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Super high throughput testing using Next Generation Sequencing

This is not a method for RNA isolation but rather the replacement of testing by qPCR by a far more high throughput method – millions of tests in one experiment . When RNA isolation problem stops being a bottle neck in testing, the method proposed below will remove the need for qPCR, which, in turn, will become the new bottle neck. The scheme of the process is shown in attached Figure. - The reverse transcription primer contains sequence standard for Illumina flow cell (blue), followed...

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RT-primers bound to streptavidin beads: No RNA extraction and No commercial kits are required; the whole testing procedure in one tube; compatible with almost any storage/transport solution

I suggest using magnetic streptavidin beads with immobilised biotinylated single-stranded DNA primers for reverse transcription (RT), which then can be used through all steps of testing.  All steps can be performed in a single tube, with almost no restriction on the composition of the storage/transport/lysis buffer.   The method can be simplified even further and made more high throughput by using streptavidin-coated micro-titre plates or tubes compatible with qPCR machines. - The...

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Nikolay Zenkin

Newcastle upon Tyne, United Kingdom

Joined this community on Apr 13, 2020

Bio CURRICULUM VITAE Full Name and Title: Nikolay Zenkin, PhD (Russian/British) Date of birth: 26/07/1979 Place of birth: Dubna (Moscow Region), Russia Title of current appointment: Professor of Molecular Biology; Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University Education and Qualification: 1996-2001 B.A., M.S. (Biochemistry). Moscow State University (Biological Faculty, Department of Molecular Biology) 2001-2004 Ph.D. (Molecular Biology). Institute of Molecular Genetics, Moscow. Appointments held: 1999-2000 Research Trainee. Engelhardt Institute of Molecular Biology, Moscow 2000-2001 Research Trainee. Institute of Molecular Genetics, Moscow 2001-2004 Ph.D. Student (Molecular Biology). Institute of Molecular Genetics, Moscow 2002-2004 Research Associate. Waksman Institute, Rutgers University, NJ 2004-2007 Post-Doctoral Research Associate. Waksman Institute, Rutgers University, NJ 2007-2009 Lecturer. Institute for Cell and Molecular Biosciences of Newcastle University. 2009-2013 Senior Lecturer. Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University. 2013-present Professor of Molecular Biology; Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University Honours and Awards: 2019 – Wellcome Trust Molecular Basis of Cell Function Expert Review Group 2019 – Wellcome Trust Investigator Award 2017 – University nominee for Blavatnik award 2017 – Organiser of 29th UK RNA polymerase workshop in Newcastle University 2014 – Philip Leverhulme Prize 2014 – Newcastle University Academic Distinction Award 2014 – Organiser of 26th UK RNA polymerase workshop in Newcastle University 2014 – Fleming Prize Lecture Award 2014 – Wellcome Trust Senior Investigator Award 2006 – Manchester University Research Fellowship (declined by applicant on account of Lecturer position) 2006 – Alexander von Humboldt Foundation Research Fellowship (declined by applicant on account of Lecturer position) 2019 – External PhD examiner York University 2016 – External PhD examiner CNRS, Montpellier University External referee for research and review papers, BBSRC, MRC, Welcome Trust, Leverhulme, Singapore NMRC research grants. Funding (sole PI unless otherwise stated): 2020 – Wellcome Trust Investigator Award: “Coupling of transcription with other cellular processes” (PI; £2.1M) 2019 – MICA MRC Grant “Novel mode of RNA polymerase inhibition by a new natural rifamycin, which is active against rifampicin-resistant RNA polymerases