RNA Ligation for Mono and Dually Labeled RNAs

Abstract

Labeled RNAs are invaluable probes for investigation of RNA function and localization. However, mRNA labeling remains challenging. Here, we developed an improved method for 3′‐end labeling of in vitro transcribed RNAs. We synthesized novel adenosine 3′,5′‐bisphosphate analogues modified at the N6 or C2 position of adenosine with an azide‐containing linker, fluorescent label, or biotin and assessed these constructs as substrates for RNA labeling directly by T4 ligase or via postenzymatic strain‐promoted alkyne‐azide cycloaddition (SPAAC). All analogues were substrates for T4 RNA ligase. Analogues containing bulky fluorescent labels or biotin showed better overall labeling yields than postenzymatic SPAAC. We successfully labeled uncapped RNAs, NAD‐capped RNAs, and 5′‐fluorescently labeled m7Gp3Am‐capped mRNAs. The obtained highly homogenous dually labeled mRNA was translationally active and enabled fluorescence‐based monitoring of decapping. This method will facilitate the use of various functionalized mRNA‐based probes.

Preparation of RNAs with non-canonical 5′ ends using novel di- and trinucleotide reagents for co-transcriptional capping

“…This purification was necessary in order to provide high efficiency DNAzyme cleavage. It has to be noted that we used a previously described HPLC purification method to purify our IVT RNAs (Depaix et al, 2021), eliminating impurities from IVT, where most capped and uncapped transcripts were co-eluting, except for FAD-RNAs. Assessing the capping efficiency required keeping uncapped and capped RNAs combined for further analysis, so the HPLC conditions were not optimized further.…”

“…In the case of NADlinked and FAD-linked RNAs, approximately 50% of produced RNA may remain uncapped, even under optimized conditions (Huang, 2003). To overcome some of these disadvantages, we and others have recently developed trinucleotide analogs of m 7 G caps and NAD caps (of Np n N′pG general structure), which provided higher RNA capping efficiencies than dinucleotides and enabled more structural variability within the position of the first transcribed nucleotide (Ishikawa et al, 2009;Sikorski et al, 2020;Depaix et al, 2021). Another class of dinucleotide analogs such as 5′-Dy-ApG has also been used as transcription initiators providing access to 5'-fluorescently labeled RNAs (Wang et al, 2013).…”

2’-O-methylation of the second transcribed nucleotide within mRNA 5’ cap impacts protein production level in a cell specific manner and contributes to RNA immune evasion

“…3A). RNAs with different cap structures were biotinylated using a pAp-biotin conjugate (25). Cell lysates were subsequently incubated with the prepared RNAs, and RNP complexes formed on capped transcripts were purified using streptavidin beads.…”

“…Microscale thermophoresis-based method, described previously in (25), with modifications, was applied to determine dissociation constant for complex encompassing eIF4E protein and capped short RNA. Fluorescent probe, murine eIF4E and capped short RNAs as ligands were utilized.…”

Chemo-enzymatische Synthese lichtaktivierbarer RNA & Synthese lichtregulierbarer Oligonukleotide zur spektroskopischen Strukturaufklärung

“…[119] Die Anwesenheit der 3'-Phosphatgruppe verhindert des Weiteren den multiplen Einbau mehrerer Bisphosphate, wodurch die Reaktion automatisch auf eine einzelne 3'-terminale Modifikation beschränkt ist. [115,118,119,121] Weitere Nukleosid-3',5'-Bisphosphate, die neben unmodifizierten Bisphosphaten auf diese Weise erfolgreich ligiert werden konnten, sind das radioaktiv-markierte [5'-32 P]pCp und p[ 125 I]Cp [120,122] , 5-Alkinuridin (pUp Alk ) [103] , 5-Fluoruridin (pFUp) [115] , das fluoreszierende Derivat N 6 -Ethenoadenosin (pƐAp) [123] , N 6 -6-Aminohexyl-Adenosin (p(A-HA)p) [124] , 8-Bromo-Adenosin (p(8-Br-A)p, Jena Bioscience) oder auch N 6 -6-Azidoethyl-Adenosin (pA Azido p) [125] (siehe Abb. 6).…”