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
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Abstract

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.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol: 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
     
 
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Materials

Basic Protocol: Introduction of LNA Oligomers into Cells

 Materials
  • Cells grown to confluence in 75-cm2 tissue culture flasks
  • Complete growth medium (see recipe)
  • 100 µM LNA stock solution (see Support Protocol 1)
  • 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% CO2 incubator
  • Additional reagents and equipment for trypsinizing and counting cells (e.g., cpmb appendix 3F)

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

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

Support Protocol 2: Determination of Tm for LNA Oligomers

 Materials
  • 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 Na2HPO4 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
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Figures

  •  FigureFigure 4.13.1 Structure of LNA.
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  •  FigureFigure 4.13.2 Preparation of LipofectAMINE-LNA complexes for cellular transfection, including subsequent dilutions of stock complexes. Vt = total volume. Volumes are sufficient for dispensing six replicates and have been optimized for COS-7 cells.
    View Image

Videos

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.
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