The Transcriptome-Wide Mapping of 2-Methylthio-N6-isopentenyladenosine at Single-Base Resolution.

Hundreds of modified bases have been identified and enzymatically modified to transfer RNAs (tRNAs) to regulate RNA function in various organisms. 2-Methylthio-N6-isopentenyladenosine (ms2i6A), a hypermodified base found at tRNA position 37, exists in both prokaryotes and eukaryotes. ms2i6A is traditionally identified by separating and digesting each tRNA from total RNA using RNA mass spectrometry. A transcriptome-wide and single-base resolution method that enables absolute mapping of ms2i6A along with analysis of its distribution in different RNAs is lacking. Here, through chemoselective methylthio group bioconjugation, we introduce a new approach (redox activated chemical tagging sequencing, ReACT-seq) to detect ms2i6A transcriptome-wide at single-base resolution. Using the chemoselectivity between the methylthio group and oxaziridine group, ms2i6A is bio-orthogonally tagged with an azide group without interference of canonical nucleotides, advancing enrichment of methylthio group modified RNAs prior to sequencing. ReACT-seq was demonstrated on nine known tRNAs and proved to be highly accurate, and the reverse transcription stop (RT-stop) character enables ReACT-seq detection at single-base resolution. In addition, ReACT-seq identified that the modification of ms2i6A is conservative and may not exist in other RNAs.

Transcriptome-wide profiling of N 6-methyladenosine via a selective chemical labeling method.

Studies of chemical modifications on RNA have ushered in the field of epitranscriptomics. N 6-Methyladenosine (m6A) is the most typical RNA modification and is indispensable for basic biological processes. This study presents a chemical pulldown method (m6A-ORL-Seq) for transcriptome-wide profiling of m6A. Moreover, chemical labeling results in a specific reverse transcription (RT) truncation signature. This study has identified four thousand high-confidence m6A sites at single-base resolution in the human transcriptome. Unlike previously reported methods based on m6A-antibody or m6A-sensitive enzymes, the antibody/enzyme-free chemical method provides a new perspective for single-base m6A detection at the transcriptome level.

Inert Pepper aptamer-mediated endogenous mRNA recognition and imaging in living cells.

The development of RNA aptamers/fluorophores system is highly desirable for understanding the dynamic molecular biology of RNAs in vivo. Peppers-based imaging systems have been reported and applied for mRNA imaging in living cells. However, the need to insert corresponding RNA aptamer sequences into target RNAs and relatively low fluorescence signal limit its application in endogenous mRNA imaging. Herein, we remolded the original Pepper aptamer and developed a tandem array of inert Pepper (iPepper) fluorescence turn-on system. iPepper allows for efficient and selective imaging of diverse endogenous mRNA species in live cells with minimal agitation of the target mRNAs. We believe iPepper would significantly expand the applications of the aptamer/fluorophore system in endogenous mRNA imaging, and it has the potential to become a powerful tool for real-time studies in living cells and biological processing.

6-Iodopurine as a Versatile Building Block for RNA Purine Architecture Modifications.

Natural modified bases in RNA were found to be indispensable for basic biological processes. In addition, artificial RNA modifications have been a versatile toolbox for the study of RNA interference, structure, and dynamics. Here, we present a chemical method for the facile synthesis of RNA containing C6-modified purine. 6-Iodopurine, as a postsynthetic building block with high reactivity, was used for metal-free construction of C-N, C-O, and C-S bonds under mild conditions and C-C bond formation by Suzuki-Miyaura cross-coupling. Our strategy provides a convenient approach for the synthesis of various RNA modifications, especially for oligonucleotides containing specific structures.

Photoactive G-Quadruplex Ligand Identifies Multiple G-Quadruplex-Related Proteins with Extensive Sequence Tolerance in the Cellular Environment.

G-Quadruplex (G4) is a noncanonical nucleic acid secondary structure with multiple biofunctions. Identifying G4-related proteins (G4RPs) is important for understanding the roles of G4 in biology. Current methods to identify G4RPs include discovery from specific biological processes or in vitro pull-down assays with specific G4 sequences. Here, we report an in vivo strategy used to identify G4RPs with extensive sequence tolerance based on G4 ligand-mediated cross-linking. Applying this method, we identified 114 and 281 G4RPs in SV589 and MM231 cells, respectively. The results successfully overlapped with all the pull-down assay literature. Through the electrophoretic mobility shift assay (EMSA), we identified some new G4-binding proteins. Moreover, enhanced cross-linking and immunoprecipitation (eCLIP) confirmed that one newly identified G4-binding protein, SERBP1, interacts with G4 in the cellular environment. The method we developed provides a new strategy for identifying proteins that interact with nucleic secondary structures in cells and benefit the study of their biological roles.

5-Formyluracil as a cornerstone for aluminum detection in vitro and in vivo: a more natural and sustainable strategy.

5-Formyluracil (5fU) based probes were designed and synthesized to detect Al3+ ions in vitro and in biological systems. These probes were synthesised using furan-2-carbohydrazide (fr) and 5fU derivatives. We also selectively labelled 5-formyluracil in DNA with the help of Al3+.