Applied Biochemistry and Biotechnology

, Volume 164, Issue 4, pp 497–513

Early In Vitro Transcription Termination in Human H5 Influenza Viral RNA Synthesis

  • Matthew B. Kerby
  • Aartik A. Sarma
  • Madhukar S. Patel
  • Andrew W. Artenstein
  • Steven M. Opal
  • Anubhav Tripathi
Article

Abstract

Rapid diagnostic identification of the human H5 influenza virus is a strategic cornerstone for outbreak prevention. We recently reported a method for direct detection of viral RNA from a highly pathogenic human H5 influenza strain (A/Hanoi/30408/2005(H5N1)), which necessarily was transcribed in vitro from non-viral sources. This article provides an in-depth analysis of the reaction conditions for in vitro transcription (IVT) of full-length influenza H5 RNA, which is needed for diagnostic RNA production, for the T7 and SP6 phage promoter systems. Gel analysis of RNA transcribed from plasmids containing the H5 sequence between a 5′ SP6 promoter and 3′ restriction site (BsmBI) showed that three sequence-verified bands at 1,776, 784, and 591 bases were consistently produced, whereas only one 1,776-base band was expected. These fragments were not observed in H1 or H3 influenza RNA transcribed under similar conditions. A reverse complement of the sequence produced only a single band at 1,776 bases, which suggested either self-cleavage or early termination. Aliquots of the IVT reaction were quenched with EDTA to track the generation of the bands over time, which maintained a constant concentration ratio. The H5 sequence was cloned with T7 and SP6 RNA polymerase promoters to allow transcription in either direction with either polymerase. The T7 transcription product from purified, restricted plasmids in the vRNA direction only produced the 1,776-base full-length sequence and the 784-base fragment, instead of the three bands generated by the SP6 system, suggesting an early termination mechanism. Additionally, the T7 system produced a higher fraction of full-length vRNA transcripts than the SP6 system did under similar reaction conditions. By sequencing we identified a type II RNA hairpin loop terminator, which forms in a transcription direction-dependent fashion. Variation of the magnesium concentration produced the greatest impact on termination profiles, where some reaction mixtures were unable to produce full-length transcripts. Optimized conditions are presented for the T7 and SP6 phage polymerase systems to minimize these early termination events during in vitro transcription of H5 influenza vRNA.

