55 research outputs found
Ribozyme‐Catalyzed Late‐Stage Functionalization and Fluorogenic Labeling of RNA
No full textSite-specific introduction of biorthogonal handles into RNAs is in high demand for decorating RNAs with fluorophores, affinity labels or other modifications. Aldehydes represent attractive functional groups for post-synthetic bioconjugation reactions. Here, we report a ribozyme-based method for the synthesis of aldehyde-functionalized RNA by directly converting a purine nucleobase. Using the methyltransferase ribozyme MTR1 as an alkyltransferase, the reaction is initiated by site-specific N1 benzylation of purine, followed by nucleophilic ring opening and spontaneous hydrolysis under mild conditions to yield a 5-amino-4-formylimidazole residue in good yields. The modified nucleotide is accessible to aldehyde-reactive probes, as demonstrated by the conjugation of biotin or fluorescent dyes to short synthetic RNAs and tRNA transcripts. Upon fluorogenic condensation with a 2,3,3-trimethylindole, a novel hemicyanine chromophore was generated directly on the RNA. This work expands the MTR1 ribozyme’s area of application from a methyltransferase to a tool for site-specific late-stage functionalization of RNA
Direct in vitro selection of trans-acting ribozymes for posttranscriptional, site-specific, and covalent fluorescent labeling of RNA
No full textGeneral 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
Ribozyme-Catalyzed Site-Specific Labeling of RNA Using O6-alkylguanine SNAP-Tag Substrates
No full textSite-specific modification of RNAs with functional handles enables studies of RNA structure, fate, function, and interactions. Ribozymes provide an elegant way to covalently modify RNA of interest (ROI). Here, we report that the methyltransferase ribozyme MTR1 can be employed as a versatile tool for RNA modification and labeling. Using O6-alkylguanine cofactors, designed in analogy to SNAP-tag substrates for protein labeling, MTR1 installs various bioorthogonal functional groups at N1 of a specific adenosine in the RNA target. In this application of ribozyme-catalyzed RNA labeling, MTR1 is now called SNAPR. In contrast to the self-labeling SNAP-tag, which is appended to the protein of interest, SNAPR is a truly intermolecular RNA catalyst (active in trans). SNAPR assembles with the ROI to the active ribozyme, allowing for the transfer of clickable tags, such as azide and alkyne moieties, as well as photolabile groups or cross-linkers from the guanine cofactor to the ROI. Moreover, we demonstrate a two-step approach to attach labels at N6 of the target adenosine: first, SNAPR generates N1A-modified RNA, followed by preparative Dimroth rearrangement to produce N6A-modified RNA. We demonstrate this strategy with p-azidobenzyl groups as photocrosslinker to generate covalent RNA-protein conjugates. Overall, this work expands the toolbox for site-specific RNA modification
Ribozyme‐Catalyzed Site‐Specific Labeling of RNA Using O6‐alkylguanine SNAP‐Tag Substrates
No full textIn vitro selektierte Ribozyme für Methylierung und Markierung von RNA
No full textThe focus of this work was the development and application of highly efficient RNA catalysts for the site-specific modification of RNA with special focus on methylation. In the course of this thesis, the first methyltransferase ribozyme (MTR1), which uses m6G as the methyl group donor was developed and further characterized. The RNA product was identified as the natural modification m1A. X-Ray crystallography was used to solve the 3D structure of the ribozyme, which directly suggested a plausible reaction meachnism. The MTR1 ribozyme was also successfully repurposed for a nucleobase transformation reaction of a purine nucleoside. This resulted in a formyl-imidazole moiety directly on the intact RNA, which was directly used for further bioconjugation reactions. Finally, additional selections and reselections led to the identification of highly active alkyltransferase ribozymes that can be used for the labeling of various RNA targetsDer Schwerpunkt dieser Arbeit lag auf der Entwicklung sowie Anwendung hocheffizienter RNA-Katalysatoren für die positionsspezifische Modifikation von RNA mit besonderem Fokus auf Methylierungen. Im Rahmen dieser Arbeit wurde das erste Methyltransferase-Ribozym (MTR1), das m6G als Methylgruppendonor verwendet, entwickelt und näher charakterisiert. Das RNA-Produkt wurde als die natürliche Modifikation m1A identifiziert. Mit Hilfe der Röntgenkristallographie wurde des Weiteren die 3D-Struktur des Ribozyms aufgeklärt, was direkt auf ein plausibles Reaktionsmuster schließen ließ. Das MTR1-Ribozym wurde zudem erfolgreich für eine Nukleobasen-Transformationsreaktion eines Purins verwendet, bei der eine Formyl-Imidazol-Einheit direkt an der intakten RNA gebildet wird. Dieses Reaktionsprodukt wurde für positionsgenaue Biokonjugationsreaktionen verwendet. Schließlich führten zusätzliche Selektionen und weitere Reselektionen zur Identifizierung hochaktiver Alkyltransferase-Ribozyme, die für die Markierung verschiedener Ziel-RNAs verwendet werden können
Structure and mechanism of the methyltransferase ribozyme MTR1
