The Investigation of DNA and RNA Structural Differences Using Ultra High Performance Liquid Chromatography

DNA and RNA chromatography is extensively used for nucleic acid analysis. To better understand the chromatographic mechanisms by which DNA and RNA oligonucleotides are separated, ion pair reverse-pair ultra-high performance liquid chromatography (IP RP UHPLC) methods were developed. 11mer and 12mer DNA and RNA oligonucleotides of various compositions were used during this study. The first part of this study analyzed 11mer DNA and RNA oligonucleotides to better understand the chromatographic separations of DNA and RNA. The results gathered through the IP RP UHPLC analysis of these oligonucleotides demonstrated the existence of structural features that affect the chromatographic separations of DNA and RNA. This led to the IP RP UHPLC analysis of DNA and RNA oligonucleotides, of equal length and sequence, which either formed a 4 base-pair or 2 base-pair tetraloop secondary structure. The purpose of this investigation is to improve the isolation and purification of nucleic acid mixtures by understanding how DNA and RNA oligonucleotides interact with the stationary support but to also illuminate the role of structural features in nucleic acid separations. The characterization and the separation of the DNA and RNA oligonucleotides were achieved through a variety of methods including temperature melting experiments. The results gathered demonstrated the effectiveness of IP RP UHPLC to analyze the differences between DNA and RNA oligonucleotide separations. The DNA oligonucleotides eluted earlier than the RNA oligonucleotides which demonstrated that RNA has a different chromatographic mechanism than DNA. Differences between nucleic acid separations of fragments with the 2 base-pair tetraloop and 4 base-pair tetraloop structural modifications were also observed. The oligonucleotides with the 4 base-pair tetraloop eluted later than the oligonucleotides with the 2 base-pair tetraloop demonstrating the influence of structural modifications on the separation mechanisms of nucleic acids. The temperature melting experiments performed also confirmed that structural modifications influence the interaction between nucleic acids and stationary support. These results demonstrate the effectiveness of IP RP UHPLC to observe structural differences between DNA and RNA and as an alternative method to traditional methods, such as gel electrophoresis, to analyze oligonucleotides

Weaker N‑Terminal Interactions for the Protective over the Causative Aβ Peptide Dimer Mutants

Knowing that abeta amyloid peptide (Aβ₄₂) 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

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

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

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 <i>K</i>_D 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 <i>K</i>_D 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

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

<p>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.</p