists of four fundamental catalytic strategies (designated α, β, γ, and δ) that contribute to rate enhancement. As more detailed mechanistic data emerge, there is need for the framework to evolve and keep pace. We develop an ontology for discussion of strategies of enzymes that catalyze RNA cleavage via 2'-O-transphosphorylation that stratifies Breaker's framework into primary (1°), secondary (2°), and tertiary (3°) contributions to enable more precise interpretation of mechanism in the context of structure and bonding. Further, we point out instances where atomic-level changes give rise to changes in more than one catalytic contribution, a phenomenon we refer to as "functional blurring". We hope that this ontology will help clarify our conversations and pave the path forward toward a consensus view of these fundamental and fascinating mechanisms. The insight gained will deepen our understanding of RNA cleavage reactions catalyzed by natural protein and RNA enzymes, as well as aid in the design of new engineered DNA and synthetic enzymes.
206645358
From the RNA world to the clinic
The study of RNA has continually emphasized the structural and functional versatility of RNA molecules. This versatility has inspired translational and clinical researchers to explore the utility of RNA-based therapeutic agents for a wide variety of medical applications. Several RNA therapeutics, with diverse modes of action, are being evaluated in large late-stage clinical trials, and many more are in early clinical development. Hundreds of patients are enrolled in large trials testing messenger RNAs to combat cancer, small interfering RNAs to treat renal and hepatic disorders, and aptamers to combat ocular and cardiovascular disease. Results from these studies are generating considerable interest among the biomedical community and the public and will be important for the future development of this emerging class of therapeutic agents.
90783038
Biomedical applications of RNA-based devices
Abstract Emergent RNA technologies employ sequence and structural information to perform a diversity of biological functions. Synthetic RNA molecules have been developed for a wide array of applications, including genetic regulation, environmental sensing, and diagnostics devices. Recent advances in chemical synthesis and computational design of RNA have enhanced our ability to program novel functions and expand upon current biomedical applications for therapeutics and diagnostics. In this review, we highlight recent advances in synthetic RNA devices that have been engineered for biomedical systems, while addressing the current limitations and challenges of translating these engineered functional RNAs to clinical applications.
12701095
Nucleic Acid Catalysis: Metals, Nucleobases, and Other Cofactors
The discovery of RNA-catalyzed phosphodiester bond cleavage by Cech and Altman in 1982–1983 shattered the paradigm of protein-dependent biological catalysis and opened up new horizons for RNA biology (reviewed in ref (1)). These discoveries ushered in a new era of high activity in RNA biochemistry and structural biology that, in conjunction with developments in genomics, established groundwork for the ensuing explosion in discoveries of other functional but noncoding RNAs. Since the initial discoveries, several classes of naturally occurring and artificially developed ribozymes have been defined. Ribozyme reactions catalyzed in nature include phosphoryl and aminoacyl transfer reactions (Figure ​(Figure1).1). RNA-catalyzed reactions obtained in vitro extend to carbon–carbon bond formation in the Diels–Alderase ribozyme. In vitro selection has also been employed to discover DNA-based enzymes that catalyze a number of different reactions, including RNA cleavage, as in Figure ​Figure11A. 
 
 
 
Figure 1 
 
Reactions catalyzed by naturally occurring ribozymes. (A) Intramolecular phosphoryl-transfer reaction catalyzed by the “nucleolytic” ribozymes including the hammerhead, hepatitis delta virus (HDV), hairpin, Neurospora VS, and GlmS RNAs. ...
32543042
How RNA acts as a nuclease: some mechanistic comparisons in the nucleolytic ribozymes.
Recent structural and mechanistic studies have shed considerable light on the catalytic mechanisms of nucleolytic ribozymes. The discovery of several new ribozymes in this class has now allowed comparisons to be made, and the beginnings of mechanistic groupings to emerge.
20888189
Structure-based mechanistic insights into catalysis by small self-cleaving ribozymes.
Small self-cleaving ribozymes are widely distributed in nature and are essential for rolling-circle-based replication of satellite and pathogenic RNAs. Earlier structure-function studies on the hammerhead, hairpin, glmS, hepatitis delta virus and Varkud satellite ribozymes have provided insights into their overall architecture, their catalytic active site organization, and the role of nearby nucleobases and hydrated divalent cations in facilitating general acid-base and electrostatic-mediated catalysis. This review focuses on recent structure-function research on active site alignments and catalytic mechanisms of the Rzb hammerhead ribozyme, as well as newly-identified pistol, twister and twister-sister ribozymes. In contrast to an agreed upon mechanistic understanding of self-cleavage by Rzb hammerhead and pistol ribozymes, there exists a divergence of views as to the cleavage site alignments and catalytic mechanisms adopted by twister and twister-sister ribozymes. One approach to resolving this conundrum would be to extend the structural studies from currently available pre-catalytic conformations to their transition state mimic vanadate counterparts for both ribozymes.
25962735
Directed evolution of nucleic acid enzymes.
Just as Darwinian evolution in nature has led to the development of many sophisticated enzymes, Darwinian evolution in vitro has proven to be a powerful approach for obtaining similar results in the laboratory. This review focuses on the development of nucleic acid enzymes starting from a population of random-sequence RNA or DNA molecules. In order to illustrate the principles and practice of in vitro evolution, two especially well-studied categories of catalytic nucleic acid are considered: RNA enzymes that catalyze the template-directed ligation of RNA and DNA enzymes that catalyze the cleavage of RNA. The former reaction, which involves attack of a 2'- or 3'-hydroxyl on the alpha-phosphate of a 5'-triphosphate, is more difficult. It requires a comparatively larger catalytic motif, containing more nucleotides than can be sampled exhaustively within a starting population of random-sequence RNAs. The latter reaction involves deprotonation of the 2'-hydroxyl adjacent to the cleavage site, resulting in cleaved products that bear a 2',3'-cyclic phosphate and 5'-hydroxyl. The difficulty of this reaction, and therefore the complexity of the corresponding DNA enzyme, depends on whether a catalytic cofactor, such as a divalent metal cation or small molecule, is present in the reaction mixture.
39111511
The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme
The RNA moieties of ribonuclease P purified from both E. coli (M1 RNA) and B. subtilis (P-RNA) can cleave tRNA precursor molecules in buffers containing either 60 mM Mg2+ or 10 mM Mg2+ plus 1 mM spermidine. The RNA acts as a true catalyst under these conditions whereas the protein moieties of the enzymes alone show no catalytic activity. However, in buffers containing 5-10 mM Mg2+ (in the absence of spermidine) both kinds of subunits are required for enzymatic activity, as shown previously. In the presence of low concentrations of Mg2+, in vitro, the RNA and protein subunits from one species can complement subunits from the other species in reconstitution experiments. When the precursor to E. coli 4.5S RNA is used as a substrate, only the enzyme complexes formed with M1 RNA from E. coli and the protein moieties from either bacterial species are active.
35082696
A general purpose RNA-cleaving DNA enzyme.
An in vitro selection procedure was used to develop a DNA enzyme that can be made to cleave almost any targeted RNA substrate under simulated physiological conditions. The enzyme is comprised of a catalytic domain of 15 deoxynucleotides, flanked by two substrate-recognition domains of seven to eight deoxynucleotides each. The RNA substrate is bound through Watson-Crick base pairing and is cleaved at a particular phosphodiester located between an unpaired purine and a paired pyrimidine residue. Despite its small size, the DNA enzyme has a catalytic efficiency (kcat/Km) of approximately 10(9) M-1.min-1 under multiple turnover conditions, exceeding that of any other known nucleic acid enzyme. Its activity is dependent on the presence of Mg2+ ion. By changing the sequence of the substrate-recognition domains, the DNA enzyme can be made to target different RNA substrates. In this study, for example, it was directed to cleave synthetic RNAs corresponding to the start codon region of HIV-1 gag/pol, env, vpr, tat, and nef mRNAs.
272539
In vitro selection, characterization, and application of deoxyribozymes that cleave RNA
Over the last decade, many catalytically active DNA molecules (deoxyribozymes; DNA enzymes) have been identified by in vitro selection from random-sequence DNA pools. This article focuses on deoxyribozymes that cleave RNA substrates. The first DNA enzyme was reported in 1994 and cleaves an RNA linkage. Since that time, many other RNA-cleaving deoxyribozymes have been identified. Most but not all of these deoxyribozymes require a divalent metal ion cofactor such as Mg2+ to catalyze attack by a specific RNA 2′-hydroxyl group on the adjacent phosphodiester linkage, forming a 2′,3′-cyclic phosphate and a 5′-hydroxyl group. Several deoxyribozymes that cleave RNA have utility for in vitro RNA biochemistry. Some DNA enzymes have been applied in vivo to degrade mRNAs, and others have been engineered into sensors. The practical impact of RNA-cleaving deoxyribozymes should continue to increase as additional applications are developed.
