Isomorphic Fluorescent Nucleosides Facilitate Real‐Time Monitoring of RNA Depurination by Ribosome Inactivating Proteins

Abstract

Ribosome‐inactivating proteins, a family of highly cytotoxic proteins, interfere with protein synthesis by depurinating a specific adenosine residue within the conserved α‐sarcin/ricin loop of eukaryotic ribosomal RNA. Besides being biological warfare agents, certain RIPs have been promoted as potential therapeutic tools. Monitoring their deglycosylation activity and their inhibition in real time have remained, however, elusive. Herein, we describe the enzymatic preparation and utility of consensus RIP hairpin substrates in which specific G residues, next to the depurination site, are surgically replaced with tzG and thG, fluorescent G analogs. By strategically modifying key positions with responsive fluorescent surrogate nucleotides, RIP‐mediated depurination can be monitored in real time by steady‐state fluorescence spectroscopy. Subtle differences observed in preferential depurination sites provide insight into the RNA folding as well as RIPs’ substrate recognition features.

Atypical Plant miRNA cal-miR2911: Robust Stability against Food Digestion and Specific Promoting Effect on Bifidobacterium in Mice

“…The high resistance in the gastric environment can be ascribed to the high GC content of miR2911, and a sequence feature provides less A nucleotide (2 out of 20), which is susceptible to depurination under acidic conditions, resulting in strandbreaking introduced digestion. 20,31 This agrees with the study by Zhou et al that the resistance of miRNA was positively correlated with its GC content. 26 One characteristic of gastric digestion is the strong acidic environment where only pepsin functions as a digestive enzyme.…”

CRISPR-Cas9 recognition of enzymatically synthesized base-modified nucleic acids

“…Previously, fluorescent th G-containing RNA oligonucleotides have been shown to function as probes in real-time assays for monitoring translation-related events (47)(48)(49). Here, we sought to exploit the unique photophysical properties of th G, such as visible light emission, high quantum yield and long excited state lifetime, in the context of sgRNA in the CRISPR-Cas9 gene-editing system.…”

Inherently Emissive Puromycin Analogues for Live Cell Labelling

“…Fluorescent, isomorphic and isofunctional purine and pyrimidine analogues derived from thieno[3,4‐ d ]‐pyrimidine and isothiazolo[4,3‐ d ]pyrimidine heterocyclic nuclei have been developed by replacing the imidazole ring with 5‐membered aromatic heterocycles [18–22] . Both emissive RNA alphabets exhibit significantly improved photophysical properties compared to their native counterparts as evidenced by their employment for analyzing enzymatic transformations and protein/nucleic acids interactions [23–28] . We have also explored the effect of exocyclic amine dialkylation on the fluorescence of both adenosine analogues [29] .…”

“…[18][19][20][21][22] Both emissive RNA alphabets exhibit significantly improved photophysical properties compared to their native counterparts as evidenced by their employment for analyzing enzymatic transformations and protein/nucleic acids interactions. [23][24][25][26][27][28] We have also explored the effect of exocyclic amine dialkylation on the fluorescence of both adenosine analogues. [29] Intriguingly, replacing the N 6 ,N 6 -dimethylamino with an azetidine dramatically increased the emission quantum yield of these purine mimics, particularly when strong electron-withdrawing groups were present at the 4-membered ring's 3-position [e.g., φ(H 2 O) = 0.64 for the 3,3-difluoroazetidine].…”