and bacteria” (PI; £800K) 2019 – EPSRC Programme Grant “The Physics of Antimicrobial Resistance” (Co-I; £3.5M) 2017 – Royal Society collaborative grant (PI; £12K) 2016 – EPSRC Programme Grant “Portabolomics” (Co-I; £7.5M) 2014 – Philip Leverhulme Prize (PI; £100K) 2014 – Wellcome Trust Senior Investigator Award: “Transcription: from catalysis to cellular regulation” (PI; £1.2M) 2013 – BBSRC Research Grant: “Mechanisms of transcription termination” (PI; £ 441K) 2012 – Russian Ministry of Education and Science Research Grant ”Bioengineering of mitochondria: directed modification of protein biosynthesis in mitochondria” (PI; ~£60K) 2012 – BBSRC Research Grant: “Regulation of elongation by RNA polymerase and ribosome via intrinsic signals and transcription-translation coupling” (PI; £692K) 2010 – System Biology in Microorganisms (SysMo) Research Grant (Funded by BBSRC): “Role of transcriptional fidelity and processivity on the noise of transcription, and its consequences for phenotypic bistability in Streptococcus pneumoniae” (PI; £360K) 2008 – European Research Council Starting Grant 2007; "Mechanisms of Transcription Proofreading" (PI; EUR 1.15M) 2008 – BBSRC Research Grant; "Transcription elongation processivity: resolution of backtracked complexes " (PI; £461K) 2008 – BBSRC Research Grant; “Characterisation of novel functions of the active centre of RNA polymerase” (PI; £600K) 2007 – Royal Society Research Grant; “Investigation of cleavage reactions by RNA polymerase” (PI; £15K) Competitive studentships: 2017 – EPSRC PhD studentship 2017 – BBSRC PhD studentship 2016 – MRC CASE PhD studentship 2013 – BBSRC PhD studentship 2009 – MRC PhD studentship 2008 – BBSRC PhD studentship Training record: PIs: -Dr Yulia Yuzenkova, Royal Society University Research Fellow, Senior Lecturer, Newcastle University -Dr Katherine James, Head of Bioinformatics, National History Museum London; Principal Fellow, Northumbria University. Post-Docs: -14, all (apart from two who went on to work in industry, and one for government) are continuing in academia in my or other labs (Birmingham, Edinburgh, Manchester, Copenhagen, Umea Universities). Post-graduate Students: -7 PhD and 1 MPhil students to completion -currently supervise 3 and directly co-supervise 3 PhD students. Invited speaker Invited lectures in Umea, Uppsala, Groningen, York, Bristol, Moscow State Universities, and Russian Academy of Sciences Federation of American Societies for Experimental Biology research conference “Mechanism & Regulation of Prokaryotic Transcription”, 2017. Saxtons River, VT, USA Zing conference “Regulating with RNA in Bacteria and Archaea” 2015 Cancun, Mexico Federation of American Societies for Experimental Biology research conference “Mechanism & Regulation of Prokaryotic Transcription”, 2015. Saxtons River, VT, USA Zing conference “Nucleic Acids” 2014 Cancun, Mexico Fleming Award Lecture, Society for General Microbiology, 2014. Liverpool, UK Federation of American Societies for Experimental Biology research conference “Mechanism & Regulation of Prokaryotic Transcription”, 2013. Saxtons River, VT, USA Invited Lecture. 2012. Bristol University, UK Society for General Microbiology Spring Conference, 2012. Dublin, Ireland Federation of American Societies for Experimental Biology research conference, “Mechanism and Regulation of Prokaryotic transcription”, 2011. Saxtons River, VT, USA 69th Harden Conference “RNAP - Structure, function and evolution of RNA polymerases”. 