17784338
The structural diversity of artificial genetic polymers
Synthetic genetics is a subdiscipline of synthetic biology that aims to develop artificial genetic polymers (also referred to as xeno-nucleic acids or XNAs) that can replicate in vitro and eventually in model cellular organisms. This field of science combines organic chemistry with polymerase engineering to create alternative forms of DNA that can store genetic information and evolve in response to external stimuli. Practitioners of synthetic genetics postulate that XNA could be used to safeguard synthetic biology organisms by storing genetic information in orthogonal chromosomes. XNA polymers are also under active investigation as a source of nuclease resistant affinity reagents (aptamers) and catalysts (xenozymes) with practical applications in disease diagnosis and treatment. In this review, we provide a structural perspective on known antiparallel duplex structures in which at least one strand of the Watson–Crick duplex is composed entirely of XNA. Currently, only a handful of XNA structures have been archived in the Protein Data Bank as compared to the more than 100 000 structures that are now available. Given the growing interest in xenobiology projects, we chose to compare the structural features of XNA polymers and discuss their potential to access new regions of nucleic acid fold space.
67876924
Hachimoji DNA and RNA: A genetic system with eight building blocks
Expanding the genetic code DNA and RNA are naturally composed of four nucleotide bases that form hydrogen bonds in order to pair. Hoshika et al. added an additional four synthetic nucleotides to produce an eight-letter genetic code and generate so-called hachimoji DNA. Coupled with an engineered T7 RNA polymerase, this expanded DNA alphabet could be transcribed into RNA. Thus, new forms of DNA that add information density to genetic biopolymers can be generated that may be useful for future synthetic biological applications. Science, this issue p. 884 DNA that includes synthetic bases expands the genetic code from four to eight nucleotide letters We report DNA- and RNA-like systems built from eight nucleotide “letters” (hence the name “hachimoji”) that form four orthogonal pairs. These synthetic systems meet the structural requirements needed to support Darwinian evolution, 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 increase 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.
3440665
Evidence of a General Acid-Base Catalysis Mechanism in the 8-17 DNAzyme.
DNAzymes are catalytic DNA molecules that can perform a variety of reactions. Although advances have been made in obtaining DNAzymes via in vitro selection and many of them have been developed into sensors and imaging agents for metal ions, bacteria, and other molecules, the structural features responsible for these enzymatic reactions are still not well understood. Previous studies of the 8-17 DNAzyme have suggested conserved guanines close to the phosphodiester transfer site may play a role in the catalytic reaction. To identify the specific guanine and functional group of the guanine responsible for the reaction, we herein report the effects of replacing G1.1 and G14 (G; p Ka,N1 = 9.4) with analogues with a different p Ka at the N1 position, such as inosine (G14I; p Ka,N1 = 8.7), 2,6-diaminopurine (G14diAP; p Ka,N1 = 5.6), and 2-aminopurine (G14AP; p Ka,N1 = 3.8) on pH-dependent reaction rates. A comparison of the pH dependence of the reaction rates of these DNAzymes demonstrated that G14 in the bulge loop next to the cleavage site, is involved in proton transfer at the catalytic site. In contrast, we did not find any evidence of G1.1 being involved in acid-base catalysis. These results support general acid-base catalysis as a feasible strategy used in DNA catalysis, as in RNA and protein enzymes.
7806344
A lead-dependent DNAzyme with a two-step mechanism.
A detailed biochemical and mechanistic study of in vitro selected variants of 8-17 DNAzymes is presented. Even though the 8-17 DNAzyme motif has been obtained through in vitro selection under three different conditions involving 10 mM Mg(2+) (called 8-17), 0.5 mM Mg(2+)/50 mM histidine (called Mg5), or 100 microM Zn(2+) (called 17E), all variants are shown to be the most active with Pb(2+) (8-17: k(obs) approximately 0.5 min(-1); Mg5: k(obs) approximately 2 min(-1); 17E: k(obs) approximately 1 min(-1) with 200 microM Pb(2+) at pH 5.0). For the 17E variant of the 8-17 DNAzyme, the single-turnover rate constants followed the order of Pb(2+) >> Zn(2+) >> Mn(2+) approximately Co(2+) > Ni(2+) > Mg(2+) approximately Ca(2+) > Sr(2+) approximately Ba(2+). The catalytic rate is half-maximal at 13.5 microM Pb(2+), 0.97 mM Zn(2+), or 10.5 mM Mg(2+), suggesting that the metal-binding affinity of the DNAzymes is in the order of Pb(2+) > Zn(2+) > Mg(2+). The Pb(2+)-dependent activity increases linearly with pH and the slope of the plot of log k(obs) versus pH is approximately 1, suggesting a single deprotonation in the rate-limiting step of the reaction. Sequence variations of the DNAzyme confirm the importance of the G*T wobble pair, the two loops and the intervening stem in maintaining the active conformation of the system. While Mg(2+) and Zn(2+) catalyze only a transesterification reaction with formation of a product containing a 2',3'-cyclic phosphate, Pb(2+) catalyzes a transesterification reaction followed by hydrolysis of the 2',3'-cyclic phosphate. Although this two-step mechanism has shown to be operative in protein ribonucleases and in the leadzyme RNAzyme, it is now demonstrated for the first time that this DNAzyme may also use the same mechanism. Therefore, the two-step mechanism is observed in metalloenzymes of all classes, and this 8-17 DNAzyme provides a simple, stable, and cost-effective model system for understanding the structure of Pb(2+)-binding sites and their roles in the two-step mechanism.
205303370
Dissecting metal ion-dependent folding and catalysis of a single DNAzyme.
Protein metalloenzymes use various modes for functions for which metal-dependent global conformational change is required in some cases but not in others. In contrast, most ribozymes require a global folding that almost always precedes enzyme reactions. Herein we studied metal-dependent folding and cleavage activity of the 8-17 DNAzyme using single-molecule fluorescence resonance energy transfer. Addition of Zn2+ and Mg2+ induced folding of the DNAzyme into a more compact structure followed by a cleavage reaction, which suggests that the DNAzyme may require metal-dependent global folding for activation. In the presence of Pb2+, however, the cleavage reaction occurred without a precedent folding step, which suggests that the DNAzyme may be prearranged to accept Pb2+ for the activity. Neither ligation reaction of the cleaved substrates nor dynamic changes between folded and unfolded states was observed. These features may contribute to the unusually fast Pb2+-dependent reaction of the DNAzyme. These results suggest that DNAzymes can use all modes of activation that metalloproteins use.
6573959
Activity, folding and Z-DNA formation of the 8-17 DNAzyme in the presence of monovalent ions.
The effect of monovalent ions on both the reactivity and global folding of the 8-17 DNAzyme is investigated, and the results are compared with those of the hammerhead ribozyme, which has similar size and secondary structure. In contrast to the hammerhead ribozyme, the 8-17 DNAzyme activity is not detectable in the presence of 4 M K(+), Rb(+), or Cs(+) or in the presence of 80 mM, [Co(NH(3))(6)](3+). Only 4 M Li(+), NH(4)(+) and, to a lesser extent, Na(+) conferred detectable activity. The observed rate constants (k(obs) approximately 10(-3) min(-1) for Li(+) and NH(4)(+)) are approximately 1000-fold lower than that in the presence of 10 mM Mg(2+), and approximately 200,000-fold slower than that in the presence of 100 microM Pb(2+). Since the hammerhead ribozyme displays monovalent ion-dependent activity that is often within approximately 10-fold of divalent metal ion-dependent activity, these results suggest that the 8-17 DNAzyme, obtained by in vitro selections, has evolved to have a more stringent divalent metal ion requirement for high activity as compared to the naturally occurring ribozymes, making the 8-17 DNAzyme an excellent choice as a Pb(2+) sensor with high selectivity. In contrast to the activity data, folding was observed in the presence of all the monovalent ions investigated, although those monovalent ions that do not support DNAzyme activity have weaker binding affinity (K(d) approximately 0.35 M for Rb(+) and Cs(+)), while those that confer DNAzyme activity possess stronger affinity (K(d) approximately 0.22 M for Li(+), Na(+) and NH(4)(+)). In addition, a correlation between metal ion charge density, binding affinity and enzyme activity was found among mono- and divalent metal ions except Pb(2+); higher charge density resulted in stronger affinity and higher activity, suggesting that the observed folding and activity is at least partially due to electrostatic interactions between ions and the DNAzyme. Finally, circular dichroism (CD) study has revealed Z-DNA formation with the monovalent metal ions, Zn(2+) and Mg(2+); the K(d) values obtained using CD were in the same range as those obtained from folding studies using FRET. However, Z-DNA formation was not observed with Pb(2+). These results indicate that Pb(2+)-dependent function follows a different mechanism from the monovalent metal ions and other divalent metal ions; in the presence of latter metal ions, metal-ion dependent folding and structural changes, including formation of Z-DNA, play an important role in the catalytic function of the 8-17 DNAzyme.
46000528
Ribozyme speed limits.
