Thioguanosine Conversion Enables mRNA-Lifetime Evaluation by RNA Sequencing Using Double Metabolic Labeling (TUC-seq DUAL)

Temporal information about cellular RNA populations is essential to understand the functional roles of RNA. We have developed the hydrazine/NH₄Cl/OsO₄-based conversion of 6-thioguanosine (6sG) into A′, where A′ constitutes a 6-hydrazino purine derivative. A′ retains the Watson–Crick base-pair mode and is efficiently decoded as adenosine in primer extension assays and in RNA sequencing. Because 6sG is applicable to metabolic labeling of freshly synthesized RNA and because the conversion chemistry is fully compatible with the conversion of the frequently used metabolic label 4-thiouridine (4sU) into C, the combination of both modified nucleosides in dual-labeling setups enables high accuracy measurements of RNA decay. This approach, termed TUC-seq DUAL, uses the two modified nucleosides in subsequent pulses and their simultaneous detection, enabling mRNA-lifetime evaluation with unprecedented precision.     

Post‐synthetic Spin‐Labeling of RNA through Click Chemistry for PELDOR Measurements

Site‐directed spin labeling of RNA based on click chemistry is used in combination with pulsed electron‐electron double resonance (PELDOR) to benchmark a nitroxide spin label, called here d? . We compare this approach with another established method that employs the rigid spin label Çm for RNA labeling. By using CD spectroscopy, thermal denaturation measurements, CW‐EPR as well as PELDOR we analyzed and compared the influence of d? and Çm on a self‐complementary RNA duplex. Our results demonstrate that the conformational diversity of d? is significantly reduced near the freezing temperature of a phosphate buffer, resulting in strongly orientation‐selective PELDOR time traces of the d? ‐labeled RNA duplex.     

Efficient Solid‐Phase Synthesis of pppRNA by Using Product‐Specific Labeling

A novel solid‐phase synthesis and purification strategy for 5′‐triphosphate oligonucleotides by using lipophilic tagging of the triphosphate moiety is reported. This is based on triphosphate synthesis with 5′‐O‐cyclotriphosphate intermediates, whereby a lipophilic tag, such as decylamine, is introduced during the ring‐opening reaction to give a linear gamma‐phosphate‐tagged species. This method enables the highly efficient synthesis of 5′‐triphosphorylated RNA derivatives and their gamma‐phosphate‐substituted analogues and will especially facilitate the advancement of therapeutic approaches that make use of 5′‐triphosphate oligonucleotides as potent activators of the cytosolic immune sensor RIG‐I.     

Profiling of Ribose Methylations in RNA by High‐Throughput Sequencing

Ribose methylations are the most abundant chemical modifications of ribosomal RNA and are critical for ribosome assembly and fidelity of translation. Many aspects of ribose methylations have been difficult to study due to lack of efficient mapping methods. Here, we present a sequencing‐based method (RiboMeth‐seq) and its application to yeast ribosomes, presently the best‐studied eukaryotic model system. We demonstrate detection of the known as well as new modifications, reveal partial modifications and unexpected communication between modification events, and determine the order of modification at several sites during ribosome biogenesis. Surprisingly, the method also provides information on a subset of other modifications. Hence, RiboMeth‐seq enables a detailed evaluation of the importance of RNA modifications in the cells most sophisticated molecular machine. RiboMeth‐seq can be adapted to other RNA classes, for example, mRNA, to reveal new biology involving RNA modifications.     

Liquid‐Phase RNA Synthesis by Using Alkyl‐Chain‐Soluble Support

Recent progress in the RNA therapeutics has increased demand for the synthesis of large quantities of oligoribonucleotides. The assembly of RNA oligomers relies mainly on solid‐phase approaches. These allow rapid product purification and the ability to drive a target reaction to completion through the use of excess reagents. Despite the known advantages of solid‐phase synthesis, some issues in the process remain to be addressed, such as low and limited scale, reagent accessibility, and the use of a very large excess of reagents. Herein, we report a highly efficient and practical method of liquid‐phase synthesis of RNA oligomers by using alkyl‐chain‐soluble support. We demonstrate the utility of the liquid‐phase method through 21‐mer RNA synthesis on a gram scale.     

