Cellular Delivery of Locked Nucleic Acids (LNAs)

Dwaine A. Braasch1, David R. Corey1

1 University of Texas Southwestern Medical Center at Dallas, Dallas, Texas
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.13
DOI:  10.1002/0471142700.nc0413s09
Online Posting Date:  August, 2002
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Locked nucleic acids (LNAs) are RNA derivatives that have an O‐methylene linkage between the 2 and 4 positions of the ribose. This leads to exceptionally high‐affinity binding to complementary sequences. They are synthesized using standard DNA/RNA synthesis methods, and have a negatively charged backbone that confers good solubility. This unit describes a method for the introduction of LNA oligomers into cells. A support protocol also describes the determination of melting temperatures for LNA oligomers.

PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Introduction of LNA Oligomers into Cells
  • Support Protocol 1: Preparation of LNA Oligomer Stock Solutions
  • Support Protocol 2: Determination of Tm for LNA Oligomers
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


Basic Protocol 1: Introduction of LNA Oligomers into Cells

  • Cells grown to confluence in 75‐cm2 tissue culture flasks
  • Complete growth medium (see recipe)
  • 100 µM LNA stock solution (see protocol 2)
  • Opti‐MEM I (Invitrogen Life Technologies; reduced‐serum medium, containing L‐glutamine and no phenol red)
  • LipofectAMINE (Invitrogen Life Technologies)
  • 48‐well tissue culture plate (Costar)
  • Repeating pipettor (e.g., Eppendorf)
  • 12.5‐mL Combitips (Eppendorf)
  • 65° and 37°C water baths or a thermal cycler
  • 12 × 75–mm round‐bottom tubes
  • 37°C, 5% CO 2 incubator
  • Additional reagents and equipment for trypsinizing and counting cells (e.g., CPMB APPENDIX )
NOTE: LipofectAMINE and Opti‐MEM I are important to the success of the experiment and should not be substituted.

Support Protocol 1: Preparation of LNA Oligomer Stock Solutions

  • Locked nucleic acid oligomers (LNAs; Proligo)
  • DNase/RNase‐free water (Life Technologies)
  • Spectrophotometer

Support Protocol 2: Determination of Tm for LNA Oligomers

  • LNA oligonucleotides
  • DNA or RNA oligomers
  • 10× Ca2+‐ and Mg2+‐free phosphate‐buffered saline (CMF‐PBS; Invitrogen Life Technologies or see recipe)
  • 0.1 M Na 2HPO 4 buffer, pH 7.5 (Fisher)
  • Mineral oil (Sigma)
  • Stoppered cuvette (1‐cm pathlength and 1.5‐cm Z dimension; Spectrosil Far UV Quartz, Uvonic Instruments)
  • Spectrophotometer with temperature‐controlled cuvette holder
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Baker, B.F., Lot, S.S., Condon, T.P., Cheng‐Flournoy, S., Lesnik, E.A., Sasmor, H.M., and Bennett, C.F. 1997. 2′‐O‐(2‐Methoxy)ethyl‐modified anti‐intercellular adhesion molecule 1 (ICAM‐1) oligonucleotides selectively increase the ICAM‐1 mRNA level and inhibit formation of the ICAM‐1 translation initiation complex in human umbilical vein endothelial cells. J. Biol. Chem. 272:11994‐12000.
   Bondensgaard, K., Petersen, M., Singh, S.K., Rajwanshi, V.K., Kumar, R., Wengel, J., and Jacobsen, J.P. 2000. Structural studies of LNA:RNA duplexes by NMR: Conformations and RNase H activity. Chem. Eur. J. 6:2687‐2695.
   Braasch, D.A. and Corey, D.R. 2001. Locked nucleic acids: Fine‐tuning nucleic acid recognition. Chem. Biol. 8:1‐7.
   Crooke, S.T. 1999. Molecular mechanisms of antisense drugs: Human RNase H. Antisense Nucl. Acid Drug Devel. 9:377‐379.
   Doyle, D.F., Braasch, D.A., Simmons, C.G., Janowski, B.A., and Corey, D.R. 2001. Inhibition of gene expression inside cells by peptide nucleic acids: Effect of mRNA target sequence, mismatched bases, and PNA length. Biochemistry 40:53‐64.
   Geary, R.S., Yu, R.Z., and Levin, A.A. 2001. Pharmacokinetics of phosphorothioate antisense oligonucleotides. Curr. Opin. Investigational New Drugs 2:562‐573.
   Kang, S.H., Cho, M.J., and Kole, R. 1998. Up‐regulation of luciferase gene expression with antisense oligonucleotides—Implications and applications in functional assay developments. Biochemistry 37:6235‐6239.
   Koshkin, A.A., Singh, S.K., Nielsen, P., Rajwanshi, V.K., Kumar, R., Meldgaard, M., Olsen, C.E., and Wengel, J. 1998. LNA (locked nucleic acids): Synthesis of the adenine, cytosine, guanine, 5‐methylcytosine, thymine, and uracil bicyclonucleoside monomers, oligomerisation and unprecedented nucleic acid recognition. Tetrahedon 54:3607‐3630.
   Kumar, R., Singh, S., Koshkin, A.A., Rajwanshi, V.K., Meldgaard, M., and Wengel, J. 1998. The first analogues of LNA (locked nucleic acids): Phosphorothioate‐LNA and 2′‐thio‐LNA. Bioorg. Med. Chem. Lett. 8:2219‐2222.
   Obika, S., Nanbu, D., Hari, Y., Andoh, J., Morio, K., Doi, T., and Imanishi, T. 1998. Stability and structural features of the duplexes containing the nucleoside analogues with a fixed N‐type conformation, 2′‐O,4′‐C‐methyleneribonucleosides. Tetrahedron Lett. 39:5401‐5404.
   Wahlestedt, C., Salmi, P., Good, L., Kela, J., Johnsson, T., Hokfelt, T., Broberger, C., Porreca, F., Lai, J., Ren, K., Ossipov, M., Koshkin, A., Jakobsen, N., Skouv, J., Oerum, H., Havsteen Jacobsen, M., and Wengel, J. 2000. Potent and nontoxic antisense oligonucleotides containing locked nucleic acids. Proc. Natl. Acad. Sci. U.S.A. 97:5633‐5638.
   Wang, G., Gunic, E., Girardet, J‐L., and Stoisavljevic, V. 1999. Conformationally locked nucleosides. Synthesis and hybridization properties of oligodeoxynucleotides containing 2′4′‐C‐bridged 2′‐deoxynucleosides. Bioorg. Med. Chem. Lett. 9:1147‐1150.
PDF or HTML at Wiley Online Library