Diazirine Photocrosslinking Recruits Activated FTO Demethylase Complexes for Specific <i>N</i>⁶‑methyladenosine Recognition

<i>N</i>⁶-methyladenosine (m⁶A) is a prevalent modification of RNAs. m⁶A exists in mRNA and plays an important role in RNA biological pathways and in RNA epigenetic regulation. We applied diazirine photocrosslinking to the event of m⁶A recognition mediated by the fat mass and obesity associated (FTO) demethylase. A highly photoreactive diazirine adjacent to m⁶A on the RNA successfully recruited activated FTO complexes with an m⁶A preference. The process of recognition of m⁶A via FTO using diazirine photocrosslinking was controlled by the α-ketoglutarate (α-KG) cosubstrate and the Fe­(II) cofactor, which are involved in m⁶A oxidative demethylation. In addition, FTO bound to ssRNAs prior to the m⁶A recognition process. Diazirine photocrosslinking contributes to increasing the chances of capturing activated FTO complexes with specific m⁶A recognition and provides new insights into the dynamic FTO oxidative demethylation process

Photoregulation of a Peptide−RNA Interaction on a Gold Surface

The discovery of riboswitching has accelerated research on the interaction between RNA and small organic compounds. It will be important for biologists to artificially and reversibly control gene expression in vivo through the interaction of RNA and small molecules. In this paper, we report that RNA aptamers obtained from in vitro selection in which a photoresponsive short peptide containing the azobenzene moiety with flanking arginine residues on both sides as a ligand provided reversible binding to the ligand peptide immobilized onto the gold surface. We designed and synthesized a photoresponsive short peptide that can interact with RNA, can convert its conformation reversibly by photoirradiation, and can be produced on a large scale for in vitro selection. The RNA pool contained N70 random sequences, and after the eighth cycle, we identified RNA aptamers showing the Kd of about a few micromolar. A surface plasmon resonance (SPR) experiment revealed that RNA aptamers could bind to the trans-isomer of the peptide immobilized on the gold surface but not to the cis-peptide isomerized by photoirradiation with 360 nm light to the gold surface. The SPR signals were recovered after photoirradiation with 430 nm light, leading to isomerization of the peptide from cis to trans

Simultaneous and Traceless Ligation of Peptide Fragments on DNA Scaffold

Peptide ligation is an indispensable step in the chemical synthesis of target peptides and proteins that are difficult to synthesize at once by a solid-phase synthesis. The ligation reaction is generally conducted with two peptide fragments at a high aqueous concentration to increase the reaction rate; however, this often causes unpredictable aggregation and precipitation of starting or resulting peptides due to their hydrophobicities. Here, we have developed a novel peptide ligation strategy harnessing the two intrinsic characteristics of oligodeoxynucleotides (ODNs), i.e., their hydrophilicity and hybridization ability, which allowed increases in the water solubility of peptides and the reaction kinetics due to the proximity effect, respectively. Peptide–ODN conjugates that can be cleaved to regenerate native peptide sequences were synthesized using novel lysine derivatives containing conjugation handles and photolabile linkers, via solid-phase peptide synthesis and subsequent conjugation to 15-mer ODNs. Two complementary conjugates were applied to carbodiimide-mediated peptide ligation on a DNA scaffold, and the subsequent DNA removal was conducted by photoirradiation in a traceless fashion. This DNA scaffold-assisted ligation resulted in a significant acceleration of the reaction kinetics and enabled ligation of a hydrophobic peptide at a micromolar concentration. On the basis of this chemistry, a simultaneous ligation of three different peptide fragments on two different DNA scaffolds has been conducted for the first time

Hybridization-Sensitive Fluorescent Oligonucleotide Probe Conjugated with a Bulky Module for Compartment-Specific mRNA Monitoring in a Living Cell

Live-cell RNA imaging at specific intracellular locations is technically limited because of the diffusive nature of small oligonucleotide probes. The bulky fluorescent light-up probes that possess streptavidin or gold nanoparticles at the end of oligonucleotides were designed and synthesized. The bulky probes allowed nucleus- and cytoplasm-selective monitoring of endogenous mRNAs through nuclear and cytoplasmic microinjection, respectively. Simultaneous use of bulky and unbulky probes conjugated with different fluorescent dyes enabled dual color imaging of mRNAs present in nucleus and cytoplasm. Furthermore, we observed that the fluorescence near the cell edge in a living HeLa cell traveled over time in coordination with the dynamic formation and deformation of the pseudopodial protrusions after lipofection of the bulky probes

Hybridization-Sensitive Fluorescent Oligonucleotide Probe Conjugated with a Bulky Module for Compartment-Specific mRNA Monitoring in a Living Cell

Live-cell RNA imaging at specific intracellular locations is technically limited because of the diffusive nature of small oligonucleotide probes. The bulky fluorescent light-up probes that possess streptavidin or gold nanoparticles at the end of oligonucleotides were designed and synthesized. The bulky probes allowed nucleus- and cytoplasm-selective monitoring of endogenous mRNAs through nuclear and cytoplasmic microinjection, respectively. Simultaneous use of bulky and unbulky probes conjugated with different fluorescent dyes enabled dual color imaging of mRNAs present in nucleus and cytoplasm. Furthermore, we observed that the fluorescence near the cell edge in a living HeLa cell traveled over time in coordination with the dynamic formation and deformation of the pseudopodial protrusions after lipofection of the bulky probes

Base-Resolution Analysis of 5‑Hydroxymethylcytosine by One-Pot Bisulfite-Free Chemical Conversion with Peroxotungstate

5-Hydroxymethylcytosine (^hmC) is an essential intermediate in the active DNA demethylation pathway. Here we report a new base-resolution method for measuring ^hmC by combining peroxotungstate-mediated oxidation and sequencing analysis. We reveal that an oxidized product of ^hmC, trihydroxylated thymine (^thT), tolerated the incorporation of dATP as a substrate in the process of DNA polymerase elongation. By comparing the results of Sanger sequencing before and after the oxidation, we observed that ^hmC sites on single-stranded DNAs could be discriminated from unmethylated cytosines. We found that a thermal cycle condition during peroxotungstate treatment enhanced the oxidation reaction of ^hmC in double-stranded DNA. Furthermore, Illumina sequencing analysis of ^hmC-containing synthetic genome fragments enabled us to identify simultaneously the positions of ^hmC in base resolution. This bisulfite-free simple ^hmC detection technique could facilitate the acquisition of epigenomic information