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.

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.

Selective Chemical Labeling and Sequencing of 5-Carboxylcytosine in DNA at Single-Base Resolution.

5-Carboxylcytosine (5caC) plays a vital role in the dynamics of DNA demethylation, and sequencing of its sites will help us dig out more biological functions of 5caC. Herein, we present a novel chemical method to efficiently label 5caC distinguished from other bases in DNA. Combined with bisulfite sequencing, 5caC sites can be located at single-base resolution, and the efficiency of 5caC labeling is 92% based on the Sanger sequencing data. Furthermore, dot blot assays have confirmed that 5caC-containing DNA isolated from HeLa cells was successfully labeled using our method. We expect that our strategy can be further applied to selectively tagging other carboxyl-modified bases and mapping their sites in RNA.

Precise Antibody-Independent m6A Identification via 4SedTTP-Involved and FTO-Assisted Strategy at Single-Nucleotide Resolution.

Innovative detection techniques to achieve precise m6A distribution within mammalian transcriptome can advance our understanding of its biological functions. We specifically introduced the atom-specific replacement of oxygen with progressively larger atoms (sulfur and selenium) at 4-position of deoxythymidine triphosphate to weaken its ability to base pair with m6A, while maintaining A-T* base pair virtually the same as the natural one. 4SedTTP turned out to be an outstanding candidate that endowed m6A with a specific signature of RT truncation, thereby making this "RT-silent" modification detectable with the assistance of m6A demethylase FTO through next-generation sequencing. This antibody-independent, 4SedTTP-involved and FTO-assisted strategy is applicable in m6A identification, even for two closely gathered m6A sites, within an unknown region at single-nucleotide resolution.

Label-free detection of pH based on the i-motif using an aggregation-caused quenching strategy.

A label-free and biocompatible pH sensor system based on the aggregation-caused quenching (ACQ) probe has been reported herein. The DNA i-motif, a kind of pH-triggered structure, affects the aggregation of PTCDI derivatives by structural switch that would provide significant fluorescence signals responding to the different pH values. Our method not only shows sensitive and reversible response to pH changes, but also could expand the detection range by allosteric control of the DNA i-motif.