In Silico Screening and Testing of FDA-Approved Small Molecules to Block SARS-CoV-2 Entry to the Host Cell by Inhibiting Spike Protein Cleavage.

The COVID-19 pandemic began in 2019, but it is still active. The development of an effective vaccine reduced the number of deaths; however, a treatment is still needed. Here, we aimed to inhibit viral entry to the host cell by inhibiting spike (S) protein cleavage by several proteases. We developed a computational pipeline to repurpose FDA-approved drugs to inhibit protease activity and thus prevent S protein cleavage. We tested some of our drug candidates and demonstrated a decrease in protease activity. We believe our pipeline will be beneficial in identifying a drug regimen for COVID-19 patients.

Physical Chemistry of a Single tRNA-Modified Nucleoside Regulates Decoding of the Synonymous Lysine Wobble Codon and Affects Type 2 Diabetes.

The 2-methylthio-modification (ms2-) of N6-threonylcarbonyladenosine (t6A37) at position-37 (ms2t6A37) in tRNAUUULys3 provides the needed stability between the tRNA anticodon and the human insulin mRNA codon AAG during translation, as determined by molecular dynamics simulation. Single-nucleoside polymorphisms of the human gene for the enzyme, Cdkal1 that post-transcriptionally modifies t6A37 to ms2t6A37 in tRNAUUULys3, correlate with type 2 diabetes mellitus. Without the ms2-modification, tRNAUUULys3 is incapable of correctly translating the insulin mRNA AAG codon for lysine at the site of protease cleavage between the A-chain and the C-peptide. By enhancing anticodon/codon cross-strand stacking, the ms2-modification adds stability through van der Waals interactions and dehydration of the ASL loop and cavity of the anticodon/codon minihelix but does not add hydrogen bonding of any consequence. Thus, the modifying enzyme Cdkal1, by adding a crucial ms2-group to tRNAUUULys3-t6A37, facilitates the decoding of the AAG codon and enables human pancreatic islets to correctly translate insulin mRNA.

Base Pairing and Functional Insights into N3-Methylcytidine (m3C) in RNA.

N3-methylcytidine (m3C) is present in both eukaryotic tRNA and mRNA and plays critical roles in many biological processes. We report the synthesis of the m3C phosphoramidite building block and its containing RNA oligonucleotides. The base-pairing stability and specificity studies show that the m3C modification significantly disrupts the stability of the Watson-Crick C:G pair. Further m3C decreases the base pairing discrimination between C:G and the other mismatched C:A, C:U, and C:C pairs. Our molecular dynamic simulation study further reveals the detailed structural insights into the m3C:G base pairing pattern in an RNA duplex. More importantly, the biochemical investigation of m3C using reverse transcription in vitro shows that N3-methylation specifies the C:A pair and induces a G to A change using HIV-1-RT, MMLV-RT, and MutiScribe-RT enzymes, all with relatively low replication fidelity. For other reverse transcriptases with higher fidelity like AMV-RT, the methylation could completely shut down DNA synthesis. Our work provides detailed insights into the thermostability of m3C in RNA and a foundation for developing new molecular tools for mapping m3C in different RNA contexts and exploring the biochemical and biomedical potentials of m3C in the design and development of RNA based therapeutics.

Terpene Chain Length Affects the Base Pairing Discrimination of S-geranyl-2-thiouridine in RNA Duplex.

Geranylation is a hydrophobic modification discovered in several bacteria tRNAs with the function of promoting codon bias during translation. However, why nature selects this C10-geranyl group remains a question. We conduct synthesis, UV-thermal denaturation, and molecular simulation studies in RNA duplexes and reveal possible reasons behind this natural selection. Among methyl-(C1), dimethylallyl-(C5), geranyl-(C10), and farnesyl-(C15) modified 2-thiouridines, only geranyl-group promotes U:G over U:A pair. Molecular simulation shows all the modified terpene groups point to the minor groove of RNA duplexes. The discrimination between U:G and U:A pairs derives from the difference in hydrogen bonding and interactions of the chain with the hydrophobic area in the minor groove. Geranyl group has perfect length to discriminate U:G and U:A pairs, whereas the others are either too long or too short to achieve the same behavior. This work indicates that geranyl group cannot be replaced by other terpene groups in promoting codon-specificity.

Base pairing, structural and functional insights into N4-methylcytidine (m4C) and N4,N4-dimethylcytidine (m42C) modified RNA.