The speed at which RNA molecules decompose is a critical determinant of many biological processes, including those directly involved in the storage and expression of genetic information. One mechanism for RNA cleavage involves internal phosphoester transfer, wherein the 2'-oxygen atom carries out an SN2-like nucleophilic attack on the adjacent phosphorus center (transesterification). In this article, we discuss fundamental principles of RNA transesterification and define a conceptual framework that can be used to assess the catalytic power of enzymes that cleave RNA. We deduce that certain ribozymes and deoxyribozymes, like their protein enzyme counterparts, can bring about enormous rate enhancements.
28707281
The expanding view of RNA and DNA function.
RNA and DNA are simple linear polymers consisting of only four major types of subunits, and yet these molecules carry out a remarkable diversity of functions in cells and in the laboratory. Each newly discovered function of natural or engineered nucleic acids enforces the view that prior assessments of nucleic acid function were far too narrow and that many more exciting findings are yet to come. This Perspective highlights just a few of the numerous discoveries over the past 20 years pertaining to nucleic acid function, focusing on those that have been of particular interest to chemical biologists. History suggests that there will continue to be many opportunities to engage chemical biologists in the discovery, creation, and manipulation of nucleic acid function in the years to come.
3484863
Catalytic DNA: Scope, Applications, and Biochemistry of Deoxyribozymes.
The discovery of natural RNA enzymes (ribozymes) prompted the pursuit of artificial DNA enzymes (deoxyribozymes) by in vitro selection methods. A key motivation is the conceptual and practical advantages of DNA relative to proteins and RNA. Early studies focused on RNA-cleaving deoxyribozymes, and more recent experiments have expanded the breadth of catalytic DNA to many other reactions. Including modified nucleotides has the potential to widen the scope of DNA enzymes even further. Practical applications of deoxyribozymes include their use as sensors for metal ions and small molecules. Structural studies of deoxyribozymes are only now beginning; mechanistic experiments will surely follow. Following the first report 21 years ago, the field of deoxyribozymes has promise for both fundamental and applied advances in chemistry, biology, and other disciplines.
52184835
GPU-Accelerated Molecular Dynamics and Free Energy Methods in Amber18: Performance Enhancements and New Features
We report progress in graphics processing unit (GPU)-accelerated molecular dynamics and free energy methods in Amber18. Of particular interest is the development of alchemical free energy algorithms, including free energy perturbation and thermodynamic integration methods with support for nonlinear soft-core potential and parameter interpolation transformation pathways. These methods can be used in conjunction with enhanced sampling techniques such as replica exchange, constant-pH molecular dynamics, and new 12-6-4 potentials for metal ions. Additional performance enhancements have been made that enable appreciable speed-up on GPUs relative to the previous software release.
11333546
Determination of Alkali and Halide Monovalent Ion Parameters for Use in Explicitly Solvated Biomolecular Simulations
Alkali (Li+, Na+, K+, Rb+, and Cs+) and halide (F−, Cl−, Br−, and I−) ions play an important role in many biological phenomena, roles that range from stabilization of biomolecular structure, to influence on biomolecular dynamics, to key physiological influence on homeostasis and signaling. To properly model ionic interaction and stability in atomistic simulations of biomolecular structure, dynamics, folding, catalysis, and function, an accurate model or representation of the monovalent ions is critically necessary. A good model needs to simultaneously reproduce many properties of ions, including their structure, dynamics, solvation, and moreover both the interactions of these ions with each other in the crystal and in solution and the interactions of ions with other molecules. At present, the best force fields for biomolecules employ a simple additive, nonpolarizable, and pairwise potential for atomic interaction. In this work, we describe our efforts to build better models of the monovalent ions within the pairwise Coulombic and 6-12 Lennard-Jones framework, where the models are tuned to balance crystal and solution properties in Ewald simulations with specific choices of well-known water models. Although it has been clearly demonstrated that truly accurate treatments of ions will require inclusion of nonadditivity and polarizability (particularly with the anions) and ultimately even a quantum mechanical treatment, our goal was to simply push the limits of the additive treatments to see if a balanced model could be created. The applied methodology is general and can be extended to other ions and to polarizable force-field models. Our starting point centered on observations from long simulations of biomolecules in salt solution with the AMBER force fields where salt crystals formed well below their solubility limit. The likely cause of the artifact in the AMBER parameters relates to the naive mixing of the Smith and Dang chloride parameters with AMBER-adapted Åqvist cation parameters. To provide a more appropriate balance, we reoptimized the parameters of the Lennard-Jones potential for the ions and specific choices of water models. To validate and optimize the parameters, we calculated hydration free energies of the solvated ions and also lattice energies (LE) and lattice constants (LC) of alkali halide salt crystals. This is the first effort that systematically scans across the Lennard-Jones space (well depth and radius) while balancing ion properties like LE and LC across all pair combinations of the alkali ions and halide ions. The optimization across the entire monovalent series avoids systematic deviations. The ion parameters developed, optimized, and characterized were targeted for use with some of the most commonly used rigid and nonpolarizable water models, specifically TIP3P, TIP4PEW, and SPC/E. In addition to well reproducing the solution and crystal properties, the new ion parameters well reproduce binding energies of the ions to water and the radii of the first hydration shells.
25563043
Rational Design of Particle Mesh Ewald Compatible Lennard-Jones Parameters for +2 Metal Cations in Explicit Solvent.
Metal ions play significant roles in biological systems. Accurate molecular dynamics (MD) simulations on these systems require a validated set of parameters. Although there are more detailed ways to model metal ions, the nonbonded model, which employs a 12-6 Lennard-Jones (LJ) term plus an electrostatic potential is still widely used in MD simulations today due to its simple form. However, LJ parameters have limited transferability due to different combining rules, various water models and diverse simulation methods. Recently, simulations employing a Particle Mesh Ewald (PME) treatment for long-range electrostatics have become more and more popular owing to their speed and accuracy. In the present work we have systematically designed LJ parameters for 24 +2 metal (M(II)) cations to reproduce different experimental properties appropriate for the Lorentz-Berthelot combining rules and PME simulations. We began by testing the transferability of currently available M(II) ion LJ parameters. The results showed that there are differences between simulations employing Ewald summation with other simulation methods and that it was necessary to design new parameters specific for PME based simulations. Employing the thermodynamic integration (TI) method and performing periodic boundary MD simulations employing PME, allowed for the systematic investigation of the LJ parameter space. Hydration free energies (HFEs), the ion-oxygen distance in the first solvation shell (IOD) and coordination numbers (CNs) were obtained for various combinations of the parameters of the LJ potential for four widely used water models (TIP3P, SPC/E, TIP4P and TIP4PEW). Results showed that the three simulated properties were highly correlated. Meanwhile, M(II) ions with the same parameters in different water models produce remarkably different HFEs but similar structural properties. It is difficult to reproduce various experimental values simultaneously because the nonbonded model underestimates the interaction between the metal ions and water molecules at short range. Moreover, the extent of underestimation increases successively for the TIP3P, SPC/E, TIP4PEW and TIP4P water models. Nonetheless, we fitted a curve to describe the relationship between ε (the well depth) and radius (Rmin/2) from experimental data on noble gases to facilitate the generation of the best possible compromise models. Hence, by targeting different experimental values, we developed three sets of parameters for M(II) cations for three different water models (TIP3P, SPC/E and TIP4PEW). These parameters we feel represent the best possible compromise that can be achieved using the nonbonded model for the ions in combination with simple water models. From a computational uncertainty analysis we estimate that the uncertainty in our computed HFEs is on the order of ±1kcal/mol. Further improvements will require more advanced non-bonded models likely with inclusion of polarization.
55356282
Particle mesh Ewald: An N⋅log(N) method for Ewald sums in large systems
An N⋅log(N) method for evaluating electrostatic energies and forces of large periodic systems is presented. The method is based on interpolation of the reciprocal space Ewald sums and evaluation of the resulting convolutions using fast Fourier transforms. Timings and accuracies are presented for three large crystalline ionic systems.
1382401
A smooth particle mesh Ewald method
The previously developed particle mesh Ewald method is reformulated in terms of efficient B‐spline interpolation of the structure factors. This reformulation allows a natural extension of the method to potentials of the form 1/rp with p≥1. Furthermore, efficient calculation of the virial tensor follows. Use of B‐splines in place of Lagrange interpolation leads to analytic gradients as well as a significant improvement in the accuracy. We demonstrate that arbitrary accuracy can be achieved, independent of system size N, at a cost that scales as N log(N). For biomolecular systems with many thousands of atoms this method permits the use of Ewald summation at a computational cost comparable to that of a simple truncation method of 10 A or less.
207162364
Ribozyme Catalysis with a Twist: Active State of the Twister Ribozyme in Solution Predicted from Molecular Simulation.
