General Approach for Introduction of Various Chemical Labels in Specific RNA Locations Based on Insertion of Amino Linkers

Abstract

Introduction of reporter groups at designed RNA sites is a widely accepted approach to gain information about the molecular environment of RNAs in their complexes with other biopolymers formed during various cellular processes. A general approach to obtain RNAs bearing diverse reporter groups at designed locations is based on site-specific insertion of groups containing primary aliphatic amine functions (amino linkers) with their subsequent selective derivatization by appropriate chemicals. This article is a brief review on methods for site-specific introduction of amino linkers in different RNAs. These methods comprise: (i) incorporation of a nucleoside carrying an amino-linker or a function that can be substituted with it into oligoribonucleotides in the course of their chemical synthesis; (ii) assembly of amino linker-containing RNAs from short synthetic fragments via their ligation; (iii) synthesis of amino linker-modified RNAs using T7 RNA polymerase; (iv) insertion of amino linkers into unmodified RNAs at functional groups of a certain type such as the 5'-phosphates and N7 of guanosine residues and (v) introduction of an amino linker into long highly structured RNAs exploiting an approach based on sequence-specific modification of nucleic acids. Particular reporter groups used for derivatization of amino linker-containing RNAs together with types of RNA derivatives obtained and fields of their application are presented.

Doubly Spin-Labeled RNA as an EPR Reporter for Studying Multicomponent Supramolecular Assemblies

“…In such structures, natural RNAs might be replaced by their derivatives bearing spin labels at various positions. For example, labels can be attached at the atoms C8 of adenosine and C5 of uridine of synthetic RNAs via amino linkers, which are easily introduced at the respective positions during automated RNA synthesis yielding RNA derivatives up to 70 nucleotides long (48). EPR data on structure and dynamics of RNAs in biologically significant multicomponent supramolecular complexes might be of great importance, helping to understand the molecular mechanisms of their functioning and opening ways for elaborating novel biomedical applications of these RNAs.…”

Evidence for a Negative Cooperativity between eIF5A and eEF2 on Binding to the Ribosome

“…By using a more physiologically relevant pre-assembled 80S complex (80S), it was possible to detect an increase by at least 3-fold in the binding affinity of eIF5A for the ribosome. Importantly, both the 40S+60S+tRNA and pre-assembled 80S complexes likely contain an enriched amount of PRE-state ribosomes containing tRNA in the P-site [ 47 ], due to the favorite stability of the tRNAs [ 48 ]. This observation strongly suggests that charged-tRNA i Met in the P-site strengthens the affinity of eIF5A to the ribosome by increasing the points of contact through the interaction eIF5A-Hyp-charged-tRNA i Met .…”

RNA editing by T7 RNA polymerase bypasses InDel mutations causing unexpected phenotypic changes

“…T7-based expression systems are broadly employed to prepare biologically active mRNA in vivo ( 3 , 4 ) and in vitro ( 5 , 6 ). Preparative quantities of a defined length of RNA is generated by run off transcription ( 7 ), often as labeled RNA probes ( 8 ) and also by amplification of a linear aRNA (amplified a ntisense) based on cDNA ( 9 , 10 ). T7 RNAP rifampicin-resistance allows directly for exclusive overexpression of the target gene, and indirectly, for specific protein radiolabeling ( 3 ).…”