The N4-methylation of cytidine (m4C and m42C) in RNA plays important roles in both bacterial and eukaryotic cells. In this work, we synthesized a series of m4C and m42C modified RNA oligonucleotides, conducted their base pairing and bioactivity studies, and solved three new crystal structures of the RNA duplexes containing these two modifications. Our thermostability and X-ray crystallography studies, together with the molecular dynamic simulation studies, demonstrated that m4C retains a regular C:G base pairing pattern in RNA duplex and has a relatively small effect on its base pairing stability and specificity. By contrast, the m42C modification disrupts the C:G pair and significantly decreases the duplex stability through a conformational shift of native Watson-Crick pair to a wobble-like pattern with the formation of two hydrogen bonds. This double-methylated m42C also results in the loss of base pairing discrimination between C:G and other mismatched pairs like C:A, C:T and C:C. The biochemical investigation of these two modified residues in the reverse transcription model shows that both mono- or di-methylated cytosine bases could specify the C:T pair and induce the G to T mutation using HIV-1 RT. In the presence of other reverse transcriptases with higher fidelity like AMV-RT, the methylation could either retain the normal nucleotide incorporation or completely inhibit the DNA synthesis. These results indicate the methylation at N4-position of cytidine is a molecular mechanism to fine tune base pairing specificity and affect the coding efficiency and fidelity during gene replication.

Silencing of the tRNA Modification Enzyme Cdkal1 Effects Functional Insulin Synthesis in NIT-1 Cells: tRNALys3 Lacking ms2- (ms2t6A37) is Unable to Establish Sufficient Anticodon:Codon Interactions to Decode the Wobble Codon AAG.

Human Genome Wide Association Studies found a significant risk of Type 2 Diabetes Mellitus (T2DM) in single nucleotide polymorphisms in the cdkal1 gene. The cdkal1 gene is remote from the insulin gene and with the surprising function of a specific tRNA modification. Population studies and case control studies acquired evidences of the connection between Cdkal1 protein and insulin production over the years. To obtain biochemical proofs directly linking potential SNPs to their roles in insulin production and availability is challenging, but the development of Cdkal1 knock out mice and knock out cell lines made it possible to extend our knowledge towards therapeutic field of diabetic research. Supporting the evidences, here we show that knock down of the cdkal1 gene using small interfering and short hairpin RNA in the NIT-1 cell line, a ß-cell line inducible for insulin resulted in reduced levels of cdkal1 and mature insulin mRNAs, increased the level of precursor insulin mRNA, decreased Cdkal1 and insulin proteins, and diminished modification of tRNALys3 from t6A37 to ms2t6A37, the specified function of Cdkal1. tRNALys3 lacking ms2- is incapable of establishing sufficient hydrogen bonding energy and hydrophobic stabilization to decode the wobble codon AAG.

Modification of messenger RNA by 2'-O-methylation regulates gene expression in vivo.

Epitranscriptomic modifications of mRNA are important regulators of gene expression. While internal 2'-O-methylation (Nm) has been discovered on mRNA, questions remain about its origin and function in cells and organisms. Here, we show that internal Nm modification can be guided by small nucleolar RNAs (snoRNAs), and that these Nm sites can regulate mRNA and protein expression. Specifically, two box C/D snoRNAs (SNORDs) and the 2'-O-methyltransferase fibrillarin lead to Nm modification in the protein-coding region of peroxidasin (Pxdn). The presence of Nm modification increases Pxdn mRNA expression but inhibits its translation, regulating PXDN protein expression and enzyme activity both in vitro and in vivo. Our findings support a model in which snoRNA-guided Nm modifications of mRNA can regulate physiologic gene expression by altering mRNA levels and tuning protein translation.

CoSIMS: An Optimized Trajectory-Based Collision Simulator for Ion Mobility Spectrometry.

