Synthesis of Poly Linear shRNA Expression Cassettes Through Branch‐PCR

Jianbing Liu1, Zhen Xi1

1 Department of Chemical Biology, State Key Laboratory of Elemento‐Organic Chemistry, National Engineering Research Center of Pesticide, Nankai University, Tianjin
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 16.5
DOI:  10.1002/cpnc.11
Online Posting Date:  September, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


A facile and universal strategy to construct the poly linear small hairpin RNA (shRNA) expression cassettes with multiple shRNA transcription templates through polymerase chain reaction with flexible branched primers (branch‐PCR) is described in this protocol. Double‐stranded RNA (dsRNA) is not stable enough for the study of RNA interference (RNAi) delivery in mammalian cells. Therefore, the more stable shRNA transcription template is employed to produce the endogenous transcribed dsRNA. Then, the covalent crosslinked linear shRNA expression cassettes are constructed through the branch‐PCR for the long‐lasting RNAi effect in this protocol. The branched primer pair is efficiently synthesized through classic click chemistry. In one step of PCR, the much more stable poly linear shRNA expression cassettes can be produced in large scale. This strategy of efficient synthesis of the poly linear gene expression cassettes can also be applied in the field for other target gene delivery. © 2016 by John Wiley & Sons, Inc.

Keywords: branched primer; branch‐PCR; click chemistry; gene vector; RNAi; shRNA

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Crosslinking Molecule 3N3
  • Basic Protocol 2: Preparation of Branched Primer Pair
  • Basic Protocol 3: Preparation of Poly Linear shRNA Expression Cassettes
  • Commentary
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


Basic Protocol 1: Preparation of Crosslinking Molecule 3N3

  • Tris(2‐hydroxyethyl)amine
  • Chloroform, anhydrous
  • Thionyl chloride
  • Dichloromethane (DCM)
  • N,N‐Dimethylformamide (DMF)
  • Sodium azide
  • Ethyl acetate (AcOEt)
  • Saturated aqueous sodium bicarbonate solution (sat. aq. NaHCO 3)
  • Brine (sat. aq. NaCl)
  • Sodium sulfate (Na 2SO 4)
  • 4 N HCl ethyl acetate solution
  • 100‐mL round‐bottomed flasks
  • Magnetic stir plate and stir bar
  • 10‐mL dropping funnels
  • Rotary evaporator equipped with a vacuum pump
  • Büchner funnel
  • 60ºC incubator
  • 200‐mL separatory funnel
  • Filter paper
  • Glass rods

Basic Protocol 2: Preparation of Branched Primer Pair

  • Tris[(1‐benzyl‐1 H‐1,2,3‐triazol‐4‐yl)methyl]amine (TBTA)
  • Methanol
  • Cupric sulfate
  • Vitamin C
  • DNA oligonucleotides with 5′ C6 alkynyl modification (Sangon Biotech)
  • Tris(2‐azidoethyl)amine (3N3; see protocol 1)
  • 15% denatured PAGE (7 M urea)
  • 1500‐ and 600‐μL centrifuge tubes
  • Vortex mixer
  • 25ºC heating block
  • Nanodrop 2000