We present results from molecular dynamics simulations and free energy calculations of the twister ribozyme at different stages along the reaction path to gain insight into its mechanism. The results, together with recent biochemical experiments, provide support for a mechanism involving general-acid catalysis by a conserved adenine residue in the active site. Although adenine has been previously implicated as a general acid acting through the N1 position in other ribozymes such as the hairpin and VS ribozymes, in the twister ribozyme there may be a twist. Biochemical experiments suggest that general acid catalysis may occur through the N3 position, which has never before been implicated in this role; however, currently, there is a lack of a detailed structural model for the active state of the twister ribozyme in solution that is consistent with these and other experiments. Simulations in a crystalline environment reported here are consistent with X-ray crystallographic data, and suggest that crystal packing contacts trap the RNA in an inactive conformation with U-1 in an extruded state that is incompatible with an in-line attack to the scissile phosphate. Simulations in solution, on the other hand, reveal this region to be dynamic and able to adopt a conformation where U-1 is stacked with G33. In this state, the nucleophile is in line with the scissile phosphate, and the N1 position of G33 and N3 position of A1 are poised to act as a general base and acid, respectively, as supported by mutational experiments. Free energy calculations further predict the electrostatic environment causes a shift of the microscopic pKa at the N3 position of A1 toward neutrality by approximately 5 pKa units. These results offer a unified interpretation of a broad range of currently available experimental data that points to a novel mode of general acid catalysis through the N3 position of an adenine nucleobase, thus expanding the repertoire of known mechanistic strategies employed by small nucleolytic ribozymes.
45719902
Convergence and reproducibility in molecular dynamics simulations of the DNA duplex d(GCACGAACGAACGAACGC).
BACKGROUND
The structure and dynamics of DNA are critically related to its function. Molecular dynamics simulations augment experiment by providing detailed information about the atomic motions. However, to date the simulations have not been long enough for convergence of the dynamics and structural properties of DNA.


METHODS
Molecular dynamics simulations performed with AMBER using the ff99SB force field with the parmbsc0 modifications, including ensembles of independent simulations, were compared to long timescale molecular dynamics performed with the specialized Anton MD engine on the B-DNA structure d(GCACGAACGAACGAACGC). To assess convergence, the decay of the average RMSD values over longer and longer time intervals was evaluated in addition to assessing convergence of the dynamics via the Kullback-Leibler divergence of principal component projection histograms.


RESULTS
These molecular dynamics simulations-including one of the longest simulations of DNA published to date at ~44μs-surprisingly suggest that the structure and dynamics of the DNA helix, neglecting the terminal base pairs, are essentially fully converged on the ~1-5μs timescale.


CONCLUSIONS
We can now reproducibly converge the structure and dynamics of B-DNA helices, omitting the terminal base pairs, on the μs time scale with both the AMBER and CHARMM C36 nucleic acid force fields. Results from independent ensembles of simulations starting from different initial conditions, when aggregated, match the results from long timescale simulations on the specialized Anton MD engine.


GENERAL SIGNIFICANCE
With access to large-scale GPU resources or the specialized MD engine "Anton" it is possible for a variety of molecular systems to reproducibly and reliably converge the conformational ensemble of sampled structures. This article is part of a Special Issue entitled: Recent developments of molecular dynamics.
53024251
Avoiding False Positive Conclusions in Molecular Simulation: The Importance of Replicas.
Molecular simulations are a computational technique used to investigate the dynamics of proteins and other molecules. The free energy landscape of these simulations is often rugged, and minor differences in the initial velocities, floating-point precision, or underlying hardware can cause identical simulations (replicas) to take different paths in the landscape. In this study we investigated the magnitude of these effects based on 310 000 ns of simulation time. We performed 100 identically parametrized replicas of 3000 ns each for a small 10 amino acid system as well as 100 identically parametrized replicas of 100 ns each for an 827 residue T-cell receptor/MHC system. Comparing randomly chosen subgroups within these replica sets, we estimated the reproducibility and reliability that can be achieved by a given number of replicas at a given simulation time. These results demonstrate that conclusions drawn from single simulations are often not reproducible and that conclusions drawn from multiple shorter replicas are more reliable than those from a single longer simulation. The actual number of replicas needed will always depend on the question asked and the level of reliability sought. On the basis of our data, it appears that a good rule of thumb is to perform a minimum of five to 10 replicas.
29300500
PTRAJ and CPPTRAJ: Software for Processing and Analysis of Molecular Dynamics Trajectory Data.
We describe PTRAJ and its successor CPPTRAJ, two complementary, portable, and freely available computer programs for the analysis and processing of time series of three-dimensional atomic positions (i.e., coordinate trajectories) and the data therein derived. Common tools include the ability to manipulate the data to convert among trajectory formats, process groups of trajectories generated with ensemble methods (e.g., replica exchange molecular dynamics), image with periodic boundary conditions, create average structures, strip subsets of the system, and perform calculations such as RMS fitting, measuring distances, B-factors, radii of gyration, radial distribution functions, and time correlations, among other actions and analyses. Both the PTRAJ and CPPTRAJ programs and source code are freely available under the GNU General Public License version 3 and are currently distributed within the AmberTools 12 suite of support programs that make up part of the Amber package of computer programs (see http://ambermd.org ). This overview describes the general design, features, and history of these two programs, as well as algorithmic improvements and new features available in CPPTRAJ.
28616617
Mechanistic insights into RNA transphosphorylation from kinetic isotope effects and linear free energy relationships of model reactions.
Phosphoryl transfer reactions are ubiquitous in biology and the understanding of the mechanisms whereby these reactions are catalyzed by protein and RNA enzymes is central to reveal design principles for new therapeutics. Two of the most powerful experimental probes of chemical mechanism involve the analysis of linear free energy relations (LFERs) and the measurement of kinetic isotope effects (KIEs). These experimental data report directly on differences in bonding between the ground state and the rate-controlling transition state, which is the most critical point along the reaction free energy pathway. However, interpretation of LFER and KIE data in terms of transition-state structure and bonding optimally requires the use of theoretical models. In this work, we apply density-functional calculations to determine KIEs for a series of phosphoryl transfer reactions of direct relevance to the 2'-O-transphosphorylation that leads to cleavage of the phosphodiester backbone of RNA. We first examine a well-studied series of phosphate and phosphorothioate mono-, di- and triesters that are useful as mechanistic probes and for which KIEs have been measured. Close agreement is demonstrated between the calculated and measured KIEs, establishing the reliability of our quantum model calculations. Next, we examine a series of RNA transesterification model reactions with a wide range of leaving groups in order to provide a direct connection between observed Brønsted coefficients and KIEs with the structure and bonding in the transition state. These relations can be used for prediction or to aid in the interpretation of experimental data for similar non-enzymatic and enzymatic reactions. Finally, we apply these relations to RNA phosphoryl transfer catalyzed by ribonuclease A, and demonstrate the reaction coordinate-KIE correlation is reasonably preserved. A prediction of the secondary deuterium KIE in this reaction is also provided. These results demonstrate the utility of building up knowledge of mechanism through the systematic study of model systems to provide insight into more complex biological systems such as phosphoryl transfer enzymes and ribozymes.
182091216
Cleaning Up Mechanistic Debris Generated by Twister Ribozymes Using Computational RNA Enzymology.
The catalytic properties of RNA have been a subject of fascination and intense research since their discovery over 30 years ago. Very recently, several classes of nucleolytic ribozymes have emerged and been characterized structurally. Among these, the twister ribozyme has been center-stage, and a topic of debate about its architecture and mechanism owing to conflicting interpretations of different crystal structures, and in some cases conflicting interpretations of the same functional data. In the present work, we attempt to clean up the mechanistic "debris" generated by twister ribozymes using a comprehensive computational RNA enzymology approach aimed to provide a unified interpretation of existing structural and functional data. Simulations in the crystalline environment and in solution provide insight into the origins of observed differences in crystal structures, and coalesce on a common active site architecture, and dynamical ensemble in solution. We use GPU-accelerated free energy methods with enhanced sampling to ascertain microscopic nucleobase pK a values of the implicated general acid and base, from which predicted activity-pH profiles can be compared directly with experiments. Next, ab initio quantum mechanical/molecular mechanical (QM/MM) simulations with full dynamic solvation under periodic boundary conditions are used to determine mechanistic pathways through multi-dimensional free energy landscapes for the reaction. We then characterize the rate-controlling transition state, and make predictions about kinetic isotope effects and linear free energy relations. Computational mutagenesis is performed to explain the origin of rate effects caused by chemical modifications and make experimentally testable predictions. Finally, we provide evidence that helps to resolve conflicting issues related to the role of metal ions in catalysis. Throughout each stage, we highlight how a conserved L-platform structural motif, to- gether with a key L-anchor residue, forms the characteristic active site scaffold enabling each of the catalytic strategies to come together not only for the twister ribozyme, but the majority of the known small nucleolytic ribozyme classes.
2157524
Geometric nomenclature and classification of RNA base pairs.