Keywords

Termination Influenza H5 In vitro transcription SP6 T7 

References

  1. 1.
    Fung, Y. W. W., Lau, L. T., & Yu, A. C. H. (2004). The necessity of molecular diagnostics for avian flu. Nature Biotechnology, 22, 267.CrossRefGoogle Scholar
  2. 2.
    Easley, C. J., Karlinsey, J. M., Bienvenue, J. M., Legendre, L. A., Roper, M. G., Feldman, S. H., et al. (2006). A fully integrated microfluidic genetic analysis system with sample-in-answer-out capability. Proceedings of the National Academy of Sciences of the United States of America, 103, 19272–19277.CrossRefGoogle Scholar
  3. 3.
    Compton, J. (1991). Nucleic-acid sequence-based amplification. Nature, 350, 91–92.CrossRefGoogle Scholar
  4. 4.
    Nygaard, V., & Hovig, E. (2006). Options available for profiling small samples: A review of sample amplification technology when combined with microarray profiling. Nucleic Acids Research, 34, 996–1014.CrossRefGoogle Scholar
  5. 5.
    Boudvillain, M., Schwartz, A., & Rahmouni, A. R. (2002). Limited topological alteration of the T7 RNA polymerase active center at intrinsic termination sites. Biochemistry, 41, 3137–3146.CrossRefGoogle Scholar
  6. 6.
    Mukherjee, S., Brieba, L. G., & Sousa, R. (2003). Discontinuous movement and conformational change during pausing and termination by T7 RNA polymerase. The EMBO Journal, 22, 6483–6493.CrossRefGoogle Scholar
  7. 7.
    Santangelo, T. J., & Roberts, J. W. (2004). Forward translocation is the natural pathway of RNA release at an intrinsic terminator. Molecular Cell, 14, 117–126.CrossRefGoogle Scholar
  8. 8.
    Brooks, E. M., Sheflin, L. G., & Spaulding, S. W. (1995). Secondary Structure in the 3′-UTR of EGF and the choice of reverse transcriptases affect the detection of message diversity by RT-PCR. Biotechniques, 19, 806–812.Google Scholar
  9. 9.
    Chan, C. H., Lin, K. L., Chan, Y., Wang, Y. L., Chi, Y. T., Tu, H. L., et al. (2006). Amplification of the entire genome of influenza A virus H1N1 and H3N2 subtypes by reverse-transcription polymerase chain reaction. Journal of Virological Methods, 136, 38–43.CrossRefGoogle Scholar
  10. 10.
    Tunitskaya, V. L., & Kochetkov, S. N. (2002). Structural–functional analysis of bacteriophage T7 RNA polymerase. Biochemistry-Moscow, 67, 1124–1135.CrossRefGoogle Scholar
  11. 11.
    Ioannou, Y., Giles, I., Lambrianides, A., Richardson, C., Pearl, L. H., Latchman, D. S., et al. (2006). A novel expression system of domain I of human beta2 glycoprotein I in Escherichia coli. BMC Biotechnology, 6, 8.CrossRefGoogle Scholar
  12. 12.
    Joshi, B. H., & Puri, R. K. (2005). Optimization of expression and purification of two biologically active chimeric fusion proteins that consist of human interleukin-13 and pseudomonas exotoxin in Escherichia coli. Protein Expression and Purification, 39, 189–198.CrossRefGoogle Scholar
  13. 13.
    Saida, F., Uzan, M., Odaert, B., & Bontems, F. (2006). Expression of highly toxic genes in E-coli: Special strategies and genetic tools. Current Protein & Peptide Science, 7, 47–56.CrossRefGoogle Scholar
  14. 14.
    Chen, J. M., Guo, L. X., Sun, C. Y., Sun, Y. X., Chen, J. W., Li, L., et al. (2006). A stable and differentiable RNA positive control for reverse transcription-polymerase chain reaction. Biotechnology Letters, 28, 1787–1792.CrossRefGoogle Scholar
  15. 15.
    Jeong, W., & Kang, C. W. (1997). The histidine-805 in motif-C of the phage SP6 RNA polymerase is essential for its activity as revealed by random mutagenesis. Biochemistry and Molecular Biology International, 42, 711–716.Google Scholar
  16. 16.
    Ma, C. Q., Lyons-Weiler, M., Liang, W. J., LaFramboise, W., Gilbertson, J. R., Becich, M. J., et al. (2006). In vitro transcription amplification and labeling methods contribute to the variability of gene expression profiling with DNA microarrays. The Journal of Molecular Diagnostics, 8, 183–192.CrossRefGoogle Scholar
  17. 17.
    Pokrovskaya, I. D., & Gurevich, V. V. (1994). In-vitro transcription—preparative RNA yields in analytical scale reactions. Analytical Biochemistry, 220, 420–423.CrossRefGoogle Scholar
  18. 18.
    Woodrow, K. A., Airen, I. O., & Swartz, J. R. (2006). Rapid expression of functional genomic libraries. Journal of Proteome Research, 5, 3288–3300.CrossRefGoogle Scholar
  19. 19.
    Li, Y., Elashoff, D., Oh, M., Sinha, U., St John, M. A. R., Zhou, X. F., et al. (2006). Serum circulating human mRNA profiling and its utility for oral cancer detection. Journal of Clinical Oncology, 24, 1754–1760.CrossRefGoogle Scholar
  20. 20.
    Rodriguez-Lazaro, D., Hernandez, M., D'Agostino, M., & Cook, N. (2006). Application of nucleic acid sequence-based amplification for the detection of viable foodborne pathogens: Progress and challenges. Journal of Rapid Methods and Automation in Microbiology, 14, 218–236.CrossRefGoogle Scholar
  21. 21.
    Romano, J. W., Williams, K. G., Shurtliff, R. N., Ginocchio, C., & Kaplan, M. (1997). NASBA technology: Isothermal RNA amplification in qualitative and quantitative diagnostics. Immunological Investigations, 26, 15–28.CrossRefGoogle Scholar
  22. 22.
