Chloroacetamide‐Modified Nucleotide and RNA for Bioconjugations and Cross‐Linking with RNA‐Binding Proteins

Abstract

Reactive RNA probes are useful for studying and identifying RNA-binding proteins. To that end, we designed and synthesized chloroacetamide-linked 7deaza-ATP which was a good substrate for T7 RNA polymerase in in vitro transcription assay to synthesize reactive RNA probes bearing one or several reactive modifications. Modified RNA probes reacted with thiolcontaining molecules as well as with cysteine-or histidine-containing peptides to form stable covalent products. They also reacted selectively with RNA-binding proteins to form cross-linked conjugates in high conversions thanks to proximity effect. Our modified nucleotide and RNA probes are promising tools for applications in RNA (bio)conjugations or RNA proteomics.

Expedient production of site specifically nucleobase-labelled or hypermodified RNA with engineered thermophilic DNA polymerases

“…Herein we elaborated an efficient route to nucleobase-modified RNA that is based on previously reported 43 – 45 engineered thermophilic primer-dependent DNA polymerases. While by means of IVT with T7 RNAP it has been possible to install smaller, dispersed modifications along whole RNA length 30 – 32 , 34 , 35 , we demonstrated superior performance of engineered thermophilic polymerases in several aspects (Fig. 3 , Supplementary Fig.…”

“…We synthesized a full set of unnatural rN X TPs bearing clickable (ethynyl, E) or hydrophobic groups of increasing bulkiness, pentyn-1-yl (Pent) and phenyl (Ph), attached to each pyrimidine or 7-deazapurine nucleotide (rA X TP, rU X TP, rC X TP, rG X TP). Further we installed reactive functionalities chloroacetamide (CA) or formylthienyl (FT) on rA X TP, rU X TP and rC X TP, which are useful for covalent capturing of RNA interacting proteins 35 . We also synthesized rC mBdp TP bearing fluorescent tag (mBdp) and also included commercial rN X TPs bearing cyanine dyes (Cy5, Cy3) and affinity tags (Bio, Dig), as well as epitranscriptomic (Me) nucleobase modifications.…”

“…Enzymatic in vitro transcription (IVT) mediated by T7 RNA polymerase (T7 RNAP) 28 allows to attain longer sequences 29 . We and others have extensively explored landscape of nucleobase-modified RNA synthesis by IVT with T7 RNAP 30 – 35 . It has been shown that this enzyme has limited tolerance to bulky or 7-deazaguanine modifications 32 , requires optimal GGG trinucleotide initiation sequence 36 and is prone to termination when transcribing heavily structured RNA 37 .…”

Artificial nucleotide codons for enzymatic DNA synthesis

“…In the case of nucleic acids, the co-polymerization of modified nucleoside triphosphates enables Darwinian selection experiments to raise functional nucleic acids with enhanced properties, 9,[20][21][22] expand the genetic alphabet with orthogonal base pairs, 12,[23][24][25] and functionally tag DNA and RNA. [26][27][28] Despite well-established protocols for the enzymatic synthesis of chemically altered oligonucleotides, most processes still rely on the stepwise incorporation of modified nucleotides by the means of natural or engineered polymerases. 29 Chemoenzymatic methods that would favor poly-rather than mono-nucleotide incorporation events would be particularly useful, especially for the production of long RNA and XNA sequences.…”

Artificial nucleotide codons for enzymatic DNA synthesis

“…In the case of nucleic acids, the co-polymerization of modified nucleoside triphosphates enables Darwinian selection experiments to raise functional nucleic acids with enhanced properties, 8,[17][18][19] expand the genetic alphabet with orthogonal base pairs, 11,20,21 and functionally tag DNA and RNA. [22][23][24] Despite well-established protocols, most processes still rely on the stepwise incorporation of modified nucleotides by polymerases (Fig. 1).…”