The mechanism of action of various viruses has been the primary focus of many studies. Yet, the data on RNA modifications in any type of virus are scarce. Methods for the sensitive analysis of RNA modifications have been developed only recently and they have not been applied to viruses. In particular, the RNA composition of HIV-1 virions has never been determined with sufficiently exact methods. Here, we reveal that the RNA of HIV-1 virions contains surprisingly high amount of the 1-methyladenosine. We are the first to use a liquid chromatography-mass spectrometry analysis (LC/MS) of virion RNA, which we combined with m1A profiling and deep sequencing. We found that m1A was present in the tRNA, but not in the genomic HIV-1 RNA and the abundant 7SL RNA. We were able to calculate that an HIV-1 virion contains per 2 copies of genomic RNA and 14 copies of 7SL RNA also 770 copies of tRNA, which is approximately 10 times more than thus far expected. These new insights into the composition of the HIV-1 virion can help in future studies to identify the role of nonprimer tRNAs in retroviruses. Moreover, we present a promising new tool for studying the compositions of virions.
- MeSH
- adenosin analogy a deriváty metabolismus MeSH
- chromatografie kapalinová metody MeSH
- genom virový genetika MeSH
- HIV-1 genetika fyziologie MeSH
- hmotnostní spektrometrie metody MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- RNA malá cytoplazmatická genetika MeSH
- RNA transferová genetika metabolismus MeSH
- RNA virová genetika metabolismus MeSH
- sekvence nukleotidů MeSH
- sestavení viru genetika MeSH
- signál-rozpoznávající částice genetika MeSH
- virion genetika metabolismus MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
An electrochemical genosensor for the detection and quantification of Karlodinium armiger is presented. The genosensor exploits tailed primers and ferrocene labelled dATP analogue to produce PCR products that can be directly hybridised on a gold electrode array and quantitatively measured using square wave voltammetry. Tailed primers consist of a sequence specific for the target, followed by a carbon spacer and a sequence specifically designed not to bind to genomic DNA, resulting in a duplex flanked by single stranded binding primers. The incorporation of the 7-(ferrocenylethynyl)-7-deaza-2'-deoxyadenosine triphosphate was optimised in terms of a compromise between maximum PCR efficiency and the limit of detection and sensitivity attainable using electrochemical detection via hybridisation of the tailed, ferrocene labelled PCR product. A limit of detection of 277aM with a linear range from 315aM to 10 fM starting DNA concentration and a sensitivity of 122 nA decade-1 was achieved. The system was successfully applied to the detection of genomic DNA in real seawater samples.
- MeSH
- biosenzitivní techniky přístrojové vybavení MeSH
- deoxyadeninnukleotidy chemie MeSH
- design vybavení MeSH
- DNA analýza MeSH
- elektrochemické techniky přístrojové vybavení MeSH
- limita detekce MeSH
- metaloceny chemie MeSH
- mikroelektrody MeSH
- mořská voda analýza MeSH
- oxidace-redukce MeSH
- polymerázová řetězová reakce přístrojové vybavení MeSH
- železnaté sloučeniny chemie MeSH
- Publikační typ
- časopisecké články MeSH
5-[(p-Carborane-2-yl)ethynyl]-2'-deoxyuridine 5'-O-triphosphate was synthesized and used as a good substrate in enzymatic construction of carborane-modified DNA or oligonucleotides containing up to 21 carborane moieties in primer extension reactions by DNA polymerases.
- MeSH
- borany chemie MeSH
- deoxyuracilnukleotidy chemie MeSH
- DNA primery chemie metabolismus MeSH
- DNA-dependentní DNA-polymerasy metabolismus MeSH
- DNA biosyntéza chemie MeSH
- oligonukleotidy biosyntéza chemie MeSH
- polymerázová řetězová reakce MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Nucleosides and 2'-deoxyribonucleoside triphosphates (dNTPs) bearing phenothiazine (PT) attached to a nucleobase (cytosine or 7-deazaadenine) either directly or through an acetylene linker were prepared through Suzuki or Sonogashira cross-coupling and triphosphorylation, and were studied as building blocks for polymerase construction of modified DNA. The directly PT-substituted dNTPs were better substrates for polymerases than the alkyne-linked dNTPs but all of them were used in enzymatic synthesis of DNA using primer extension, nicking enzyme amplification, PCR or 3'-tail labelling by terminal deoxynucleotidyl transferase. The phenothiazine served as an oxidizable redox label (giving two analytically useful signals of oxidation on electrode) for nucleosides and DNA and was also used in orthogonal combination with previously developed benzofurazane or nitrophenyl labels for redox coding of DNA bases. Therefore, the title PT-linked dNTPs are useful additions to the portfolio of nucleotides for enzymatic synthesis of redox-labelled DNA for electrochemical analysis.
2.vyd. 159 s.
- Klíčová slova
- výtvarná činnost, tvořivé hry,
- MeSH
- dítě MeSH
- Check Tag
- dítě MeSH