Non-Watson-Crick base pairs mediate specific interactions responsible for RNA-RNA self-assembly and RNA-protein recognition. An unambiguous and descriptive nomenclature with well-defined and nonoverlapping parameters is needed to communicate concisely structural information about RNA base pairs. The definitions should reflect underlying molecular structures and interactions and, thus, facilitate automated annotation, classification, and comparison of new RNA structures. We propose a classification based on the observation that the planar edge-to-edge, hydrogen-bonding interactions between RNA bases involve one of three distinct edges: the Watson-Crick edge, the Hoogsteen edge, and the Sugar edge (which includes the 2'-OH and which has also been referred to as the Shallow-groove edge). Bases can interact in either of two orientations with respect to the glycosidic bonds, cis or trans relative to the hydrogen bonds. This gives rise to 12 basic geometric types with at least two H bonds connecting the bases. For each geometric type, the relative orientations of the strands can be easily deduced. High-resolution examples of 11 of the 12 geometries are presently available. Bifurcated pairs, in which a single exocyclic carbonyl or amino group of one base directly contacts the edge of a second base, and water-inserted pairs, in which single functional groups on each base interact directly, are intermediate between two of the standard geometries. The nomenclature facilitates the recognition of isosteric relationships among base pairs within each geometry, and thus facilitates the recognition of recurrent three-dimensional motifs from comparison of homologous sequences. Graphical conventions are proposed for displaying non-Watson-Crick interactions on a secondary structure diagram. The utility of the classification in homology modeling of RNA tertiary motifs is illustrated.
35343204
The non-Watson-Crick base pairs and their associated isostericity matrices.
RNA molecules exhibit complex structures in which a large fraction of the bases engage in non-Watson-Crick base pairing, forming motifs that mediate long-range RNA-RNA interactions and create binding sites for proteins and small molecule ligands. The rapidly growing number of three-dimensional RNA structures at atomic resolution requires that databases contain the annotation of such base pairs. An unambiguous and descriptive nomenclature was proposed recently in which RNA base pairs were classified by the base edges participating in the interaction (Watson-Crick, Hoogsteen/CH or sugar edge) and the orientation of the glycosidic bonds relative to the hydrogen bonds (cis or trans). Twelve basic geometric families were identified and all 12 have been observed in crystal structures. For each base pairing family, we present here the 4 x 4 'isostericity matrices' summarizing the geometric relationships between the 16 pairwise combinations of the four standard bases, A, C, G and U. Whenever available, a representative example of each observed base pair from X-ray crystal structures (3.0 A resolution or better) is provided or, otherwise, theoretically plausible models. This format makes apparent the recurrent geometric patterns that are observed and helps identify isosteric pairs that co-vary or interchange in sequences of homologous molecules while maintaining conserved three-dimensional motifs.
90373638
Elucidation of Catalytic Strategies of Small Nucleolytic Ribozymes From Comparative Analysis of Active Sites.
A number of small, self-cleaving ribozyme classes have been identified including the hammerhead, hairpin, hepatitis delta virus (HDV), Varkud satellite (VS), glmS, twister, hatchet, pistol, and twister sister ribozymes. Within the active sites of these ribozymes, myriad functional groups contribute to catalysis. There has been extensive structure-function analysis of individual ribozymes, but the extent to which catalytic devices are shared across different ribozyme classes is unclear. As such, emergent catalytic principles for ribozymes may await discovery. Identification of conserved catalytic devices can deepen our understanding of RNA catalysis specifically and of enzymic catalysis generally. To probe similarities and differences amongst ribozyme classes, active sites from more than 80 high-resolution crystal structures of self-cleaving ribozymes were compared computationally. We identify commonalities amongst ribozyme classes pertaining to four classic catalytic devices: deprotonation of the 2'OH nucleophile (γ), neutralization of the non-bridging oxygens of the scissile phosphate (β), neutralization of the O5' leaving group (δ), and in-line nucleophilic attack (α). In addition, we uncover conservation of two catalytic devices, each of which centers on the activation of the 2'OH nucleophile by a guanine: one to acidify the 2'OH by hydrogen bond donation to it (γ') and one to acidify the 2'OH by releasing it from non-productive interactions by competitive hydrogen bonding (γ''). Our findings reveal that the amidine functionalities of G, A, and C are especially important for these strategies, and help explain absence of U at ribozyme active sites. The identified γ' and γ'' catalytic strategies help unify the catalytic strategies shared amongst catalytic RNAs and may be important for large ribozymes, as well as protein enzymes that act on nucleic acids.
11570769
Threshold occupancy and specific cation binding modes in the hammerhead ribozyme active site are required for active conformation.
The relationship between formation of active in-line attack conformations and monovalent (Na(+)) and divalent (Mg(2+)) metal ion binding in hammerhead ribozyme (HHR) has been explored with molecular dynamics simulations. To stabilize repulsions between negatively charged groups, different requirements of the threshold occupancy of metal ions were observed in the reactant and activated precursor states both in the presence and in the absence of a Mg(2+) in the active site. Specific bridging coordination patterns of the ions are correlated with the formation of active in-line attack conformations and can be accommodated in both cases. Furthermore, simulation results suggest that the HHR folds to form an electronegative recruiting pocket that attracts high local concentrations of positive charge. The present simulations help to reconcile experiments that probe the metal ion sensitivity of HHR catalysis and support the supposition that Mg(2+), in addition to stabilizing active conformations, plays a specific chemical role in catalysis.
25533952
Bridging the gap between theory and experiment to derive a detailed understanding of hammerhead ribozyme catalysis.
Herein we summarize our progress toward the understanding of hammerhead ribozyme (HHR) catalysis through a multiscale simulation strategy. Simulation results collectively paint a picture of HHR catalysis: HHR first folds to form an electronegative active site pocket to recruit a threshold occupation of cationic charges, either a Mg(2+) ion or multiple monovalent cations. Catalytically active conformations that have good in-line fitness are supported by specific metal ion coordination patterns that involve either a bridging Mg(2+) ion or multiple Na(+) ions, one of which is also in a bridging coordination pattern. In the case of a single Mg(2+) ion bound in the active site, the Mg(2+) ion undergoes a migration that is coupled with deprotonation of the nucleophile (C17:O2'). As the reaction proceeds, the Mg(2+) ion stabilizes the accumulating charge of the leaving group and significantly increases the general acid ability of G8:O2'. Further computational mutagenesis simulations suggest that the disruptions due to mutations may severely impact HHR catalysis at different stages of the reaction. Catalytic mechanisms supported by the simulation results are consistent with available structural and biochemical experiments, and together they advance our understanding of HHR catalysis.
34668443
Assessment of metal-assisted nucleophile activation in the hepatitis delta virus ribozyme from molecular simulation and 3D-RISM.
The hepatitis delta virus ribozyme is an efficient catalyst of RNA 2'-O-transphosphorylation and has emerged as a key experimental system for identifying and characterizing fundamental features of RNA catalysis. Recent structural and biochemical data have led to a proposed mechanistic model whereby an active site Mg(2+) ion facilitates deprotonation of the O2' nucleophile, and a protonated cytosine residue (C75) acts as an acid to donate a proton to the O5' leaving group as noted in a previous study. This model assumes that the active site Mg(2+) ion forms an inner-sphere coordination with the O2' nucleophile and a nonbridging oxygen of the scissile phosphate. These contacts, however, are not fully resolved in the crystal structure, and biochemical data are not able to unambiguously exclude other mechanistic models. In order to explore the feasibility of this model, we exhaustively mapped the free energy surfaces with different active site ion occupancies via quantum mechanical/molecular mechanical (QM/MM) simulations. We further incorporate a three-dimensional reference interaction site model for the solvated ion atmosphere that allows these calculations to consider not only the rate associated with the chemical steps, but also the probability of observing the system in the presumed active state with the Mg(2+) ion bound. The QM/MM results predict that a pathway involving metal-assisted nucleophile activation is feasible based on the rate-controlling transition state barrier departing from the presumed metal-bound active state. However, QM/MM results for a similar pathway in the absence of Mg(2+) are not consistent with experimental data, suggesting that a structural model in which the crystallographically determined Mg(2+) is simply replaced with Na(+) is likely incorrect. It should be emphasized, however, that these results hinge upon the assumption of the validity of the presumed Mg(2+)-bound starting state, which has not yet been definitively verified experimentally, nor explored in depth computationally. Thus, further experimental and theoretical study is needed such that a consensus view of the catalytic mechanism emerges.
27169717
A Two-Metal-Ion-Mediated Conformational Switching Pathway for HDV Ribozyme Activation.
RNA enzymes serve as a potentially powerful platform from which to design catalysts and engineer new biotechnology. A fundamental understanding of these systems provides insight to guide design. The hepatitis delta virus ribozyme (HDVr) is a small, self-cleaving RNA motif widely distributed in nature, that has served as a paradigm for understanding basic principles of RNA catalysis. Nevertheless, questions remain regarding the precise roles of divalent metal ions and key nucleotides in catalysis. In an effort to establish a reaction mechanism model consistent with available experimental data, we utilize molecular dynamics simulations to explore different conformations and metal ion binding modes along the HDVr reaction path. Building upon recent crystallographic data, our results provide a dynamic model of the HDVr reaction mechanism involving a conformational switch between multiple non-canonical G25:U20 base pair conformations in the active site. These local nucleobase dynamics play an important role in catalysis by modulating the metal binding environments of two Mg2+ ions that support catalysis at different steps of the reaction pathway. The first ion plays a structural role by inducing a base pair flip necessary to obtain the catalytic fold in which C75 moves towards to the scissile phosphate in the active site. Ejection of this ion then permits a second ion to bind elsewhere in the active site and facilitate nucleophile activation. The simulations collectively describe a mechanistic scenario that is consistent with currently available experimental data from crystallography, phosphorothioate substitutions, and chemical probing studies. Avenues for further experimental verification are suggested.