No full textRNA-catalysed RNA methylation was recently shown to be part of the catalytic repertoire of ribozymes. The methyltransferase ribozyme MTR1 catalyses the site-specific synthesis of 1-methyladenosine (mA) in RNA, using O-methylguanine (mG) as methyl group donor. Here we report the crystal structure of MTR1 at a resolution of 2.8 Å, which reveals a guanine binding site reminiscent of natural guanine riboswitches. The structure represents the postcatalytic state of a split ribozyme in complex with the m1A-containing RNA product and the demethylated cofactor guanine. The structural data suggest the mechanistic involvement of a protonated cytidine in the methyl transfer reaction. A synergistic effect of two 2'-O-methylated ribose residues in the active site results in accelerated methyl group transfer. Supported by these results, it seems plausible that modified nucleotides may have enhanced early RNA catalysis and that metabolite-binding riboswitches may resemble inactivated ribozymes that have lost their catalytic activity during evolution
New deoxyribozymes for the native ligation of RNA
No full textDeoxyribozymes (DNAzymes) are small, synthetic, single-stranded DNAs capable of catalysing chemical reactions, including RNA ligation. Herein, we report a novel class of RNA ligase deoxyribozymes that utilize 5’-adenylated RNA (5’-AppRNA) as the donor substrate, mimicking the activated intermediates of protein-catalyzed RNA ligation. Four new DNAzymes were identified by in vitro selection from an N40 random DNA library and were shown to catalyze the intermolecular linear RNA-RNA ligation via the formation of a native 3’-5’-phosphodiester linkage. The catalytic activity is distinct from previously described RNA-ligating deoxyribozymes. Kinetic analyses revealed the optimal incubation conditions for high ligation yields and demonstrated a broad RNA substrate scope. Together with the smooth synthetic accessibility of 5’-adenylated RNAs, the new DNA enzymes are promising tools for the protein-free synthesis of long RNAs, for example containing precious modified nucleotides or fluorescent labels for biochemical and biophysical investigations
Specific and General Information Sharing Among Academic Scientists
No full textWe provide theoretical and empirical evidence on the factors that influence the willingness of academic scientists to share research results. We distinguish between two types of sharing, specific sharing in which a researcher shares her data or materials with another and general sharing in which scientists report results to the entire community (as in conference presentations). We present two simple games in which scientists research a problem of scientific merit (with an associated prize of academic and/or commercial value). In both cases, the scientists have intermediate research results but none has solved the entire problem.We test these models using a unique survey of bio-scientists in the UK and Germany regarding their willingness to "share." Our results generally support both models. In both, sharing is negatively related to competition and the importance of patents. In other respects they differ markedly. For example, large teams are more likely to share specifically but less likely to share generally. Rank does not matter for general sharing, but it does for specific sharing, where untenured faculty are less likely to share. One important implication is that policies designed to enhance sharing must be tailored to the type of sharing.
Author Correction: The copy number and mutational landscape of recurrent ovarian high-grade serous carcinoma
No full textThe original version of this Article mistakenly misspelled “Carolin M. Sauer” from the author list as “Carolyn Sauer,” who is from the ‘CRUK Cambridge Institute, University of Cambridge, Cambridge, UK.’ This has been corrected in both the PDF and HTML versions of the Article. The original version of this Article mistakenly misspelled “Ionut-Gabriel Funingana” from the author list as “Ionat-Gabriel Funingana,” who is from the ‘CRUK Cambridge Institute, University of Cambridge, Cambridge, UK’ and ‘Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK.’ This has been corrected in both the PDF and HTML versions of the Article. The original version of this Article contained an error in Fig. 1a, in which a panel indicating copy number event analysis was missing from the study workflow diagram. This has been corrected in both the PDF and HTML versions of the Article. The original version of this Article contained an error in the Methods section which incorrectly read ‘Fastq sequencing reads were aligned to GRCh37 (g1kp2) using bwa samse.’ The correct version states ‘hs37d5’ in place of ‘glkp2.’ This has been corrected in both the PDF and HTML versions of the Article. The original version of this Article contained an error in the Data Availability Statement stating ‘The pre-processed single nucleotide variant and copy number data are available through a Zenodo data repository and can be downloaded here’ which linked to an incorrect version of the Zenodo repository. The correct link is as follows:. This has been corrected in both the PDF and HTML versions of the Article.</p
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