A new, multithreaded, trajectory method based software platform, CoSIMS, is revealed and compared to reference MOBCAL collision cross sections (CCS). CoSIMS employs various molecular mechanics algorithms to lessen the computational resources required to simulate thousands of buffer gas-ion collisions, including the neglect of London dispersion interactions at long distances and the removal of trajectories that insignificantly contribute to the total CCS via an ellipsoidal projection approximation. The showcased program is used to calculate the collision cross sections of carbon fullerenes, proteins, and DNA strands of various lengths, sizes, and molecular weights, and these are compared against the CCSs calculated by MOBCAL. Through this analysis, it is shown that the application of the aforementioned algorithms enables both faster and more reasonable CCS calculations than MOBCAL for highly elongated molecules such as nucleic acids; for all other molecules, CoSIMS is able to reproduce the CCSs generated by MOBCAL's trajectory method within a few percent. Overall, CoSIMS is able to calculate nearly identical CCSs as MOBCAL in nearly 2 orders of magnitude less CPU time due to the various numerical methods implemented into the software, even when run on a single CPU core.

Construction and structure studies of DNA-bipyridine complexes as versatile scaffolds for site-specific incorporation of metal ions into DNA.

The facile construction of metal-DNA complexes using 'Click' reactions is reported here. A series of 2'-propargyl-modified DNA oligonucleotides were initially synthesized as structure scaffolds and were then modified through 'Click' reaction to incorporate a bipyridine ligand equipped with an azido group. These metal chelating ligands can be placed in the DNA context in site-specific fashion to provide versatile templates for binding various metal ions, which are exchangeable using a simple EDTA washing-and-filtration step. The constructed metal-DNA complexes were found to be thermally stable. Their structures were explored by solving a crystal structure of a propargyl-modified DNA duplex and installing the bipyridine ligands by molecular modeling and simulation. These metal-DNA complexes could have wide applications as novel organometallic catalysts, artificial ribonucleases, and potential metal delivery systems.

Cyano Modification on Uridine Decreases Base-Pairing Stability and Specificity through Neighboring Disruption in RNA Duplex.

5-Cyanomethyluridine (cnm5 U) and 5-cyanouridine (cn5 U), the two uridine analogues, were synthesized and incorporated into RNA oligonucleotides. Base-pairing stability and specificity studies in RNA duplexes indicated that cnm5 U slightly decreased the stability of the duplex but retained the base-pairing preference. In contrast, cn5 U dramatically decreased both base-pairing stability and specificity between U:A and other noncanonical U:G, U:U, and U:C pairs. In addition, the cn5 U:G pair was found to be stronger than the cn5 U:A pair and the other mismatched pairs in the context of a RNA duplex; this implied that cn5 U might slightly prefer to recognize G over A. Our mechanistic studies by molecular simulations showed that the cn5 U modification did not directly affect the base pairing of the parent nucleotide; instead, it weakened the neighboring base pair in the 5' side of the modification in the RNA duplexes. Consistent with the simulation data, replacing the Watson-Crick A:U pair to a mismatched C:U pair in the 5'-neighboring site did not affect the overall stability of the duplex. Our work reveals the significance of the electron-withdrawing cyano group in natural tRNA systems and provides two novel building blocks for constructing RNA-based therapeutics.

Weaker N-Terminal Interactions for the Protective over the Causative Aß Peptide Dimer Mutants.

Knowing that abeta amyloid peptide (Aß42) dimers are the smallest and most abundant neurotoxic oligomers for Alzheimer's disease (AD), we used molecular simulations with advanced sampling methods (replica-exchange) to characterize and compare interactions between the N-termini (residues 1-16) of wild type (WT-WT) and five mutant dimers under constrained and unconstrained conditions. The number of contacts and distances between the N-termini, and contact maps of their conformational landscape illustrate substantial differences for a single residue change. The N-terminal contacts are significantly diminished for the dimers containing the monomers that protect against (WT-A2T) as compared with those that predispose toward (A2V-A2V) AD and for the control WT-WT dimers. The reduced number of N-terminal contacts not only occurs at or near the second residue mutations but also is distributed through to the 10th residue. These findings provide added support to the accumulating evidence for the "N-terminal hypothesis of AD" and offer an alternate mechanism for the cause of protection from the A2T mutant.

Celebrating wobble decoding: Half a century and still much is new.

A simple post-transcriptional modification of tRNA, deamination of adenosine to inosine at the first, or wobble, position of the anticodon, inspired Francis Crick's Wobble Hypothesis 50 years ago. Many more naturally-occurring modifications have been elucidated and continue to be discovered. The post-transcriptional modifications of tRNA's anticodon domain are the most diverse and chemically complex of any RNA modifications. Their contribution with regards to chemistry, structure and dynamics reveal individual and combined effects on tRNA function in recognition of cognate and wobble codons. As forecast by the Modified Wobble Hypothesis 25 years ago, some individual modifications at tRNA's wobble position have evolved to restrict codon recognition whereas others expand the tRNA's ability to read as many as four synonymous codons. Here, we review tRNA wobble codon recognition using specific examples of simple and complex modification chemistries that alter tRNA function. Understanding natural modifications has inspired evolutionary insights and possible innovation in protein synthesis.

