DOI QR코드

DOI QR Code

Recyclable single-stranded DNA template for synthesis of siRNAs

  • Ali, Mussa M. ;
  • Obregon, Demian ;
  • Agrawal, Krishna C. ;
  • Mansour, Mahmoud ;
  • Abdel-Mageed, Asim B.
  • 투고 : 2010.09.02
  • 심사 : 2010.09.23
  • 발행 : 2010.11.30

초록

RNA interference is a post-transcriptional silencing mechanism triggered by the bioavailability and/or exogenous introduction of double-stranded RNA (dsRNA) into cells. Here we describe a novel method for the synthesis of siRNA in a single vessel. The method employs in vitro transcription and a single-stranded DNA (ssDNA) template and design, which incorporates upon self-annealing, two promoters, two templates, and three loop regions. Using this method of synthesis we generated efficacious siRNAs designed to silence both exogenous and endogenous genes in mammalian cells. Due to its unique design the single-stranded template is easily amenable to adaptation for attachment to surface platforms for synthesis of siRNAs. A siRNA synthesis platform was generated using a 3' end-biotinylated ssDNA template tethered to a streptavidin coated surface that generates stable siRNAs under multiple cycles of production. Together these data demonstrate a unique and robust method for scalable siRNA synthesis with potential application in RNAi-based array systems.

키워드

Recyclable;Single-stranded DNA template;siRNA;Synthesis

참고문헌

  1. Tomari, Y. and Zamore, P. D. (2005) Perspective: machines for RNAi. Genes Dev. 15, R61-R64.
  2. Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. and Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 441, 494-498. https://doi.org/10.1038/nature04762
  3. Sohail, M., Doran, G., Riedemann, J., Macaulay, V. and Southern, E. M. (2003) A simple and cost-effective method for producing small interfering RNAs with high efficacy. Nucleic. Acids. Res. 31, e38. https://doi.org/10.1093/nar/gng038
  4. Reynolds, A., Leake, D., Boese, Q., Scaringe, S., Marshall, W. S. and Khvorova, A. (2004) Rational siRNA design for RNA interference. Nature Biotechnol. 3, 326-330.
  5. Donze, O. and Picard, D. (2002) RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase. Nucleic. Acids. Res. 30, e46. https://doi.org/10.1093/nar/30.10.e46
  6. Oleinikov, A. V., Zhao, J. and Gray, M. D. (2005) RNA interference by mixtures of siRNAs prepared using custom oligonucleotide arrays. Nucleic. Acids. Res. 33, e92. https://doi.org/10.1093/nar/gni091
  7. Yu, J. Y., DeRuiter, S. L. and Turner, D. L. (2002) RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc. Natl. Acad. Sci. U.S.A 99, 6047-6052. https://doi.org/10.1073/pnas.092143499
  8. Luo, B., Heard, A. D. and Lodish, H. F. (2004) Small interfering RNA production by enzymatic engineering of DNA (SPEED). Proc. Natl. Acad. Sci. U.S.A. 101, 5494-5499. https://doi.org/10.1073/pnas.0400551101
  9. Rose, S. D., Kim, D. H., Amarzguioui, M., Heidel, J. D., Collingwood, M. A., Davis, M. E., Rossi, J. J. and Behlke, M. A. (2005) Functional polarity is introduced by dicer processing of short substrate RNAs. Nucleic. Acids. Res. 33, 4140-4156. https://doi.org/10.1093/nar/gki732
  10. Jiang, M., Arzumanov, A. A., Gait, M. J., Milner, J. A. (2005) Bi-functional siRNA construct induces RNA interference and also primes PCR amplification for its own quantification. Nucleic. Acids. Res. 33, e151. https://doi.org/10.1093/nar/gni144
  11. Castanotto, D. and Rossi, J. J. (2004) Construction and transfection of PCR products expressing siRNAs or shRNAs in mammalian cells. Methods Mol. Biol. 252, 509-514.
  12. Kim, J., Kim, H., Lee, Y., Yang, K., Byun, S. and Han, K. A. (2006) Simple and economical short-oligonucleotidebased approach to shRNA generation. J. Biochem. Mol. Biol. 39, 329-334. https://doi.org/10.5483/BMBRep.2006.39.3.329
  13. MacDonald, L. E., Zhou, Y. and McAllister, W. T. (1993) Termination and slippage by bacteriophage T7 RNA polymerase. J. Mol. Biol. 232, 1030-1047. https://doi.org/10.1006/jmbi.1993.1458
  14. Donze, O. and Picard, D. (2002) RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase. Nucleic. Acids. Res. 30, e46. https://doi.org/10.1093/nar/30.10.e46
  15. Strat, A., Gao, L., Utsuki, T., Cheng, B., Nuthalapaty, S. Mathis, J. M., Odaka, Y. and Giordano, T. (2006) Specific and nontoxic silencing in mammalian cells with expressed long dsRNAs. Nucleic. Acids. Res. 34, 3803-3810. https://doi.org/10.1093/nar/gkl532
  16. Milligan, J. F., Groebe, D. R., Witherell, G. W. and Uhlenbeck, O. C. (1987) Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic. Acids. Res. 15, 8783-8798. https://doi.org/10.1093/nar/15.21.8783
  17. Davis, R., Jia, D., Cinar, B., Sikka, S. C., Moparty, K., Zhau, H. E., Chung, L. W., Agrawal, K. C. and Abdel-Mageed, A. B. (2003) Functional androgen receptor confers sensitization of androgen independent prostate cancer cells to anticancer therapy via caspase activation. Biochem. Biophy. Res. Commun. 309, 937-945. https://doi.org/10.1016/j.bbrc.2003.08.096
  18. Caplen, N. J., Parrish, S., Imani, F., Fire, A. and Morgan, R. A. (2001) Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc. Natl. Acad. Sci. U.S.A. 98, 9742-9747. https://doi.org/10.1073/pnas.171251798
  19. Abdel-Mageed, A. B. and Agrawal, K. C. (1998) Activation of nuclear factor Kappa B: potential role in metallothionein-mediated mitogenic response. Cancer Res. 58, 2335-2338.
  20. Honore, B., Vorum, H. and Baandrup, U. (1999) hnRNPs H, H' and F behave differently with respect to posttranslational cleavage and subcellular localization. FEBS Lett. 456, 274-280. https://doi.org/10.1016/S0014-5793(99)00911-4
  21. Abdel-Mageed, A. B. and Agrawal, K. C. (1997) Antisense down-regulation of metallothionein induces growth arrest and apoptosis in human breast carcinoma cells. Cancer Gene Ther. 4, 199-207.