58666009
Predicting Site-Binding Modes of Ions and Water to Nucleic Acids Using Molecular Solvation Theory.
Site binding of ions and water shapes nucleic acids folding, dynamics, and biological function, complementing the more diffuse, nonspecific "territorial" ion binding. Unlike territorial binding, prediction of site-specific binding to nucleic acids remains an unsolved challenge in computational biophysics. This work presents a new toolset based on the 3D-RISM molecular solvation theory and topological analysis that predicts cation and water site binding to nucleic acids. 3D-RISM is shown to accurately capture alkali cations and water binding to the central channel, transversal loops, and grooves of the Oxytricha nova's telomeres' G-quadruplex ( Oxy-GQ), in agreement with high-resolution crystallographic data. To improve the computed cation occupancy along the Oxy-GQ central channel, it was necessary to refine and validate new cation-oxygen parameters using structural and thermodynamic data available for crown ethers and ion channels. This single set of parameters that describes both localized and delocalized binding to various biological systems is used to gain insight into cation occupancy along the Oxy-GQ channel under various salt conditions. The paper concludes with prospects for extending the method to predict divalent cation binding to nucleic acids. This work advances the forefront of theoretical methods able to provide predictive insight into ion atmosphere effects on nucleic acids function.
8827124
Rational evolution of Cd2+-specific DNAzymes with phosphorothioate modified cleavage junction and Cd2+ sensing
In vitro selection of RNA-cleaving DNAzymes is a powerful method for isolating metal-specific DNA. A few successful examples are known, but it is still difficult to target some thiophilic metals such as Cd2+ due to limited functional groups in DNA. While using modified bases expands the chemical functionality of DNA, a single phosphorothioate modification might boost its affinity for thiophilic metals without complicating the selection process or using bases that are not commercially available. In this work, the first such in vitro selection for Cd2+ is reported. After using a blocking DNA and negative selections to rationally direct the library outcome, a highly specific DNAzyme with only 12 nucleotides in the catalytic loop is isolated. This DNAzyme has a cleavage rate of 0.12 min−1 with 10 μM Cd2+ at pH 6.0. The Rp form of the substrate is cleaved ∼100-fold faster than the Sp form. The DNAzyme is most active with Cd2+ and its selectivity against Zn2+ is over 100 000-fold. Its application in detecting Cd2+ is also demonstrated. The idea of introducing single modifications in the fixed region expands the scope of DNA/metal interactions with minimal perturbation of DNA structure and property.
11011159
A stability concept for metal ion coordination to single-stranded nucleic acids and affinities of individual sites.
The three-dimensional architecture and function of nucleic acids strongly depend on the presence of metal ions, among other factors. Given the negative charge of the phosphate-sugar backbone, positively charged species, mostly metal ions, are necessary for compensation. However, these ions also allow and induce folding of complicated RNA structures. Furthermore, metal ions bind to specific sites, stabilizing local motifs and positioning themselves correctly to aid (or even enable) a catalytic mechanism, as, for example, in ribozymes. Many nucleic acids thereby exhibit large differences in folding and activity depending not only on the concentration but also on the kind of metal ion involved. As a consequence, understanding the role of metal ions in nucleic acids requires knowing not only the exact positioning and coordination sphere of each specifically bound metal ion but also its intrinsic site affinity. However, the quantification of metal ion affinities toward certain sites in a single-stranded (though folded) nucleic acid is a demanding task, and few experimental data exist. In this Account, we present a new tool for estimating the binding affinity of a given metal ion, based on its ligating sites within the nucleic acid. To this end, we have summarized the available affinity constants of Mg(2+), Ca(2+), Mn(2+), Cu(2+), Zn(2+), Cd(2+), and Pb(2+) for binding to nucleobase residues, as well as to mono- and dinucleotides. We have also estimated for these ions the stability constants for coordinating the phosphodiester bridge. In this way, stability increments for each ligand site are obtained, and a clear selectivity of the ligating atoms, as well as their discrimination by different metal ions, can thus be recognized. On the basis of these data, we propose a concept that allows one to estimate the intrinsic stabilities of nucleic acid-binding pockets for these metal ions. For example, the presence of a phosphate group has a much larger influence on the overall affinity of Mg(2+), Ca(2+), or Mn(2+) compared with, for example, that of Cd(2+) or Zn(2+). In the case of Cd(2+) and Zn(2+), the guanine N7 position is the strongest intrinsic binding site. By adding up the individual increments like building blocks, one derives an estimate not only for the overall stability of a given coordination sphere but also for the most stable complex if an excess of ligating atoms is available in a binding pocket saturating the coordination sphere of the metal ion. Hence, this empirical concept of adding up known intrinsic stabilities, like building blocks, to an estimated overall stability will help in understanding the accelerating or inhibiting effects of different metal ions in ribozymes and DNAzymes.
207056675
Computational mutagenesis studies of hammerhead ribozyme catalysis.
Computational studies of the mutational effects at the C3, G8, and G5 positions of the hammerhead ribozyme (HHR) are reported, based on a series of twenty-four 100-ns molecular dynamics simulations of the native and mutated HHR in the reactant state and in an activated precursor state (G8:2'OH deprotonated). Invoking the assumptions that G12 acts as the general base while the 2'OH of G8 acts as a general acid, the simulations are able to explain the origins of experimentally observed mutational effects, including several that are not easily inferred from the crystal structure. Simulations suggest that the Watson-Crick base-pairing between G8 and C3, the hydrogen bond network between C17 and G5, and the base stacking interactions between G8 and C1.1, collectively, are key to maintaining an active site structure conducive for catalytic activity. Mutation-induced disruption of any of these interactions will adversely affect activity. The simulation results predict that the C3U/G8D double mutant, where D is 2,6-diaminopurine, will have a rescue effect relative to the corresponding single mutations. Two general conclusions about the simulations emerge from this work. First, mutation simulations may require 30 ns or more to suitably relax such that the mutational effects become apparent. Second, in some cases, it is necessary to look beyond the reactant state in order to interpret mutational effects in terms of catalytically active structure. The present simulation results lead to better understanding of the origin of experimental mutational effects and provide insight into the key conserved features necessary to maintain the integrity of the active site architecture.
46848028
The GlcN6P cofactor plays multiple catalytic roles in the glmS ribozyme.
RNA enzymes (ribozymes) have remarkably diverse biological roles despite having limited chemical diversity. Protein enzymes enhance their reactivity through recruitment of cofactors; likewise, the naturally occurring glmS ribozyme uses the glucosamine-6-phosphate (GlcN6P) organic cofactor for phosphodiester bond cleavage. Prior structural and biochemical studies have implicated GlcN6P as the general acid. Here we describe new catalytic roles of GlcN6P through experiments and calculations. Large stereospecific normal thio effects and a lack of metal-ion rescue in the holoribozyme indicate that nucleobases and the cofactor play direct chemical roles and align the active site for self-cleavage. Large stereospecific inverse thio effects in the aporibozyme suggest that the GlcN6P cofactor disrupts an inhibitory interaction of the nucleophile. Strong metal-ion rescue in the aporibozyme reveals that this cofactor also provides electrostatic stabilization. Ribozyme organic cofactors thus perform myriad catalytic roles, thereby allowing RNA to compensate for its limited functional diversity.
42575162
Activation of the glmS Ribozyme Nucleophile via Overdetermined Hydrogen Bonding.
RNA enzymes, or ribozymes, catalyze internal phosphodiester bond cleavage using diverse catalytic strategies. These include the four classic strategies: in-line nucleophilic attack, deprotonation of the 2'-OH nucleophile, protonation of the 5'-O leaving group, and stabilization of developing charge on the nonbridging oxygen atoms of the scissile phosphate. In addition, we recently identified two additional ribozyme strategies: acidification of the 2'-OH and release of the 2'-OH from inhibitory interactions. Herein, we report inverse thio effects in the presence of glmS ribozyme variants and a 1-deoxyglucosamine 6-phosphate cofactor analogue and demonstrate that activation of the 2'-OH nucleophile is promoted by competitive hydrogen bonding among diverse ribozyme moieties for the pro-RP nonbridging oxygen. We conclude that the glmS ribozyme uses an overdetermined set of competing hydrogen bond donors in its active site to ensure potent activation and regulation by the cofactor. Nucleophile activation through competitive, overdetermined hydrogen bonding could be a general strategy for ribozyme activation and may be applicable for controlling the function of ribozymes and riboswitches in the laboratory.
