Locked Analog Oligos
Locked analog bases are bicyclic nucleic acid where a ribonucleoside is linked between the 2’-oxygen and the 4’-carbon atoms with a methylene unit. Locked Nucleic Acid (LNA) was first described by Wengel and co-workers in 1998 as a novel class of conformationally restricted oligonucleotide analogues (1,2,3).
LNA base containing aptamers have been used and their properties studied (4). The aim was to improve its in vivo stability and target binding without compromising their binding properties. To achieve maximal thermal stem stabilization melting experiments with model hexanucleotide duplexes consisting of unmodified RNA, 2’-O-methyl RNA (2’-OMe), 2’-Fluoro RNA (2’-F) or Locked Nucleic Acids (LNAs) were initially carried out. Extremely high melting temperatures have been found for an LNA/LNA duplex.
Prerequisites for a successful in vivo application of aptamers as molecular target imaging agents are represented by high affinity and selectivity to their target as well as by adequate stability against in vivo degradation.
The design and ability of oligos containing locked nucleic acids (LNAs) to bind supercoiled, double-stranded plasmid DNA in a sequence-specific manner has been described by Hertoghs et al (3). The main mechanism for LNA oligos binding plasmid DNA is demonstrated to be by strand displacement. LNA oligos are more stably bound to plasmid DNA than similar peptide nucleic acid (PNA) `clamps' for procedures such as particle mediated DNA delivery (gene gun). It is shown that LNA oligos remain associated with plasmid DNA after cationic lipid-mediated transfection into mammalian cells. LNA oligos can bind to DNA in a sequence-specific manner so that binding does not interfere with plasmid conformation or gene expression (3).
LNA Oligonucleotides exhibit unprecedented thermal stabilities towards complementary DNA and RNA, which allow excellent mismatch discrimination (6). The high binding affinity of LNA oligos allows for the use of short probes in antisense protocols and LNA is recommended for use in any hybridization assay that requires high specificity and/or reproducibility, e.g., dual labeled probes, in situ hybridization probes, molecular beacons and PCR primers. Furthermore, LNA offers the possibility to adjust Tm values of primers and probes in multiplex assays. Each LNA base addition in an oligo increases the Tm by approximately 2-4oC. As a result of these significant characteristics, the use of LNA-modified oligos in antisense drug development is now coming under investigation (5-8), and recently the therapeutic potential of LNA has been reviewed (10).
The synthesis and incorporation of LNA bases can be achieved by using standard DNA synthesis chemistry. Detailed research results have not yet concluded as to the amount of LNA bases and regular DNA base combination in successful aptamer design. The investigator can elect to substitute individual bases in the oligo to LNA bases or use a combination. Due to the high affinity and thermal stability of the LNA: DNA duplex it is not advised to have more than 15 LNA bases in an oligo; this induces strong self-hybridization
The use of LNA C base requires special synthesis and post synthesis protocols. LNA-containing oligonucleotides can be purified and analyzed using the same methods employed for standard DNA. LNA can be mixed with DNA and RNA, as well as other nucleic acid analogues, modifiers and labels. LNA oligonucleotides are water soluble, and can be separated by gel electrophoresis and precipitated by ethanol.
LNA Oligonucleotide Melting Temperature
LNA is a unique base modification due to its higher Tm. This extreme duplex stability restricts the use of to many LNA bases especially in short oligos. Introduction of more than 4 LNA bases consecutively induces irreversible intra oligo LNA base binding and should be avoided. A maximum of 8 LNA bases is recommended in a 18mer oligo.
Prediction of Melting Temperature (Tm) for DNA and LNA Modified Oligonucleotides
In order to design optimal probes and primers it is important to predict the melting temperature (Tm) of the LNA modified oligonucleotides. Tm is a necessary, but not sufficient, factor for determining the efficiency of capture probes and primers. Other important factors are the secondary structure, the discrimination ability and the steepness of the melting curve. LNA gives a higher degree of freedom with regard to optimising the melting temperature range of the oligonucleotides.
Parameter estimation in the Tm model
LNA modified oligonucleotides have eight possible monomers as opposed to the four DNA nucleotides, therefore the number of possible nearest neighbour pairs is 64 instead of 16. Since both entropy (?S) and enthalpy (?H) are estimated using statistical learning, up to 128 parameters needs to be estimated from the experimental data. Because of experimental error, more than one sample per parameter is necessary, thus in the order of 300 measurements of hybridisations are used, in order to get good parameter estimates. To get even better parameter estimates several parameter reduction techniques have been used. The model parameters were trained by optimising a sum-of-squared-residual error function using gradient descent (16).
General LNA Aptamer Oligonucleotide Design Guidelines
1. LNA’s should be introduced at the positions where specificity and discrimination is needed.
2. Avoid stretches of more than 4 LNA bases. LNA hybridizes very tightly when several consecutive residues are substituted with LNA bases.
3. Avoid LNA self-complimentarity and complementarity to other LNA containing oligonucleotides in the assay. LNA binds very tightly to other LNA residues.
4. Typical aptamer length of 40mer should not contain more than 15 LNA bases.
5. Each LNA bases increases the Tm by approximately 2-4oC.
6. Do not use blocks of LNA near the 3’ end.
7. Keep the GC-content between 30-60 %.
8. Avoid stretches of more than 3 G’s DNA or LNA bases.
For very specific or novel assay settings, design rules may have to be established empirically, but following the above recommendations will provide a good start.
All fluorescent dyes offered by Gene Link can be conjugated to LNA containing probes.
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2. Petersen, M and Wengel, J. (2003) Trends in Biotechnology 21(2): 74-81.
3. Hertoghs, K.M.L., Ellis, J.H. and Catchpole, I.R (2003) Use of locked nucleic acid oligonucleotides to add functionality to plasmid DNA. Nucl. Acids Res.31 (20): 5817-5830.
4. Schmidt, K.S., Borkowski, S., Kurreck, J., Stephens, A.W., Bald, R., Hecht, M., Friebe, M., Dinkelborg, L. and Erdmann, V.A. (2004). Application of locked nucleic acids to improve aptamer in vivo stability and targeting function. Nucleic Acids Research, 2004, Vol. 32, No. 19 5757–5765.
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11. John SantaLucia, Jr. (1998) Proc. Natl. Acad. Sci. 95 1460 – 1465.