Nucleic Acids Research, 2006, Vol. 34, No. 8, pp e60.

Design of LNA probes that improve mismatch discrimination

Locked nucleic acids (LNA) show remarkable affinity and specificity against native DNA targets. Effects of LNA modifications on mismatch discrimination were studied as a function of sequence context and identity of the mismatch using ultraviolet (UV) melting experiments. A triplet of LNA residues centered on the mismatch was generally found to have the largest discriminatory power. An exception was observed for G-T mismatches, where discrimination decreased when the guanine nucleotide at the mismatch site or even the flanking nucleotides were modified. Fluorescence experiments using 2-aminopurine suggest that LNA modifications enhance base stacking of perfectly matched base pairs and decrease stabilizing stacking interactions of mismatched base pairs. LNAs do not change the amount of counterions (Na⁺) that are released when duplexes denature. New guidelines are suggested for design of LNA probes, which significantly improve mismatch discrimination in comparison with unmodified DNA probes.

• McErlean C., Gonzalez A.A., Cunningham R., Meenagh A., Shovlin T., Middleton D. (2010) Differential RNA expression of KIR alleles. Immunogenetics 62, 431-440.
• Satterfield B.C., Bartosiewicz M., West J.A.A., Caplan M.R. (2010) Surpassing specificity limits of nucleic acid probes via cooperativity. J. Mol. Diagnostics 12, 359-367.
• Lezar S., Barros E. (2010) Oligonucleotide microarray for the identification of potential mycotoxigenic fungi. BMC Microbiology 10, Art. No. 87.
• Paris C., Moreau V., Deglane G., Voirin E., Erbacher P., Lenne-Samuel N. (2010) Zip nucleic acids are potent hydrolysis probes for quantitative PCR. Nucleic Acids Res. 38, Art. No. e95.
• Halm A., Wagner M., Kofer J., Hein I. (2010) Novel real-time PCR assay for simultaneous detection and differentiation of Clostridium chauvoei and Clostridium septicum in Clostridial myonecrosis. J. Clin. Microbiol. 48, 1093-1098.
• Lin L.Q., Chen J., Lin Q.H., Chen W., Chen J.H., Yao H., Liu A.L., Lin X.H., Chen Y.Z. (2010) Electrochemical biosensor based on nanogold-modified poly-eriochrome black T film for BCR/ABL fusion gene assay by using hairpin LNA probe. Talanta 80, 2113-2119.
• Han W.H., Liao J.M., Chen K.L., Wu S.M., Chiang Y.W., Lo S.T., Chen C.L., Chiang C.M. (2010) Enhanced recognition of single-base mismatch using locked nucleic acid-integrated hairpin DNA probes revealed by atomic force microscopy nanolithography. Anal. Chem. 82, 2395-2400.
• Muradrasoli S., Mohamed N., Belak S., Czifra G., Herrmann B., Berencsi G., Blomberg J. (2010) Broadly targeted triplex real-time PCR detection of influenza A, B and C viruses based on the nucleoprotein gene and a novel "MegaBeacon" probe strategy. J. Virol. Methods 163, 313-322.
• Choi J.J., Jang M., Kim J., Park H. (2010) Highly sensitive PNA array platform technology for single nucleotide mismatch discrimination. J. Microbiol. & Biotech. 20, 287-293.
• Kawakami S., Kubota K., Imachi H., Yamaguchi T., Harada H., Ohashi A. (2010) Detection of single copy genes by two-pass tyramide signal amplification fluorescence in situ hybridization (Two-Pass TSA-FISH) with single oligonucleotide probes. Microbes and Environments 25, 15-21.
• van der Vries E., Jonges M., Herfst S., Maaskant J., Van der Linden A., Guldemeester J., Aron G.I., Bestebroer T.M., Koopmans M., Meijer A., Fouchier R.A.M., Osterhaus A.D.M.E., Boucher C.A., Schutten M. (2010) Evaluation of a rapid molecular algorithm for detection of pandemic influenza A (H1N1) 2009 virus and screening for a key oseltamivir resistance (H275Y) substitution in neuraminidase. J. Clin. Virol. 47, 34-37.
• Bruge F., Littarru G.P., Silvestrini L., Mancuso T., Tiano L. (2009) A novel real time PCR strategy to detect SOD3 SNP using LNA probes. Mutation Research-Fundamental and Molecular Mechanisms of Mutagenesis 669, 80-84.