Basic Protocol 3: Preparation of Poly Linear shRNA Expression Cassettes

  • shRNA transcription template (422 bp)
  • Linear primer pair (F1 and R1)
  • La‐Taq DNA polymerase, 5 U/μL (TaKaRa)
  • 5× PCR reaction buffer (TaKaRa)
  • Branched primer pair (F3 and R3)
  • 2% agarose gel
  • 10 mM dNTP mix (TaKaRa)
  • PCR purification kit (Thermo)
  • 0.2‐mL PCR reaction tubes
  • Thermal cycler
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
  Brunner, K., Harder, J., Halbach, T., Willibald, J., Spada, F., Gnerlich, F., Sparrer, K., Beil, A., Möckl, L., Bräuchle, C., Conzelmann, K.‐K., and Carell, T. 2015. Cell‐penetrating and neurotargeting dendritic siRNA nanostructures. Angew. Chem. Int. Ed. 54:1946‐1949. doi: 10.1002/anie.201409803.
  Bumcrot, D., Manoharan, M., Koteliansky, V., and Sah, D.W.Y. 2006. RNAi therapeutics: A potential new class of pharmaceutical drugs. Nat. Chem. Biol. 2:711‐719. doi: 10.1038/nchembio839.
  Castanotto, D., Li, H., and Rossi, J.J. 2002. Functional siRNA expression from transfected PCR products. RNA 8:1454‐1460. doi: 10.1017/S1355838202021362.
  De Fougerolles, A., Vornlocher, H.‐P., Maraganore, J., and Lieberman, J. 2007. Interfering with disease: A progress report on siRNA‐based therapeutics. Nat. Rev. Drug. Discov. 6:443‐453. doi: 10.1038/nrd2310.
  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 411:494‐498. doi: 10.1038/35078107.
  Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C. 1998. Potent and specific genetic interference by double‐stranded RNA in Caenorhabditis elegans. Nature 391:806‐811. doi: 10.1038/35888.
  Hartman, M.R., Yang, D.Y., Tran, T.N.N., Lee, K., Kahn, J.S., Kiatwuthinon, P., Yancey, K.G., Trotsenko, O., Minko, S., and Luo, D. 2013. Thermostable branched DNA nanostructures as modular primers for polymerase chain reaction. Angew. Chem. Int. Ed. 52:8699‐8702. doi: 10.1002/anie.201302175.
  Hong, C.A., Eltoukhy, A.A., Lee, H., Langer, R., Anderson, D.G., and Nam, Y.S. 2015. Dendrimeric siRNA for efficient gene silencing. Angew. Chem. Int. Ed. 54:6740‐6744. doi: 10.1002/anie.201412493.
  Hong, C.A., Lee, S.H., Kim, J.S., Park, J.W., Bae, K.H., Mok, H., Park, T.G., and Lee, H. 2011. Gene silencing by siRNA microhydrogels via polymeric nanoscale condensation. J. Am. Chem. Soc. 133:13914‐13917. doi: 10.1021/ja2056984.
  Keller, S., Wang, J., Chandra, M., Berger, R., and Marx, A. 2008. DNA polymerase‐catalyzed DNA network growth. J. Am. Chem. Soc. 130:13188‐13189. doi: 10.1021/ja8045348.
  Kuang, H., Ma, W., Xu, L.G., Wang, L.B., and Xu, C.L. 2013. Nanoscale superstructures assembled by polymerase chain reaction (PCR): Programmable construction, structural diversity, and emerging applications. Accounts. Chem. Res. 46:2341‐2354. doi: 10.1021/ar300206m.
  Lares, M.R., Rossi, J.J., and Ouellet, D.L. 2010. RNAi and small interfering RNAs in human disease therapeutic applications. Trends Biotechnol. 28:570‐579. doi: 10.1016/j.tibtech.2010.07.009.
  Li, J., Zheng, C., Cansiz, S., Wu, C., Xu, J., Cui, C., Liu, Y., Hou, W., Wang, Y., Zhang, L., Teng, I.t., Yang, H.‐H., and Tan, W. 2015. Self‐assembly of DNA nanohydrogels with controllable size and stimuli‐responsive property for targeted gene regulation therapy. J. Am. Chem. Soc. 137:1412‐1415. doi: 10.1021/ja512293f.
  Liu, J., Wang, R., Ma, D., Li, Y., Wei, C., and Xi, Z. 2016. Branch‐PCR constructed stable shRNA transcription nanoparticles have long‐lasting RNAi effect. Chembiochem doi: 10.1002/cbic.201600047.
  Nakashima, Y., Abe, H., Abe, N., Aikawa, K., and Ito, Y. 2011. Branched RNA nanostructures for RNA interference. Chem. Commun. 47:8367‐8369. doi: 10.1039/c1cc11780g.
  Park, N., Um, S.H., Funabashi, H., Xu, J., and Luo, D. 2009. A cell‐free protein‐producing gel. Nat. Mater. 8:432‐437. doi: 10.1038/nmat2419.
  Pecot, C.V., Calin, G.A., Coleman, R.L., Lopez‐Berestein, G., and Sood, A.K. 2011. RNA interference in the clinic: Challenges and future directions. Nat. Rev. Cancer 11:59‐67. doi: 10.1038/nrc2966.
  Sui, G., Soohoo, C., Affar el, B., Gay, F., Shi, Y., and Forrester, W.C. 2002. A DNA vector‐based RNAi technology to suppress gene expression in mammalian cells. Proc. Natl. Acad Sci. 99:5515‐5520. doi: 10.1073/pnas.082117599.
  Um, S.H., Lee, J.B., Park, N., Kwon, S.Y., Umbach, C.C., and Luo, D. 2006. Enzyme‐catalysed assembly of DNA hydrogel. Nat. Mater. 5:797‐801. doi: 10.1038/nmat1741.
  Whitehead, K.A., Langer, R., and Anderson, D.G. 2009. Knocking down barriers: Advances in siRNA delivery. Nat. Rev. Drug. Discov. 8:129‐138. doi: 10.1038/nrd2742.
  Yan, M., Liang, M., Wen, J., Liu, Y., Lu, Y., and Chen, I.S.Y. 2012. Single siRNA nanocapsules for enhanced RNAi delivery. J. Am. Chem. Soc. 134:13542‐13545. doi: 10.1021/ja304649a.
  Yang, D., Hartman, M.R., Derrien, T.L., Hamada, S., An, D., Yancey, K.G., Cheng, R., Ma, M., and Luo, D. 2014. DNA materials: Bridging nanotechnology and biotechnology. Accounts Chem. Res. 47:1902‐1911. doi: 10.1021/ar5001082.
PDF or HTML at Wiley Online Library