36560149
Metal Ion Modeling Using Classical Mechanics
Metal ions play significant roles in numerous fields including chemistry, geochemistry, biochemistry, and materials science. With computational tools increasingly becoming important in chemical research, methods have emerged to effectively face the challenge of modeling metal ions in the gas, aqueous, and solid phases. Herein, we review both quantum and classical modeling strategies for metal ion-containing systems that have been developed over the past few decades. This Review focuses on classical metal ion modeling based on unpolarized models (including the nonbonded, bonded, cationic dummy atom, and combined models), polarizable models (e.g., the fluctuating charge, Drude oscillator, and the induced dipole models), the angular overlap model, and valence bond-based models. Quantum mechanical studies of metal ion-containing systems at the semiempirical, ab initio, and density functional levels of theory are reviewed as well with a particular focus on how these methods inform classical modeling efforts. Finally, conclusions and future prospects and directions are offered that will further enhance the classical modeling of metal ion-containing systems.
4935419
Integration of kinetic isotope effect analyses to elucidate ribonuclease mechanism.
The well-studied mechanism of ribonuclease A is believed to involve concerted general acid-base catalysis by two histidine residues, His12 and His119. The basic features of this mechanism are often cited to explain rate enhancement by both protein and RNA enzymes that catalyze RNA 2'-O-transphosphorylation. Recent kinetic isotope effect analyses and computational studies are providing a more chemically detailed description of the mechanism of RNase A and the rate limiting transition state. Overall, the results support an asynchronous mechanism for both solution and ribonuclease catalyzed reactions in which breakdown of a transient dianoinic phosphorane intermediate by 5'OP bond cleavage is rate limiting. Relative to non-enzymatic reactions catalyzed by specific base, a smaller KIE on the 5'O leaving group and a less negative βLG are observed for RNase A catalysis. Quantum mechanical calculations consistent with these data support a model in which electrostatic and H-bonding interactions with the non-bridging oxygens and proton transfer from His119 render departure of the 5'O less advanced and stabilize charge buildup in the transition state. Both experiment and computation indicate advanced 2'OP bond formation in the rate limiting transition state. However, this feature makes it difficult to resolve the chemical steps involved in 2'O activation. Thus, modeling the transition state for RNase A catalysis underscores those elements of its chemical mechanism that are well resolved, as well as highlighting those where ambiguity remains. This article is part of a Special Issue entitled: Enzyme Transition States from Theory and Experiment.
1359736
Interpretation of pH-activity profiles for acid-base catalysis from molecular simulations.
The measurement of reaction rate as a function of pH provides essential information about mechanism. These rates are sensitive to the pK(a) values of amino acids directly involved in catalysis that are often shifted by the enzyme active site environment. Experimentally observed pH-rate profiles are usually interpreted using simple kinetic models that allow estimation of "apparent pK(a)" values of presumed general acid and base catalysts. One of the underlying assumptions in these models is that the protonation states are uncorrelated. In this work, we introduce the use of constant pH molecular dynamics simulations in explicit solvent (CpHMD) with replica exchange in the pH-dimension (pH-REMD) as a tool to aid in the interpretation of pH-activity data of enzymes and to test the validity of different kinetic models. We apply the methods to RNase A, a prototype acid-base catalyst, to predict the macroscopic and microscopic pK(a) values, as well as the shape of the pH-rate profile. Results for apo and cCMP-bound RNase A agree well with available experimental data and suggest that deprotonation of the general acid and protonation of the general base are not strongly coupled in transphosphorylation and hydrolysis steps. Stronger coupling, however, is predicted for the Lys41 and His119 protonation states in apo RNase A, leading to the requirement for a microscopic kinetic model. This type of analysis may be important for other catalytic systems where the active forms of the implicated general acid and base are oppositely charged and more highly correlated. These results suggest a new way for CpHMD/pH-REMD simulations to bridge the gap with experiments to provide a molecular-level interpretation of pH-activity data in studies of enzyme mechanisms.
11709639
Two Divalent Metal Ions and Conformational Changes Play Roles in the Hammerhead Ribozyme Cleavage Reaction.
The hammerhead ribozyme is a self-cleaving RNA broadly dispersed across all kingdoms of life. Although it was the first of the small, nucleolytic ribozymes discovered, the mechanism by which it catalyzes its reaction remains elusive. The nucleobase of G12 is well positioned to be a general base, but it is unclear if or how this guanine base becomes activated for proton transfer. Metal ions have been implicated in the chemical mechanism, but no interactions between divalent metal ions and the cleavage site have been observed crystallographically. To better understand how this ribozyme functions, we have solved crystal structures of wild-type and G12A mutant ribozymes. We observe a pH-dependent conformational change centered around G12, consistent with this nucleotide becoming deprotonated. Crystallographic and kinetic analysis of the G12A mutant reveals a Zn(2+) specificity switch suggesting a direct interaction between a divalent metal ion and the purine at position 12. The metal ion specificity switch and the pH-rate profile of the G12A mutant suggest that the minor imino tautomer of A12 serves as the general base in the mutant ribozyme. We propose a model in which the hammerhead ribozyme rearranges prior to the cleavage reaction, positioning two divalent metal ions in the process. The first metal ion, positioned near G12, becomes directly coordinated to the O6 keto oxygen, to lower the pKa of the general base and organize the active site. The second metal ion, positioned near G10.1, bridges the N7 of G10.1 and the scissile phosphate and may participate directly in the cleavage reaction.
46625505
Two Active Site Divalent Ions in the Crystal Structure of the Hammerhead Ribozyme Bound to a Transition State Analogue.
The crystal structure of the hammerhead ribozyme bound to the pentavalent transition state analogue vanadate reveals significant rearrangements relative to the previously determined structures. The active site contracts, bringing G10.1 closer to the cleavage site and repositioning a divalent metal ion such that it could, ultimately, interact directly with the scissile phosphate. This ion could also position a water molecule to serve as a general acid in the cleavage reaction. A second divalent ion is observed coordinated to O6 of G12. This metal ion is well-placed to help tune the pKA of G12. On the basis of this crystal structure as well as a wealth of biochemical studies, we propose a mechanism in which G12 serves as the general base and a magnesium-bound water serves as a general acid.
3671395
Divalent Metal Ion Activation of a Guanine General Base in the Hammerhead Ribozyme: Insights from Molecular Simulations.
The hammerhead ribozyme is a well-studied nucleolytic ribozyme that catalyzes the self-cleavage of the RNA phosphodiester backbone. Despite experimental and theoretical efforts, key questions remain about details of the mechanism with regard to the activation of the nucleophile by the putative general base guanine (G12). Straightforward interpretation of the measured activity-pH data implies the pKa value of the N1 position in the G12 nucleobase is significantly shifted by the ribozyme environment. Recent crystallographic and biochemical work has identified pH-dependent divalent metal ion binding at the N7/O6 position of G12, leading to the hypothesis that this binding mode could induce a pKa shift of G12 toward neutrality. We present computational results that support this hypothesis and provide a model that unifies the interpretation of available structural and biochemical data and paints a detailed mechanistic picture of the general base step of the reaction. Experimentally testable predictions are made for mutational and rescue effects on G12, which will give further insights into the catalytic mechanism. These results contribute to our growing knowledge of the potential roles of divalent metal ions in RNA catalysis.
14013611
Identification of the hammerhead ribozyme metal ion binding site responsible for rescue of the deleterious effect of a cleavage site phosphorothioate.
The hammerhead ribozyme crystal structure identified a specific metal ion binding site referred to as the P9/G10.1 site. Although this metal ion binding site is approximately 20 A away from the cleavage site, its disruption is highly deleterious for catalysis. Additional published results have suggested that the pro-R(P) oxygen at the cleavage site is coordinated by a metal ion in the reaction's transition state. Herein, we report a study on Cd(2+) rescue of the deleterious phosphorothioate substitution at the cleavage site. Under all conditions, the Cd(2+) concentration dependence can be accounted for by binding of a single rescuing metal ion. The affinity of the rescuing Cd(2+) is sensitive to perturbations at the P9/G10.1 site but not at the cleavage site or other sites in the conserved core. These observations led to a model in which a metal ion bound at the P9/G10.1 site in the ground state acquires an additional interaction with the cleavage site prior to and in the transition state. A titration experiment ruled out the possibility that a second tight-binding metal ion (< 10 microM) is involved in the rescue, further supporting the single metal ion model. Additionally, weakening Cd(2+) binding at the P9/G10.1 site did not result in the biphasic binding curve predicted from other models involving two metal ions. The large stereospecific thio-effects at the P9/G10.1 and the cleavage site suggest that there are interactions with these oxygen atoms in the normal reaction that are compromised by replacement of oxygen with sulfur. The simplest interpretation of the substantial rescue by Cd(2+) is that these atoms interact with a common metal ion in the normal reaction. Furthermore, base deletions and functional group modifications have similar energetic effects on the transition state in the Cd(2+)-rescued phosphorothioate reaction and the wild-type reaction, further supporting the model that a metal ion bridges the P9/G10.1 and the cleavage site in the normal reaction (i.e., with phosphate linkages rather than phosphorothioate linkages). These results suggest that the hammerhead undergoes a substantial conformational rearrangement to attain its catalytic conformation. Such rearrangements appear to be general features of small functional RNAs, presumably reflecting their structural limitations.
