547 Organische Chemie
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- Analysis of RNA Modifications (1)
- Helicene diimide (1)
- High-Throughput Sequencing Method, DZ-seq (1)
- Methyltransferase Ribozyme (1)
- Photoresponsive DNA Crosslinker (1)
- RNA Aptamer (1)
- RNA Enzymes (1)
- RNA-Cleaving Deoxyribozymes (1)
- Ribozyme-catalyzed RNA labeling (1)
- Tolane-Modified Fluorescent Nucleosides (1)
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- 682586 (4)
In this communication we describe a helically chiral push-pull molecule named 9,10-dimethoxy-[7]helicene diimide, displaying fluorescence (FL) and circularly polarised luminescence (CPL) over nearly the entire visible spectrum dependent on solvent polarity. The synthesised molecule exhibits an unusual solvent polarity dependence of FL quantum yield and nonradiative rate constant, as well as remarkable gabs and glum values along with high configurational stability.
A tolane-modified 5-ethynyluridine as a universal and fluorogenic photochemical DNA crosslinker
(2023)
We report the fluorescent nucleoside ToldU and its application as a photoresponsive crosslinker in three different DNA architectures with enhanced fluorescence emission of the crosslinked products. The fluorogenic ToldU crosslinking reaction enables the assembly of DNA polymers in a hybridization chain reaction for the concentration-dependent detectio of a specific DNA sequence.
RNA-cleaving deoxyribozymes have found broad application as useful tools for RNA biochemistry. However, tedious in vitro selection procedures combined with laborious characterization of individual candidate catalysts hinder the discovery of novel catalytic motifs. Here, we present a new high-throughput sequencing method, DZ-seq, which directly measures activity and localizes cleavage sites of thousands of deoxyribozymes. DZ-seq exploits A-tailing followed by reverse transcription with an oligo-dT primer to capture the cleavage status and sequences of both deoxyribozyme and RNA substrate. We validated DZ-seq by conventional analytical methods and demonstrated its utility by discovery of novel deoxyribozymes that allow for cleaving challenging RNA targets or the analysis of RNA modification states.
Nearly all classes of coding and non-coding RNA undergo post-transcriptional modification including RNA methylation. Methylated nucleotides belong to the evolutionarily most conserved features of tRNA and rRNA.1,2 Many contemporary methyltransferases use the universal cofactor S-adenosylmethionine (SAM) as methyl group donor. This and other nucleotide-derived cofactors are considered as evolutionary leftovers from an RNA World, in which ribozymes may have catalysed essential metabolic reactions beyond self-replication.3 Chemically diverse ribozymes seem to have been lost in Nature, but may be reconstructed in the laboratory by in vitro selection. Here, we report a methyltransferase ribozyme that catalyses the site-specific installation of 1-methyladenosine (m1A) in a substrate RNA, utilizing O6-methylguanine (m6G) as a small-molecule cofactor. The ribozyme shows a broad RNA sequence scope, as exemplified by site-specific adenosine methylation in tRNAs. This finding provides fundamental insights into RNA’s catalytic abilities, serves a synthetic tool to install m1A in RNA, and may pave the way to in vitro evolution of other methyltransferase and demethylase ribozymes.
General and efficient tools for site-specific fluorescent or bioorthogonal labeling of RNA are in high demand. Here, we report direct in vitro selection, characterization, and application of versatile trans-acting 2'-5' adenylyl transferase ribozymes for covalent and site-specific RNA labeling. The design of our partially structured RNA pool allowed for in vitro evolution of ribozymes that modify a predetermined nucleotide in cis (i.e. intramolecular reaction), and were then easily engineered for applications in trans (i.e. in an intermolecular setup). The resulting ribozymes are readily designed for specific target sites in small and large RNAs and accept a wide variety of N6-modified ATP analogues as small molecule substrates. The most efficient new ribozyme (FH14) shows excellent specificity towards its target sequence also in the context of total cellular RNA.
Large Stokes shift (LSS) fluorescent proteins (FPs) exploit excited state proton transfer pathways to enable fluorescence emission from the phenolate intermediate of their internal 4 hydroxybenzylidene imidazolone (HBI) chromophore. An RNA aptamer named Chili mimics LSS FPs by inducing highly Stokes-shifted emission from several new green and red HBI analogs that are non-fluorescent when free in solution. The ligands are bound by the RNA in their protonated phenol form and feature a cationic aromatic side chain for increased RNA affinity and reduced magnesium dependence. In combination with oxidative functional-ization at the C2 position of the imidazolone, this strategy yielded DMHBO\(^+\), which binds to the Chili aptamer with a low-nanomolar K\(_D\). Because of its highly red-shifted fluorescence emission at 592 nm, the Chili–DMHBO\(^+\) complex is an ideal fluorescence donor for Förster resonance energy transfer (FRET) to the rhodamine dye Atto 590 and will therefore find applications in FRET-based analytical RNA systems.