2010. Hinxton, Cambridge, UK Federation of American Societies for Experimental Biology research conference “Nucleic Acid Enzymes”, 2010. Snowmass Village, Colorado, USA Federation of American Societies for Experimental Biology research conference, “Mechanism and Regulation of Prokaryotic transcription”, 2009. Saxtons River, VT, USA American Society for Microbiology 108th General Meeting, 2008 UK RNA polymerase workshop, York University, 2008. Federation of American Societies for Experimental Biology research conference, “Mechanism and Regulation of Prokaryotic transcription”, 2007. Saxtons River, VT, USA (n/a) UK RNA polymerase workshop, Imperial College of London, 2007. Federation of American Societies for Experimental Biology research conference, “Mechanism and Regulation of Prokaryotic transcription”, 2005. Saxtons River, VT, USA Federation of American Societies for Experimental Biology research conference “Mechanism and Regulation of Prokaryotic transcription”, 2003. Saxtons River, VT, USA Book chapters: Castro-Roa D and Zenkin N. Relations Between Replication and Transcription. In: Kušić-Tišma, J, ed. Fundamental Aspects of DNA Replication. Rijeka, Croatia: InTech Open, 2011, pp.289-306 Nechaev S., Zenkin N. and Severinov K. Regulation of RNA polymerase through its active center. In: Buc, H., and Strick, T. ed. RNA polymerases as molecular motors. Cambridge, UK: RSC Pub., 2009, pp 236-259 Patents: “Compounds for treating infections caused by rifampicin-resistant bacteria” GB Initial filing-GB201812078, PCT Filing-WO2020/021252 Publications (*corresponding) 1. Stevenson-Jones F., Woodgate J., Castro-Roa D, Zenkin N*. (2020) Ribosome reactivates transcription by physically pushing RNA polymerase out of transcription arrest. Proc Natl Acad Sci U S A. pii: 201919985. doi: 10.1073/pnas.1919985117 2. Mosaei H., Molodtsov, V., Kepplinger B., Harbottle J., Moon C., Jeeves R., Ceccaroni L., Shin, Y., Morton-Laing S., Marrs M., Wills C., Clegg W., Yuzenkova Y., Perry J., Bacon J., Errington J., Allenby N., Hall M., Murakami K., Zenkin N*.(2018) Mode of Action of Kanglemycin A, an Ansamycin Natural Product that Is Active against Rifampicin-Resistant Mycobacterium tuberculosis. Molecular Cell 72(2):263 doi: 10.1016/j.molcel.2018.08.028 3. Talavera A, Hendrix J, Versées W, Jurėnas D, Van Nerom K, Vandenberk N, Singh RK, Konijnenberg A, De Gieter S, Castro-Roa D, Barth A, De Greve H, Sobott F, Hofkens J, Zenkin N, Loris R, Garcia-Pino A. (2018) Phosphorylation decelerates conformational dynamics in bacterial translation elongation factors. Science Advances. 4:eaap9714. doi: 10.1126/sciadv.aap9714. 4. Gamba, P., Zenkin, N*. (2018) Transcription fidelity and its roles in the cell. Current Opinion in Microbiology. 42:13. doi: 10.1016/j.mib.2017.08.004. 5. Forrest, D., James, K., Yuzenkova, Y., Zenkin, N*. (2017) Single-peptide DNA-dependent RNA polymerase homologous to multi-subunit RNA polymerase. Nature Communications. 8:15774. doi: 10.1038/ncomms15774. 6. James, K., Cockell, S.J., Zenkin, N*. (2017) Deep sequencing approaches for the analysis of prokaryotic transcriptional boundaries and dynamics. Methods. 120:76-84. doi: 10.1016/j.ymeth.2017.04.016. 7. Tyler, A.R., Mosaei, H., Morton, S., Waddell, P.G., Wills, C., McFarlane, W., Gray, J., Goodfellow, M., Errington, J., Allenby, N., Zenkin, N., Hall, M.J. Structural Reassignment and Absolute Stereochemistry of Madurastatin C1 (MBJ-0034) and the Related Aziridine Siderophores: Madurastatins A1, B1, and MBJ-0035. J Nat Prod. (2017). 80:558-1562 doi: 10.1021/acs.jnatprod.7b00082 8. Gamba, P., James, K., Zenkin, N*. A link between transcription fidelity and pausing in vivo. Transcription. 2017 8:99-105. doi: 10.1080/21541264.2016.1274812. Epub 2017 Jan 10. 9. James, K., Gamba, P., Cockell, S.J., Zenkin, N*. (2017) Misincorporation by RNA polymerase is a major source of transcription pausing in vivo. Nucleic Acids Res. 45:1105-1113. doi: 10.1093/nar/gkw969. 10. van Nues, R. W., Castro-Roa, D., Yuzenkova, Y., Zenkin, N*.(2016) Ribonucleoprotein particles of bacterial small non-coding RNA IsrA (IS61 or McaS) and its interaction with RNA polymerase core may link transcription to mRNA fate. Nucleic Acids Res, 44(6):2577-92 doi:10.1093/nar/gkv1302. 11. Castro-Roa, D., Zenkin, N*. (2015) Methodology for the analysis of transcription and translation in transcription-coupled-to-translation systems in vitro. Methods 86, 51-59, doi:10.1016/j.ymeth.2015.05.029. 