ABSTRACT
A polymerase ribozyme can be used to label the 3' end of RNA or DNA molecules by incorporating a variety of functionalized nucleotide analogs. Guided by a complementary template, the ribozyme adds a single nucleotide that may contain a fluorophore, biotin, azide or alkyne moiety, thus enabling the detection and/or capture of selectively labeled materials. Employing a variety of commercially available nucleotide analogs, efficient labeling was demonstrated for model RNAs and DNAs, human microRNAs and natural tRNA.
Subject(s)
3' Flanking Region , DNA-Directed RNA Polymerases/metabolism , Nucleic Acids/metabolism , RNA, Catalytic/metabolism , Staining and Labeling/methods , Biotin/chemistry , Biotin/metabolism , DNA/chemistry , DNA/metabolism , Fluorescein/chemistry , Fluorescein/metabolism , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , MicroRNAs/chemistry , MicroRNAs/metabolism , Nucleic Acid Conformation , Nucleic Acids/chemistry , Nucleotidyltransferases/metabolism , RNA/chemistry , RNA/metabolism , RNA, Transfer/chemistry , RNA, Transfer/metabolism , Rhodamines/chemistry , Rhodamines/metabolismABSTRACT
A highly evolved RNA polymerase ribozyme was found to also be capable of functioning as a reverse transcriptase, an activity that has never been demonstrated before for RNA. This activity is thought to have been crucial for the transition from RNA to DNA genomes during the early history of life on Earth, when it similarly could have arisen as a secondary function of an RNA-dependent RNA polymerase. The reverse transcriptase ribozyme can incorporate all four dNTPs and can generate products containing up to 32 deoxynucleotides. It is likely that this activity could be improved through evolution, ultimately enabling the synthesis of complete DNA genomes. DNA is much more stable compared to RNA and thus provides a larger and more secure repository for genetic information.
Subject(s)
DNA/biosynthesis , RNA, Catalytic/metabolism , Reverse Transcription , RNA/metabolism , RNA, Catalytic/geneticsABSTRACT
N(2)-Furfuryl-deoxyguanosine (fdG) is carcinogenic DNA adduct that originates from furfuryl alcohol. It is also a stable structural mimic of the damage induced by the nitrofurazone family of antibiotics. For the structural and functional studies of this model N(2)-dG adduct, reliable and rapid access to fdG-modified DNAs are warranted. Toward this end, here we report the synthesis of fdG-modified DNAs using phosphoramidite chemistry involving only three steps. The functional integrity of the modified DNA has been verified by primer extension studies with DNA polymerases I and IV from E. coli. Introduction of fdG into a DNA duplex decreases the Tm by â¼1.6 °C/modification. Molecular dynamics simulations of a DNA duplex bearing the fdG adduct revealed that though the overall B-DNA structure is maintained, this lesion can disrupt W-C H-bonding, stacking interactions, and minor groove hydrations to some extent at the modified site, and these effects lead to slight variations in the local base pair parameters. Overall, our studies show that fdG is tolerated at the minor groove of the DNA to a better extent compared with other bulky DNA damages, and this property will make it difficult for the DNA repair pathways to detect this adduct.
Subject(s)
DNA Adducts/chemistry , DNA, B-Form/chemistry , DNA/chemistry , Deoxyguanosine/analogs & derivatives , Escherichia coli/chemistry , Base Pairing , DNA/metabolism , DNA Adducts/metabolism , DNA, B-Form/metabolism , Deoxyguanosine/chemistry , Hydrogen Bonding , Magnetic Resonance Spectroscopy , Molecular Dynamics SimulationABSTRACT
5-Formylcytosine (5fC) and 5-formyluracil (5fU) are natural nucleobase modifications that are generated by oxidative modification of 5-methylcytosine and thymine (or 5-methyluracil). Herein, we describe chemoselective labeling of 5-formylpyrimidine nucleotides in DNA and RNA by fluorogenic aldol-type condensation reactions with 2,3,3-trimethylindole derivatives. Mild and specific reaction conditions were developed for 5fU and 5fC to produce hemicyanine-like chromophores with distinct photophysical properties. Residue-specific detection was established by fluorescence readout as well as primer-extension assays. The reactions were optimized on DNA oligonucleotides and were equally suitable for the modification of 5fU- and 5fC-modified RNA. This direct labeling approach of 5-formylpyrimidines is expected to help in elucidating the occurrence, enzymatic transformations, and functional roles of these epigenetic/epitranscriptomic nucleobase modifications in DNA and RNA.
Subject(s)
DNA/chemistry , Fluorescent Dyes/chemistry , Pyrimidine Nucleotides/chemistry , RNA/chemistryABSTRACT
BACKGROUND: In this study, the measurement of job stress of electric overhead traveling crane operators and quantification of the effects of operator and workplace characteristics on job stress were assessed. METHODS: Job stress was measured on five subscales: employee empowerment, role overload, role ambiguity, rule violation, and job hazard. The characteristics of the operators that were studied were age, experience, body weight, and body height. The workplace characteristics considered were hours of exposure, cabin type, cabin feature, and crane height. The proposed methodology included administration of a questionnaire survey to 76 electric overhead traveling crane operators followed by analysis using analysis of variance and a classification and regression tree. RESULTS: The key findings were: (1) the five subscales can be used to measure job stress; (2) employee empowerment was the most significant factor followed by the role overload; (3) workplace characteristics contributed more towards job stress than operator's characteristics; and (4) of the workplace characteristics, crane height was the major contributor. CONCLUSION: The issues related to crane height and cabin feature can be fixed by providing engineering or foolproof solutions than relying on interventions related to the demographic factors.
ABSTRACT
The reduction in the efficacy of therapeutic antibiotics represents a global problem of increasing intensity and concern. Nitrofuran antibiotics act primarily through the formation of covalent adducts at the N(2) atom of the deoxyguanosine nucleotide in genomic DNA. These adducts inhibit replicative DNA polymerases (dPols), leading to the death of the prokaryote. N(2)-furfuryl-deoxyguanosine (fdG) represents a stable structural analog of the nitrofuran-induced adducts. Unlike other known dPols, DNA polymerase IV (PolIV) from E. coli can bypass the fdG adduct accurately with high catalytic efficiency. This property of PolIV is central to its role in reducing the sensitivity of E. coli toward nitrofuran antibiotics such as nitrofurazone (NFZ). We present the mechanism used by PolIV to bypass NFZ-induced adducts and thus improve viability of E. coli in the presence of NFZ. Our results can be used to develop specific inhibitors of PolIV that may potentiate the activity of nitrofuran antibiotics.