Nature's Selection of Geranyl Group as a tRNA Modification: The Effects of Chain Length on Base-Pairing Specificity.

The recently discovered geranyl modification on the 2-thio position of wobble U34 residues in tRNAGlu, tRNALys, and tRNAGln in several bacteria has been found to enhance the U:G pairing specificity and reduce the frameshifting error during translation. It is a fundamentally interesting question why nature chose a C10 terpene group in tRNA systems. In this study, we explore the significance of the terpene length on base-paring stability and specificity using a series of 2-thiouridine analogues containing different lengths of carbon chains, namely, methyl- (C1), dimethylallyl- (C5), and farnesyl-modified (C15) 2-thiothymidines in a DNA duplex. Our thermal denaturation studies indicate that the relatively long chain length of ≥ C10 is required to maintain the base-pairing discrimination of thymidine between G and A. The results from our molecular dynamics simulations show that in the T:G-pair-containing duplex, the geranyl and farnesyl groups fit into the minor groove and stabilize the overall duplex stability. This effect cannot be achieved by the shorter carbon chains such as methyl and dimethylallyl groups. For a duplex containing a T:A pair, the terpene groups disrupt both hydrogen bonding and stacking interactions by pushing the opposite A out of the helical structure. Overall, as the terpene chain length increases, the xT:G pair stabilizes the duplex, whereas the xT:A pair causes destabilization, indicating the evolutionary significance of the long terpene group on base-pairing specificity and codon recognition.

tRNA Modification Detection Using Graphene Nanopores: A Simulation Study.

There are over 100 enzyme-catalyzed modifications on transfer RNA (tRNA) molecules. The levels and identity of wobble uridine (U) modifications are affected by environmental conditions and diseased states, making wobble U detection a potential biomarker for exposures and pathological conditions. The current detection of RNA modifications requires working with nucleosides in bulk samples. Nanopore detection technology uses a single-molecule approach that has the potential to detect tRNA modifications. To evaluate the feasibility of this approach, we have performed all-atom molecular dynamics (MD) simulation studies of a five-layered graphene nanopore by localizing canonical and modified uridine nucleosides. We found that in a 1 M KCl solution with applied positive and negative biases not exceeding 2 V, nanopores can distinguish U from 5-carbonylmethyluridine (cm5U), 5-methoxycarbonylmethyluridine (mcm5U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), and 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um) based on changes in the resistance of the nanopore. Specifically, we observed that in nanopores with dimensions less than 3 nm diameter, a localized mcm5Um and mcm5U modifications could be clearly distinguished from the canonical uridine, while the other modifications showed a modest yet detectable decrease in their respective nanopore conductance. We have compared the results between nanopores of various sizes to aid in the design, optimization, and fabrication of graphene nanopores devices for tRNA modification detection.

Advances in RNA molecular dynamics: a simulator's guide to RNA force fields.

Molecular simulations have become an essential tool for biochemical research. When they work properly, they are able to provide invaluable interpretations of experimental results and ultimately provide novel, experimentally testable predictions. Unfortunately, not all simulation models are created equal, and with inaccurate models it becomes unclear what is a bona fide prediction versus a simulation artifact. RNA models are still in their infancy compared to the many robust protein models that are widely in use, and for that reason the number of RNA force field revisions in recent years has been rapidly increasing. As there is no universally accepted 'best' RNA force field at the current time, RNA simulators must decide which one is most suited to their purposes, cognizant of its essential assumptions and their inherent strengths and weaknesses. Hopefully, armed with a better understanding of what goes inside the simulation 'black box,' RNA biochemists can devise novel experiments and provide crucial thermodynamic and structural data that will guide the development and testing of improved RNA models. WIREs RNA 2017, 8:e1396. doi: 10.1002/wrna.1396 For further resources related to this article, please visit the WIREs website.

Synthesis, base pairing and structure studies of geranylated RNA.