    Starkey, W. G., Millar, R. M., Jenkins, M. E., Ireland, J. H., Muir, K. F., & Richards, R. H. (2004). Detection of piscine nodaviruses by real-time nucleic acid sequence based amplification (NASBA). Diseases of Aquatic Organisms, 59, 93–100.CrossRefGoogle Scholar
  23. 23.
    Morisset, D., Dobnik, D., Hamels, S., Zel, J., & Gruden, K. (2008). NAIMA: Target amplification strategy allowing quantitative on-chip detection of GMOs. Nucleic Acids Research, 36, e118.CrossRefGoogle Scholar
  24. 24.
    Kerby, M. B., Freeman, S., Prachanronarong, K., Artenstein, A. W., Opal, S. M., & Tripathi, A. (2008). Direct sequence detection of structured H5 influenza viral RNA. The Journal of Molecular Diagnostics, 10, 225–235.CrossRefGoogle Scholar
  25. 25.
    Toulokhonov, I., & Landick, R. (2003). The flap domain is required for pause RNA hairpin inhibition of catalysis by RNA polymerase and can modulate intrinsic termination. Molecular Cell, 12, 1125–1136.CrossRefGoogle Scholar
  26. 26.
    Komissarova, N., & Kashlev, M. (1997). Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3′ end of the RNA intact and extruded. Proceedings of the National Academy of Sciences of the United States of America, 94, 1755–1760.CrossRefGoogle Scholar
  27. 27.
    Hartvig, L., & Christiansen, J. (1996). Intrinsic termination of T7 RNA polymerase mediated by either RNA or DNA. The EMBO Journal, 15, 4767–4774.Google Scholar
  28. 28.
    Dunn, J. J., & Studier, F. W. (1983). Complete nucleotide-sequence of bacteriophage-T7 DNA and the locations of T7 genetic elements. Journal of Molecular Biology, 166, 477–535.CrossRefGoogle Scholar
  29. 29.
    Sousa, R., & Mukherjee, S. (2003). T7 RNA polymerase. Progress in Nucleic Acid Research and Molecular Biology, 73(73), 1–41.CrossRefGoogle Scholar
  30. 30.
    Yarnell, W. S., & Roberts, J. W. (1999). Mechanism of intrinsic transcription termination and antitermination. Science, 284, 611–615.CrossRefGoogle Scholar
  31. 31.
    Sasaki, N., Izawa, M., Sugahara, Y., Tanaka, T., Watahiki, M., Ozawa, K., et al. (1998). Identification of stable RNA hairpins causing band compression in transcriptional sequencing and their elimination by use of inosine triphosphate. Gene, 222, 17–24.CrossRefGoogle Scholar
  32. 32.
    Dobbins, A. T., George, M., Basham, D. A., Ford, M. E., Houtz, J. M., Pedulla, M. L., et al. (2004). Complete genomic sequence of the virulent salmonella bacteriophage SP6. Journal of Bacteriology, 186, 1933–1944.CrossRefGoogle Scholar
  33. 33.
    He, B., Kukarin, A., Temiakov, D., Chin-Bow, S. T., Lyakhov, D. L., Rong, M. Q., et al. (1998). Characterization of an unusual, sequence-specific termination signal for T7 RNA polymerase. The Journal of Biological Chemistry, 273, 18802–18811.CrossRefGoogle Scholar
  34. 34.
    Lyakhov, D. L., He, B., Zhang, X., Studier, F. W., Dunn, J. J., & McAllister, W. T. (1998). Pausing and termination by bacteriophage T7 RNA polymerase. Journal of Molecular Biology, 280, 201–213.CrossRefGoogle Scholar
  35. 35.
    Mead, D. A., Skorupa, E. S., & Kemper, B. (1985). Single stranded DNA Sp6 promoter plasmids for engineering mutant RNAs and proteins—synthesis of a stretched preproparathyroid hormone. Nucleic Acids Research, 13, 1103–1118.CrossRefGoogle Scholar
  36. 36.
    Mead, D. A., Skorupa, E. S., & Kemper, B. (1986). DNA ‘blue’ promoter plasmids: A versatile tandem promoter system for cloning and protein engineering. Protein Engineering, 1, 67–74.CrossRefGoogle Scholar
  37. 37.
    Portugal, J., & Rodriguez-Campos, A. (1996). T7 RNA polymerase cannot transcribe through a highly knotted DNA template. Nucleic Acids Research, 24, 4890–4894.CrossRefGoogle Scholar
  38. 38.
    Neumann, G., Brownlee, G. G., Fodor, E., & Kawaoka, Y. (2004). Orthomyxovirus replication, transcription, and polyadenylation. Biology of Negative Strand RNA Viruses: The Power of Reverse Genetics, 283, 121–143.Google Scholar
  39. 39.
    Zuker, M. (2003). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Research, 31, 3406–3415.CrossRefGoogle Scholar
  40. 40.
    Collins, R. A., Ko, L. S., So, K. L., Ellis, T., Lau, L. T., & Yu, A. C. H. (2003). A NASBA method to detect high- and low-pathogenicity H5 avian influenza viruses. Avian Diseases, 47, 1069–1074.CrossRefGoogle Scholar
  41. 41.
    Lanciotti, R. S., & Kerst, A. J. (2001). Nucleic acid sequence-based amplification assays for rapid detection of West Nile and St. Louis encephalitis viruses. Journal of Clinical Microbiology, 39, 4506–4513.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Matthew B. Kerby
    • 1
    • 4
  • Aartik A. Sarma
    • 1
    • 5
  • Madhukar S. Patel
    • 1
    • 6
  • Andrew W. Artenstein
    • 2
    • 3
  • Steven M. Opal
    • 2
    • 3
  • Anubhav Tripathi
    • 1
    • 2
    • 3
  1. 1.School of Engineering and Division of Biology and Medicine, Biomedical Engineering, Center for Biomedical EngineeringBrown UniversityProvidenceUSA
  2. 2.Department of Medicine & Center for Biodefense and Emerging PathogensMemorial Hospital of RIPawtucketUSA
  3. 3.Warren Alpert School of MedicineBrown UniversityProvidenceUSA
  4. 4.Department of BioengineeringStanford UniversityStanfordUSA
  5. 5.Harvard Medical SchoolBostonUSA
  6. 6.School of MedicineUniversity of California, IrvineIrvineUSA

Personalised recommendations