27121383
Insight into the role of Mg in hammerhead ribozyme catalysis from X-ray crystallography and molecular dynamics simulation.
Results of a series of 12 ns molecular dynamics (MD) simulations of the reactant state (with and without a Mg(2+) ion), early and late transition state mimics are presented based on a recently reported crystal structure of a full-length hammerhead RNA. The simulation results support a catalytically active conformation with a Mg(2+) ion bridging the A9 and scissile phosphates. In the reactant state, the Mg(2+) spends significant time closely associated with the 2'OH of G8, but remains fairly distant from the leaving group O(5') position. In the early TS mimic simulation, where the nucleophilic O(2') and leaving group O(5') are equidistant from the phosphorus, the Mg(2+) ion remains tightly coordinated to the 2'OH of G8, but is positioned closer to the O(5') leaving group, stabilizing the accumulating charge. In the late TS mimic simulation, the coordination around the bridging Mg(2+) ion undergoes a transition whereby the coordination with the 2'OH of G8 is replace by the leaving group O(5') that has developed significant charge. At the same time, the 2'OH of G8 forms a hydrogen bond with the leaving group O(5') and is positioned to act as a general acid catalyst. This work represents the first reported simulations of the full-length hammerhead structure and TS mimics, and provides direct evidence for the possible role of a bridging Mg(2+) ion in catalysis that is consistent with both crystallographic and biochemical data.
44679246
Ground-state coordination of a catalytic metal to the scissile phosphate of a tertiary-stabilized Hammerhead ribozyme.
Although the Hammerhead ribozyme (HHRz) has long been used as a model system in the field of ribozyme enzymology, several details of its mechanism are still not well understood. In particular, significant questions remain concerning the disposition and role of catalytic metals in the HHRz. Previous metal-rescue experiments using a "minimal" HHRz resulted in prediction of a catalytic metal that is bound in the A9/G10.1 site in the ground state of the reaction and that bridges to the scissile phosphate further along the reaction pathway. "Native" or extended HHRz constructs contain tertiary contacts that stabilize a more compact structure at moderate ionic strength. We performed Cd(2+) rescue experiments on an extended HHRz from Schistosoma mansoni using stereo-pure scissile phosphorothioate-substituted substrates in order to determine whether a metal ion makes contact with the scissile phosphate in the ground state or further along the reaction coordinate. Inhibition in Ca(2+)/Mg(2+) and rescue by thiophilic Cd(2+) was specific for the R(p)-S stereoisomer of the scissile phosphate. The affinity of the rescuing Cd(2+), measured in two different ionic strength backgrounds, increased fourfold to 17-fold when the pro-R(p) oxygen is replaced by sulfur. These data support a model in which the rescuing metal ion makes a ground-state interaction with the scissile phosphate in the native HHRz. The resulting model for Mg(2+) activation in the HHRz places a metal ion in contact with the scissile phosphate, where it may provide ground-state electrostatic activation of the substrate.
23750787
Cooperative Interactions in the Hammerhead Ribozyme Drive pKa Shifting of G12 and Its Stacked Base C17.
General acid-base catalysis is a key mechanistic strategy in protein and RNA enzymes. Ribozymes use hydrated metal ions, nucleobases, and organic cofactors to carry this out. In most small ribozymes, a guanosine is positioned to participate in proton transfer with the nucleophilic 2'-OH. The unshifted pKa values for nucleobases and solvated metal ions are far from neutrality, however, and thus nonideal for general acid-base catalysis. Herein, evidence is provided for cooperative interaction in the hammerhead ribozyme among the guanine that interacts with the nucleophilic 2'-OH, G12, the -1 nucleobase C17, and Mg2+ ions. We introduce global fitting for analyzing ribozyme rate-pH data parametric in Mg2+ concentration and benchmark this method on data from the hepatitis delta virus ribozyme. We then apply global fitting to new rate-pH data for the hammerhead ribozyme using a minimal three-dimensional, four-channel cooperative model. The value for the pKa of G12 that we obtain is channel-dependent and varies from 8.1 to 9.9, shifting closest toward neutrality in the presence of two cationic species: C17H+ and a Mg2+ ion. The value for the pKa of the -1 nucleotide, C17, is increased a remarkable 3.5-5 pKa units toward neutrality. Shifting of the pKa of C17 appears to be driven by an electrostatic sandwich of C17 between carbonyl groups of the 5'-neighboring U and of G12 and involves cation-π interactions. Rate-pH profiles reveal that the major reactive channel under biological Mg2+ and pH involves a cationic C17 rather than a second metal ion. Substitution of a cationic base for a metal underscores the versatility of RNA.
207196211
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.
207124345
Detection of innersphere interactions between magnesium hydrate and the phosphate backbone of the HDV ribozyme using Raman crystallography.
A Raman microscope and Raman difference spectroscopy are used to detect the vibrational signature of RNA-bound magnesium hydrate in crystals of hepatitis delta virus (HDV) ribozyme and to follow the effects of magnesium hydrate binding to the nonbridging phosphate oxygens in the phosphodiester backbone. There is a correlation between the Raman intensity of the innersphere magnesium hydrate signature peak, near 322 cm-1, and the intensity of the PO2- symmetric stretch, near 1100 cm-1, perturbed by magnesium binding, demonstrating direct observation of -PO2-...Mg2+(H2O)x innersphere complexes. The complexes may be pentahydrates (x = 5) and tetrahydrates (x = 4). The assignment of the Raman feature near 322 cm-1 to a magnesium hydrate species is confirmed by isotope shifts observed in D2O and H218O that are semiquantitatively reproduced by calculations. The standardized intensity changes in the 1100 cm-1 PO2- feature seen upon magnesium hydrate binding indicates that there are approximately 5 innersphere Mg2+...-O2P contacts per HDV molecule when the crystal is exposed to a solution containing 20 mM magnesium.
19433371
A catalytic metal ion interacts with the cleavage Site G.U wobble in the HDV ribozyme.
The HDV ribozyme self-cleaves by a chemical mechanism involving general acid-base catalysis to generate 2',3'-cyclic phosphate and 5'-hydroxyl termini. Biochemical studies from several laboratories have implicated C75 as the general acid and hydrated magnesium as the general base. We have previously shown that C75 has a pK(a) shifted >2 pH units toward neutrality [Gong, B., Chen, J. H., Chase, E., Chadalavada, D. M., Yajima, R., Golden, B. L., Bevilacqua, P. C., and Carey, P. R. (2007) J. Am. Chem. Soc. 129, 13335-13342], while in crystal structures, it is well-positioned for proton transfer. However, no evidence for a hydrated magnesium poised to serve as a general base in the reaction has been observed in high-resolution crystal structures of various reaction states and mutants. Herein, we use solution kinetic experiments and parallel Raman crystallographic studies to examine the effects of pH on the rate and Mg(2+) binding properties of wild-type and 7-deazaguanosine mutants of the HDV ribozyme. These data suggest that a previously unobserved hydrated magnesium ion interacts with N7 of the cleavage site G.U wobble base pair. Integrating this metal ion binding site with the available crystal structures provides a new three-dimensional model for the active site of the ribozyme that accommodates all available biochemical data and appears competent for catalysis. The position of this metal is consistent with a role of a magnesium-bound hydroxide as a general base as dictated by biochemical data.
13692098
Evidence That Nucleophile Deprotonation Exceeds Bond Formation in the HDV Ribozyme Transition State.
Steric constraints imposed by the active sites of protein and RNA enzymes pose major challenges to the investigation of structure-function relationships within these systems. As a strategy to circumvent such constraints in the HDV ribozyme, we have synthesized phosphoramidites from propanediol derivatives and incorporated them at the 5'-termini of RNA and DNA oligonucleotides to generate a series of novel substrates with nucleophiles perturbed electronically through geminal fluorination. In nonenzymatic, hydroxide-catalyzed intramolecular transphosphorylation of the DNA substrates, pH-rate profiles revealed that fluorine substitution reduces the maximal rate and the kinetic p Ka, consistent with the expected electron-withdrawing effect. In HDV ribozyme reactions, we observed that the RNA substrates undergo transphosphorylation relatively efficiently, suggesting that the conformational constraints imposed by a ribofuranose ring are not strictly required for ribozyme catalysis. In contrast to the nonenzymatic reactions, however, substrate fluorination modestly increases the ribozyme reaction rate, consistent with a mechanism in which (1) the 2'-hydroxyl nucleophile exists predominantly in its neutral, protonated form in the ground state and (2) the 2'-hydroxyl bears some negative charge in the rate-determining step, consistent with a transition state in which the extent of 2'-OH deprotonation exceeds the extent of P-O bond formation.