12. Zenkin, N., Yuzenkova Y.(2015) New Insights into the Functions of Transcription Factors that Bind the RNA Polymerase Secondary Channel. Biomolecules. 25;5(3):1195-209. doi: 10.3390/biom5031195. 13. Zenkin, N., Severinov, K., Yuzenkova, Y. (2015) Bacteriophage Xp10 anti-termination factor p7 induces forward translocation by host RNA polymerase. Nucleic Acids Res. 27;43(13):6299-308. doi: 10.1093/nar/gkv586. 14. Castro-Roa D, Zenkin, N*. (2015) Methods for the Assembly and Analysis of In Vitro Transcription-Coupled-to-Translation Systems. Methods Mol Biol. 2015;1276:81-99. doi: 10.1007/978-1-4939-2392-2_5. 15. Roghanian, M., Zenkin, N*., Yuzenkova, Y. (2015) Bacterial global regulators DksA/ppGpp increase fidelity of transcription. Nucleic Acids Res. 43(3):1529-36. 16. Yuzenkova, Y., Gamba, P., Herber, M., Attaiech, L., Shafeeq, S., Kuipers, O.P., Klumpp, S., Zenkin, N., and Veening, J-W. (2014) The main function of the bacterial cleavage factor GreA is to prevent transcription traffic jams. Nucleic Acids Res 42:10987-99 17. Zenkin, N*. (2014) Multiple personalities of the RNA polymerase active centre. Microbiology. 160:1316-20 18. Sustarsic M, Plochowietz A, Aigrain L, Yuzenkova Y, Zenkin N, Kapanidis A. (2014) Optimized delivery of fluorescently labeled proteins in live bacteria using electroporation. Histochem Cell Biol. [Epub ahead of print] 19. Zenkin N*.(2014) Ancient RNA stems that terminate transcription. RNA Biol. 5;11 20. Kuzmenko, A., Atkinson, G., Levitskii, S., Zenkin, N., Tenson, T., Hauryliuk, V., Kamenski, P. (2014) Mitochondrial translation initiation machinery: conservation and diversification. Biochimie 100:132-40 doi: 10.1016/j.biochi.2013.07.024. 21. Garcia-Pino A, Zenkin N, Loris R. (2014) The many faces of Fic: structural and functional aspects of Fic enzymes. Trends Biochem Sci. 39:121-9. 22. Zorov, S., Yuzenkova, Y., Nikiforov, V., Severinov, K. and Zenkin, N*. (2014) Antibiotic streptolydigin requires non-catalytic Mg2+ for binding to RNA polymerase. Antimicrob Agents Chemother. 58:1420-4 23. Zenkin, N*. (2014) RNA secondary structure-dependent termination of transcription. Cell Cycle. 13: 3-4. doi: 10.4161/cc.27018 24. Nielsen, S., and Zenkin, N*. (2013) Transcript assisted phosphodiester bond hydrolysis by eukaryotic RNA polymerase II. Transcription; 4:8-7; doi: 10.4161/trns.27062 25. Castro-Roa, D., Garcia-Pino, A., van Nuland, N. A. J., Loris, R., and Zenkin, N*. (2013) The Fic protein Doc uses an inverted substrate to phosphorylate and inactivate EF-Tu. Nat Chem Biol 9:811-7 26. Germain, E., Castro-Roa, D., Zenkin, N*., and Gerdes, K. (2013) Molecular Mechanism of Bacterial Persistence by HipA. Molecular Cell 52:248-54 27. Kuzmenko, A., Levitskii, S., Vinogradova, E., Atkinson, G., Hauryliuk, V., Zenkin, N., and Kamenski, P. (2013) Protein Biosynthesis in Mitochondria Biochemistry (Moscow), 78: 855-866. 28. Yuzenkova, Y., Roghanian, M., Bochkareva, A., Zenkin, N*. (2013) Tagetitoxin inhibits transcription by stabilizing pre-translocated state of the elongation complex. Nucleic Acids Res 41:9257-65 29. Nielsen, S.U., Yuzenkova, Y., and Zenkin, N*. (2013). Mechanism of RNA polymerase III transcription termination Science, 340: 1577-1580. 30. Bochkareva, A., Zenkin, N*. (2013) The sigma70 region 1.2 regulates promoter escape by unwinding DNA downstream of the transcription start site. Nucleic Acids Res 41: 4565-4572 31. Zenkin, N*. (2012). Hypothesis: emergence of translation as a result of RNA helicase evolution. J Mol Evol 74, 249-256 32. Yuzenkova, Y., Roghanian, M., and Zenkin, N*. (2012). Multiple active centers of multi-subunit RNA polymerases. Transcription 3, 115-118. 