• MacAskill A., Crawford. D, Graham D., Faulds K. (2009) DNA sequence detection using surface-enhanced resonance Raman spectroscopy in a homogeneous multiplexed assay. Anal. Chem. 81, 8134-8140.
• Laschi S., Palchetti I., Marrazza G., Mascini M. (2009) Enzyme-amplified electrochemical hybridization assay based on PNA, LNA and DNA probe-modified micro-magnetic beads. Bioelectrochemistry 76, 214-220.
• Lin L.Q., Lin X.H., Chen J.H., Chen W., He M., Chen Y.Z. (2009) Electrochemical biosensor for detection of BCR/ABL fusion gene based on hairpin locked nucleic acids probe. Electrochemistry Communications 11, 1650-1653.
• Lu J., Tsourkas A. (2009) Imaging individual microRNAs in single mammalian cells in situ. Nucleic Acids Res. 37, e100.
• Chen W., Seifert K.A., Levesque C.A. (2009) A high density COX1 barcode oligonucleotide array for identification and detection of species of Penicillium subgenus Penicillium. Mol. Ecol. Res. 9, 114-129.
• Petri A., Lindow M., Kauppinen S. (2009) MicroRNA silencing in primates: Towards development of novel therapeutics. Cancer Res. 69, 393-395.
• Madani M., Ward L.J., De Boer S.H. (2008) Multiplex real-time polymerase chain reaction for identifying potato cyst nematodes, Globodera pallida and Globodera rostochiensis, and the tobacco cyst nematode, Globodera tabacum. Can. J. Plant Pathol. 30, 554-564.
• Kutyavin I.V. (2008) Use of base-modified duplex-stabilizing deoxynucleoside 5 '-triphosphates to enhance the hybridization properties of primers and probes in detection polymerase chain reaction. Biochemistry 47, 13666-13673.
• Owczarzy R., Tataurov A.V., Wu Y., Manthey J.A., McQuisten K.A., Almabrazi H.G., Pedersen K.F., Lin Y., Garretson J., McEntaggart N.O., Sailor C.A., Dawson R.B., Peek A.S. (2008) IDT SciTools: a suite for analysis and design of nucleic acid oligomers. Nucleic Acids Res. 36, Suppl. S, W163-W169.
• Morgan J.R., Lyon R.P., Maeda D.Y., Zebala J.A. (2008) Snap-to-it probes: chelate-constrained nucleobase oligomers with enhanced binding specificity. Nucleic Acids Res. 36, 3522-3530.
• Tan Z.J., Chen S.J. (2008) Salt dependence of nucleic acid hairpin stability. Biophys. J. 95, 738-752.
• Guterstam P., Lindgren M., Johansson H., Tedebark U., Wengel J., El Andaloussi S., Langel U. (2008) Splice-switching efficiency and specificity for oligonucleotides with locked nucleic acid monomers. Biochem. J. 412, Part 2, 307-313.
• Ballantyne K.N., van Oorschot R.A.H., Mitchell R.J. (2008) Locked nucleic acids in PCR primers increase sensitivity and performance. Genomics 91, 301-305.
• Umemoto T., Hrdlicka P.J., Babu B.R., Wengel J. (2007) Sensitive SNP dual-probe assays based on pyrene-functionalized 2 '-amino-LNA: Lessons to be learned. ChemBiochem 8, 2240-2248.
• Ahlborn C., Siegmund K., Richert C. (2007) Isostable DNA. J. Am. Chem. Soc. 129, 15218-15232.
• Donini S., Clerici M., Wengel J., Vester B., Peracchi A. (2007) The advantages of being locked - Assessing the cleavage of short and long RNAs by locked nucleic acid-containing 8-17 deoxyribozymes. J. Biol. Chem. 282, 35510-35518.
• Nakitandwe J., Trognitz F., Trognitz B. (2007) Reliable allele detection using SNP-based PCR primers containing locked nucleic acid: application in genetic mapping. Plant Methods 3, Art. No. 2.
• Prepens S., Kreuzer K.A., Leendertz F., Nitsche A., Ehlers B. (2007) Discovery of herpesviruses in multi-infected primates using locked nucleic acids (LNA) and a bigenic PCR approach. Virology J. 4, Art. No. 84.
• Di Primo C., Rudloff I., Reigadas. S, Arzumanov A.A., Gait M.J., Toulme J.J. (2007) Systematic screening of LNA/2'-O-methyl chimeric derivatives of a TAR RNA aptamer. FEBS Lett. 581, 771-774.
• Politz J.C.R., Zhang F., Pederson T. (2006) MicroRNA-206 colocalizes with ribosome-rich regions in both the nucleolus and cytoplasm of rat myogenic cells. Proc. Nat. Acad. Sci. USA 103, 18957-18962.
• Levin J.D., Fiala D., Samala M.F., Kahn J.D., Peterson R.J. (2006) Position-dependent effects of locked nucleic acid (LNA) on DNA sequencing and PCR primers. Nucleic Acids Res. 34, e142.