Premium
RNA Ligation for Mono and Dually Labeled RNAs
Author(s) -
Depaix Anaïs,
MlynarskaCieslak Agnieszka,
Warminski Marcin,
Sikorski Pawel J.,
Jemielity Jacek,
Kowalska Joanna
Publication year - 2021
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.202101909
Subject(s) - rna , linker , dna ligase , chemistry , messenger rna , biotin , fluorescence , biotinylation , biochemistry , rna ligase , microbiology and biotechnology , biophysics , biology , enzyme , gene , physics , quantum mechanics , computer science , operating system
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 m 7 Gp 3 A m ‐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.