33. Castro-Roa, D. and Zenkin, N*. (2012) In vitro experimental system for analysis of transcription-translation coupling. Nucleic Acids Res 40, e45 34. Bochkareva, A., Yuzenkova, Y., Tadigotla, V. and Zenkin, N*. (2012). Factor-independent transcription pausing caused by recognition of the RNA-DNA hybrid sequence. EMBO J 31, 630-639 35. Yuzenkova, Y., Tadigotla, V.R., Severinov, K., and Zenkin, N*. (2011). A new basal promoter element recognized by RNA polymerase core enzyme. EMBO J 30, 3766-3775. 36. Roghanian, M., Yuzenkova, Y., and Zenkin, N*. (2011). Controlled interplay between trigger loop and Gre factor in the RNA polymerase active centre. Nucleic Acids Res 39, 4352-4359. 37. Yuzenkova, Y., Zenkin, N*. (2010) Central role of the RNA polymerase trigger loop in intrinsic RNA hydrolysis Proc Natl Acad Sci U S A 107(24):10878-83 38. Yuzenkova, Y., Bochkareva, A., Tadigotla, V., Roghanian, M., Zorov, S., Severinov, K., Zenkin, N*. (2010) Stepwise mechanism for transcription fidelity. BMC Biology 8:54 39. Zenkin, N*. and Severinov, K. (2008). RNA polymerase - the third class of primases. Cell Mol Life Sci. 65, 2280-8. 40. Yuzenkova, Y., Zenkin, N., and Severinov, K. (2008). Mapping of RNA polymerase residues that interact with bacteriophage Xp10 transcription antitermination factor p7. J Mol Biol 375, 29-35 41. Zenkin, N., Kulbachinskiy, A., Yuzenkova, Y., Mustaev, A., Bass, I., Severinov, K. and Brodolin, K. (2007). Region 1.2 of the RNA polymerase sigma subunit controls recognition of the -10 promoter element. EMBO J 26, 955-64. 42. Zenkin, N*., Yuzenkova, Y. and Severinov, K. (2006) Transcript-assisted transcriptional proofreading. Science, 313, 518-20. 43. Zenkin, N*., Naryshkina, T., Kuznedelov, K. and Severinov, K. (2006) The mechanism of DNA primer synthesis by RNA polymerase. Nature, 439, 617 44. Brodolin, K., Zenkin, N. and Severinov, K. (2005). Remodeling of the sigma70 subunit non-template DNA strand contacts during the final step of transcription initiation. J Mol Biol 350, 930-7. 45. Temiakov, D., Zenkin, N., Vassylyeva, M. N., Perederina, A., Tahirov, T. H., Kashkina, E., Savkina, M., Zorov, S., Nikiforov, V., Igarashi, N., Matsugaki, N., Wakatsuki, S., Severinov, K. and Vassylyev, D. G. (2005). Structural basis of transcription inhibition by antibiotic streptolydigin. Molecular Cell 19, 655-66. 46. Zenkin, N., Kulbachinskiy, A., Bass, I. and Nikiforov, V. (2005). Different rifampin sensitivities of Escherichia coli and Mycobacterium tuberculosis RNA polymerases are not explained by the difference in the beta-subunit rifampin regions I and II. Antimicrob Agents Chemother 49, 1587-90. 47. Campbell, E. A., Pavlova, O., Zenkin, N., Leon, F., Irschik, H., Jansen, R., Severinov, K. and Darst, S. A. (2005). Structural, functional, and genetic analysis of sorangicin inhibition of bacterial RNA polymerase. EMBO J 24, 674-82. 48. Wigneshweraraj, S. R., Burrows, P. C., Nechaev, S., Zenkin, N., Severinov, K. and Buck, M. (2004). Regulated communication between the upstream face of RNA polymerase and the beta' subunit jaw domain. EMBO J 23, 4264-74. 49. Budarina, Z. I., Nikitin, D. V., Zenkin, N., Zakharova, M., Semenova, E., Shlyapnikov, M. G., Rodikova, E. A., Masyukova, S., Ogarkov, O., Baida, G. E., Solonin, A. S. and Severinov, K. (2004). A new Bacillus cereus DNA-binding protein, HlyIIR, negatively regulates expression of B. cereus haemolysin II. Microbiology 150, 3691-701. 50. Adelman, K., Yuzenkova, J., La Porta, A., Zenkin, N., Lee, J., Lis, J. T., Borukhov, S., Wang, M. D. and Severinov, K. (2004). Molecular mechanism of transcription inhibition by peptide antibiotic Microcin J25. Molecular Cell 14, 753-62. 51. Brodolin, K., Zenkin, N., Mustaev, A., Mamaeva, D. and Heumann, H. (2004). The sigma 70 subunit of RNA polymerase induces lacUV5 promoter-proximal pausing of transcription. Nat Struct Mol Biol 11, 551-7. 52. Zenkin, N. and Severinov, K. (2004). The role of RNA polymerase sigma subunit in promoter-independent initiation of transcription. Proc Natl Acad Sci U S A 101, 4396-400.

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