Repurpsing Antiviral Drugs for Orthogonal RNA-Catalyzed Labeling

In vitro selected ribozymes are promising tools for site-specific labeling of RNA. Previously known nucleic acid catalysts attached fluorescently labeled adenosine or guanosine derivatives through 2’,5’-branched phosphodiester bonds to the RNA of interest. Herein, we report new ribozymes that use orthogonal substrates, derived from the antiviral drug tenofovir, and attach bioorthogonal functional groups, as well as affinity handles and fluorescent reporter units through a hydrolytically more stable phosphonate ester linkage. The tenofovir transferase ribozymes were identified by in vitro selection and are orthogonal to nucleotide transferase ribozymes. As genetically encodable functional RNAs, these ribozymes may be developed for potential cellular applications. The orthogonal ribozymes addressed desired target sites in large RNAs in vitro, as shown by fluorescent labeling of E. coli 16S and 23S RNAs in total cellular RNA

Direct in vitro selection of trans-acting ribozymes for posttranscriptional, site-specific, and covalent fluorescent labeling of RNA

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

Site-specific RNA methylation by a methyltransferase ribozyme

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

Chromosomal radiosensitivity in lymphocytes of head and neck cancer patients as determind by mironucleus and G2 assays

Background: It is reported that high frequency of chromosomal aberrations in peripheral blood lymphocytes of individuals is a marker of cancer predisposition. The aim of this study was to investigate the in vitro frequency of chromosomal damage in lymphocytes of patients with head and neck cancer against gamma irradiation compared with those in healthy individuals. Methods: In a case and control study, peripheral blood lymphocytes of 101 patients with head and neck cancer were collected before the onset of radiotherapy. Lymphocytes of 40 healthy individuals were also collected as controls. Head and neck cancer patients and the control group were consecutively recruited between April 2012 and February 2015 from Clinics of Cancer Institute, Imam Khomeini Hospital, Tehran, Iran. Lymphocytes of patients or control group were cultured and exposed to gamma radiation in G2- and G0- phase of the cell cycle. The induced chromosomal aberrations such as chromosome and chromatid breakages, chromosome and chromatid gaps, chromatid exchanges and micronuclei were scored in one-hundred metaphase cells of each individual. The mean of each chromosomal aberration was compared in patient and control groups. Early and late tissue reactions were scored during radiotherapy treatment or thereafter. Results: There was no significant difference in demographic characterization between the two study groups. The frequency of radiation- induced G2 aberrations in lymphocytes of patients was significantly higher than in those of healthy donors (P= 0.001 for chromosomal breaks). The frequency of radiation-induced micronuclei in G0 assay was also higher in patients than in those in controls (P= 0.05). The results also indicate that there is no correlation between the two assays. No significant correlation was also observed between aberration frequencies in lymphocytes and the degree of both early and late normal tissue reactions. Conclusion: The results indicate that the in vitro chromosomal radiosensitivity of peripheral blood lymphocytes of patients with head and neck cancer against gamma irradiation was significantly higher than that in healthy individuals