Chemical Deprenylation of N6‐Isopentenyladenosine (i6A) RNA

N⁶‐isopentenyladenosine (i⁶A) is an RNA modification found in cytokinins, which regulate plant growth/differentiation, and a subset of tRNAs, where it improves the efficiency and accuracy of translation. The installation and removal of this modification is mediated by prenyltransferases and cytokinin oxidases, and a chemical approach to selective deprenylation of i⁶A has not been developed. We show that a selected group of oxoammonium cations function as artificial deprenylases to promote highly selective deprenylation of i⁶A in nucleosides, oligonucleotides, and live cells. Importantly, other epigenetic modifications, amino acid residues, and natural products were not affected. Moreover, a significant phenotype difference in the Arabidopsis thaliana shoot and root development was observed with incubation of the cation. These results establish these small organic molecules as direct chemical regulators/artificial deprenylases of i⁶A.     

Naphthyridine‐Benzoazaquinolone: Evaluation of a Tricyclic System for the Binding to (CAG)n Repeat DNA and RNA

The expansion of CAG repeats in the human genome causes the neurological disorder Huntington's disease. The small‐molecule naphthyridine‐azaquinolone NA we reported earlier bound to the CAG/CAG motif in the hairpin structure of the CAG repeat DNA. In order to investigate and improve NA ‐binding to the CAG repeat DNA and RNA, we conducted systematic structure‐binding studies of NA to CAG repeats. Among the five new NA derivatives we synthesized, surface plasmon resonance (SPR) assay showed that all of the derivatives modified from amide linkages in NA to a carbamate linkage failed to bind to CAG repeat DNA and RNA. One derivative, NBzA , modified by incorporating an additional ring to the azaquinolone was found to bind to both d(CAG)₉ and r(CAG)₉. NBzA binding to d(CAG)₉ was similar to NA binding in terms of large changes in the SPR assay and circular dichroism (CD) as well as pairwise binding, as assessed by electron spray ionization time‐of‐flight (ESI‐TOF) mass spectrometry. For the binding to r(CAG)₉, both NA and NBzA showed stepwise binding in ESI‐TOF MS, and NBzA ‐binding to r(CAG)₉ induced more extensive conformational change than NA ‐binding. The tricyclic system in NBzA did not show significant effects on the binding, selectivity, and translation, but provides a large chemical space for further modification to gain higher affinity and selectivity. These studies revealed that the linker structure in NA and NBzA was suitable for the binding to CAG DNA and RNA, and that the tricyclic benzoazaquinolone did not interfere with the binding.     

Differentiate RNA Single‐Stranded Region of the Branched Structures and Hairpin Loops by an Octahedral Cobalt(II) Complex

Single‐stranded RNA molecules usually include secondary structural elements such as bulges, internal loops, and hairpin loops. These RNA secondary structural elements are often essential for the biological activity and functions of the RNA molecule. Chemical probe Co^II(HAPP)(TFA)₂ in the presence of H₂O₂ is found to differentiate single‐stranded RNA from branched structures and hairpin loops. This study uses Co^II(HAPP)(TFA)2 to analyze the structures of two RNA molecules: a fragment of HIV TAR RNA (TAR‐27) and the catalytic domain 5 of group II intron (D5‐29). The electrophoretic mobility of TAR‐27 does not shift in the presence of Co^II(HAPP)(TFA)₂, suggesting that the reagent does not change the conformation of RNA substrate. Cleavage of the RNA substrates by Co^II(HAPP)(TFA)₂ unambiguously differentiated RNA internal looped structures from hairpin loops. The results show that Co^II(HAPP)(TFA)2 is a sensitive, informative and convenient tool for analysis of RNA secondary structures.