subject: Comparison Of Lna And Pna Probes [print this page] Introduction Introduction
Detection of nucleic acid sequences is widely used in many biological assays, with increasing penetration into clinical sciences and other areas of life sciences. Quite often fluorescent tagged DNA sequences, either from naturally occurring sequences or synthetic oligonucleotides, are used to probe a sample of interest. Synthetic non-natural analogs have also been found useful in increasing specificity of detection. Here we take a look at two synthetic analogs: Locked Nucleic Acids (LNAs) and Peptide Nucleic Acids (PNAs).
Thermodynamic comparison of LNA and PNA probes
LNAs are a class of conformationally restricted nucleotide analogs, in which the ribose ring is constrained by a methylene linkage between the 2oxygen and the 4 carbon resulting in the locked 3endo formation.(figure1) (Saenger, 1984).
The incorporation of LNA in an oligonucleotide increases the affinity of that oligonucleotide for its complementary RNA or DNA target by increasing the melting temperature (Tm) of the duplex. Additionally, the Tm difference between a perfectly matched target and a mismatched target is substantially higher than that observed when a DNA-based oligonucleotide is used. The properties like high Tm and excellent mismatch discriminationmake LNA-modified probes ideal for analysis of short and very similar targets like miRNAs. Furthermore, by adjusting the LNA content and probe length, it is possible to design Tm-normalized probes, thereby allowing hybridization conditions that are optimal for all probes used on, for example, an array.
LNAs were seen to display increased thermodynamic stability and enhanced nucleic acid recognition. Initial investigations of LNA melting temperatures (Tm) by Koshkin et al. (1998) revealed increased values per LNA monomer(Tm) of +3 to +5oC and +4 to +8oC against complementary DNA and RNA oligonucleotides respectively.
