Therapeutic oligonucleotides (ONs) commonly incorporate phosphorothioate (PS) modifications. These introduce chiral centers and generate ON diastereomers. The increasing number of ONs undergoing clinical trials reaching the market has led to a growing interest better characterize diastereomer composition, especially for small interfering ribonucleic acids (siRNAs). In this study, first time, we identify higher-order structures as major cause separation in hydrophilic interaction...

The analytical identification of positional isomers (e.g., 3-, N4-, 5-methylcytidine) within the > 160 different post-transcriptional modifications found in RNA can be challenging. Conventional liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) approaches rely on chromatographic separation for accurate because collision-induced dissociation (CID) spectra these nearly exclusively yield identical nucleobase ions (BH2+) from same molecular ion (MH+). Here, we have explored...

tRNAs play a critical role in mRNA decoding, and posttranscriptional modifications within drive decoding efficiency accuracy. The types positions of tRNA model bacteria have been extensively studied, few eukaryotic organisms also characterized localized to particular sequences. However, far less is known regarding archaea. While the identities determined for multiple archaeal organisms, Haloferax volcanii only organism which specific To improve our understanding modification patterns...

The tRNA m1R9 methyltransferase (Trm10) family is conserved throughout Eukarya and Archaea. Despite the presence of a single Trm10 gene in Archaea most single-celled eukaryotes, metazoans encode up to three homologs Trm10. Several disease states correlate with deficiency human homolog TRMT10A, despite another cytoplasmic enzyme, TRMT10B. Here we investigate these phenomena demonstrate that TRMT10A (hTRMT10A) TRMT10B (hTRMT10B) are not biochemically redundant. In vitro activity assays...

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the gold-standard technique to study RNA and its various modifications. While most research on nucleosides been focused their biological roles, discovery of new modifications remains interest. With state-of-the-art technology, presence artifacts can confound identification Here, we report characterization a non-natural mcm5 isoC ribonucleoside in S. cerevisiae total tRNA hydrolysate by higher-energy collisional dissociation...

Oxidative stress triggered by the Fenton reaction (chemical) or UVR exposure (photo) can damage cellular biomolecules including RNA through oxidation of nucleotides. Besides such xenobiotic chemical modifications, also contains several post-transcriptional nucleoside modifications that are installed enzymes to modulate structure, RNA-protein interactions, and biochemical functions. We examined extent oxidative naturally modified which is required for protein synthesis under two different...

Ultraviolet radiation (UVR) is a known genotoxic agent. Although its effects on DNA have been well-documented, impact RNA and modifications less studied. By using Escherichia coli tRNA (tRNA) as model system, we identify the UVA (370 nm) susceptible chemical groups bonds in large variety of modified nucleosides. We use liquid chromatography tandem mass spectrometry to specific nucleoside photoproducts under vitro vivo conditions, which were then verified by employing stable-isotope labeled...

Short interfering RNA (siRNA) represents a rapidly expanding class of marketed oligonucleotide therapeutics. Due to its double-stranded nature, the characterization siRNA is twofold: (i) at single-strand (denaturing) level for impurity profiling and (ii) intact (nondenaturing) confirm duplex formation quantify excess single strands (including strand-derived impurities). While denaturing analysis can be carried out using conventional ion-pair reversed-phase liquid chromatography (IP-RPLC),...

Higher-energy collisional dissociation (HCD) of modified ribonucleosides generates characteristic and highly reproducible nucleoside-specific tandem mass spectra (MS/MS). Here, we demonstrate the capability HCD in combination with spectral matching for semi-automated characterization ribonucleosides. This process involved generation an library establishment a network rapid detection high sensitivity specificity retention time-independent fashion. Systematic analysis MS/MS tRNA hydrolysates...

Cryptococcus neoformans is a fungus that able to survive abnormally high levels of ionizing radiation (IR). The radiolysis water by IR generates reactive oxygen species (ROS) such as H 2 O and OH - . C withstands the damage caused ROS through antioxidant production enzyme-catalyzed breakdown ROS. Given these particular cellular protein needs, questions arise whether transfer ribonucleic acids molecules (tRNAs) undergo unique chemical modifications maintain their structure, stability, and/or...

Due to their potential for gene regulation, oligonucleotides have moved into focus as one of the preferred modalities modulating currently undruggable disease-associated targets. In course synthesis and storage a significant number compound-related impurities can be generated. Purification protocols analytical methods become crucial therapeutic application any oligonucleotides, they antisense (ASOs), small interfering ribonucleic acids (siRNAs) or conjugates. Ion-pair chromatography is...

There has been recent interest in trying to understand the connection between transfer RNA (tRNA) posttranscriptional modifications and changes in-cellular environmental conditions. Here, we report on identification of modified nucleoside 5-methylcytidine (m 5 C) Escherichia coli tRNAs. This modification was determined be present at position 49 tRNA Tyr-QUA-II. Moreover, m C levels this are significantly elevated under high reactive oxygen specieis (ROS) conditions E. cells. We identified...

Heterologous tRNA:aminoacyl tRNA synthetase pairs are often employed for noncanonical amino acid incorporation in the quest an expanded genetic code. In this work, we investigated one possible mechanism by which directed evolution can improve orthogonal behavior a suite of Methanocaldococcus jannaschii (Mj) tRNATyr-derived amber suppressor tRNAs. Northern blotting demonstrated that reduced expression heterologous variants correlated with improved orthogonality. We suspected likely minimized...

Abstract Liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) has become the gold‐standard technique to study RNA and its various modifications. While most research on nucleosides been focused their biological roles, discovery of new modifications remains interest. With state‐of‐the‐art technology, presence artifacts can confound identification Here, we report characterization a non‐natural mcm 5 isoC ribonucleoside in S. cerevisiae total tRNA hydrolysate by higher‐energy collisional...

Cryptococcus neoformans is a radiation resistant fungus that has been found growing at the site of nuclear reactor meltdown in Chernobyl, Ukraine. The Defense Threat Reduction Agency (DTRA) funds research on this organism to gain fundamental knowledge survival. Many DTRA projects cause soldiers be exposed radiation, therefore, gaining information may have future therapeutic implications. Radiation causes cellular damage through radiolysis water into reactive oxygen species (ROS) – such as...

The goal of this work was to profile the oxidative damage exerted by ultraviolet‐A radiation (UVA, 370 nm) or H 2 O on RNA modifications liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Oxidative impacts biological systems due its association with metabolic, mitochondrial, neurological diseases and cancer.1,2 In presence photosensitizer ( e.g. , riboflavin), UVA induces production reactive oxygen species (ROS, • HOO HO ) in vivo .3 Likewise, generates ROS under physiological...

Cryptococcus neoformans is a radiotrophic fungi capable of surviving in extreme environments. Understanding post‐transcriptional RNA modifications may lead to insight on translational regulation and protein synthesis. Little known about the role transfer (tRNA) C. . Modification profiles change during oxidative stress play biological roles cell survival. Here, impact peroxide exposure tRNA investigated through liquid chromatography coupled with mass spectrometry. Support or Funding...

Fungi have been found in extreme environments all over the world. Of particular interest, Cladosporium sphaerospermum is a filamentous fungus to grow around nuclear reactor at Chernobyl. Thus far, little known of mechanistic details that allow these fungi survive such harsh oxidative conditions. The current hypothesis pigment melanin contributes growth through enhancing energy production mechanisms. In this work, we aim identify effect presence on post‐transcriptional modifications transfer...