Natural RNAs utilize extensive chemical modifications to diversify their structures and functions. 2-Thiouridine geranylation is a special hydrophobic tRNA modification that has been discovered very recently in several bacteria, such as Escherichia coli, Enterobacter aerogenes, Pseudomonas aeruginosa and Salmonella Typhimurium The geranylated residues are located in the first anticodon position of tRNAs specific for lysine, glutamine and glutamic acid. This big hydrophobic terpene functional group affects the codon recognition patterns and reduces frameshifting errors during translation. We aimed to systematically study the structure, function and biosynthesis mechanism of this geranylation pathway, as well as answer the question of why nature uses such a hydrophobic modification in hydrophilic RNA systems. Recently, we have synthesized the deoxy-analog of S-geranyluridine and showed the geranylated T-G pair is much stronger than the geranylated T-A pair and other mismatched pairs in the B-form DNA duplex context, which is consistent with the observation that the geranylated tRNA(Glu) UUC recognizes GAG more efficiently than GAA. In this manuscript we report the synthesis and base pairing specificity studies of geranylated RNA oligos. We also report extensive molecular simulation studies to explore the structural features of the geranyl group in the context of A-form RNA and its effect on codon-anticodon interaction during ribosome binding.

Complex Thermodynamic Behavior of Single-Stranded Nucleic Acid Adsorption to Graphene Surfaces.

In just over a decade since its discovery, research on graphene has exploded due to a number of potential applications in electronics, materials, and medicine. In its water-soluble form of graphene oxide, the material has shown promise as a biosensor due to its preferential absorption of single-stranded polynucleotides and fluorescence quenching properties. The rational design of these biosensors, however, requires an improved understanding of the binding thermodynamics and ultimately a predictive model of sequence-specific binding. Toward these goals, here we directly measured the binding of nucleosides and oligonucleotides to graphene oxide nanoparticles using isothermal titration calorimetry and used the results to develop molecular models of graphene-nucleic acid interactions. We found individual nucleosides binding KD values lie in the submillimolar range with binding order of rG < rA < rC < dT < rU, while 5mer and 15mer oligonucleotides had markedly higher binding affinities in the range of micromolar and submicromolar KD values, respectively. The molecular models developed here are calibrated to quantitatively reproduce the above-mentioned experimental results. For oligonucleotides, our model predicts complex binding features such as double-stacked bases and a decrease in the fraction of graphene stacked bases with increasing oligonucleotide length until plateauing beyond ∼10-15 nucleotides. These experimental and computational results set the platform for informed design of graphene-based biosensors, further increasing their potential and application.

Modular calibrant sets for the structural analysis of nucleic acids by ion mobility spectrometry mass spectrometry.

This study explored the use of modular nucleic acid (NA) standards to generate calibration curves capable of translating primary ion mobility readouts into corresponding collision cross section (CCS) data. Putative calibrants consisted of single- (ss) and double-stranded (ds) oligo-deoxynucleotides reaching up to ∼40 kDa in size (i.e., 64 bp) and ∼5700 Å(2) in CCS. To ensure self-consistency among reference CCS values, computational data obtained in house were preferred to any experimental or computational data from disparate sources. Such values were obtained by molecular dynamics (MD) simulations and either the exact hard sphere scattering (EHSS) or the projection superposition approximation (PSA) methods, and then plotted against the corresponding experimental values to generate separate calibration curves. Their performance was evaluated on the basis of their correlation coefficients and ability to provide values that matched the CCS of selected test samples mimicking typical unknowns. The results indicated that the predictive power benefited from the exclusion of higher charged species that were more susceptible to the destabilizing effects of Coulombic repulsion. The results revealed discrepancies between EHSS and PSA data that were ascribable to the different approximations used to describe the ion mobility process. Within the boundaries defined by these approximations and the challenges of modeling NA structure in a solvent-free environment, the calibrant sets enabled the experimental determination of CCS with excellent reproducibility (precision) and error (accuracy), which will support the analysis of progressively larger NA samples of biological significance.

Synthesis and base pairing studies of geranylated 2-thiothymidine, a natural variant of thymidine.

The synthesis and base pairing of DNA duplexes containing the geranylated 2-thiothymidine have been investigated. This naturally existing hydrophobic modification could grant better base pairing stability to the T-G pair over normal T-A and other mismatched pairs in the duplex context. This study provides a potential explanation for the different codon recognition preferences of the geranylated tRNAs.