Enhanced recognition of non-complementary hybridization by single-LNA-modified oligonucleotide probes.
ABSTRACT Locked nucleic acid (LNA) is a deoxyribonucleotide analogue with an unusual 'locked' furanose conformation. LNA-modified oligonucleotide probes have demonstrated an enhanced binding affinity towards their complementary strands; however, their potential to discriminate non-complementary hybridization of mismatches has not been explored. In this study, we investigated the effect of the chemical nature of LNA nucleobases on the hybridization stability and the capability of LNA-modified oligonucleotides to discriminate the LNA:DNA mismatched base pairs. It was observed that LNA modification indeed improves the discrimination capability of oligonucleotides by increasing the melting temperature differences between the complementary duplexes and hybrids containing mismatches. Particularly, LNA purines offer a greater potential to recognize the mismatches than LNA pyrimidines and DNA purines. Real-time PCR experiments further confirmed that LNA modifications at the 3'-end are more effective. The results and conclusions in this study provide useful information for hybridization-based nucleic acid analysis where designing sound oligonucleotide probes is crucial to the success of the analyses.
SourceAvailable from: Sourav Mishra[Show abstract] [Hide abstract]
ABSTRACT: Several investigations on DNA-based nucleic acid sensors performed in the last few years point towards the requirement of an alternative nucleic acid that can detect target DNA strands more efficiently, i.e., with higher sensitivity and selectivity, and can be more robust compared to the DNA sensor probes. Locked Nucleic Acid (LNA), a conformationally restricted DNA analogue, is potentially a better alternative than DNA, since it is nuclease-resistant, it can form more stable duplex with DNA in sequence-specific manner, and it interacts less with substrate surface due to presence of a rigid backbone. In this work, we probed solid-phase dehybridization of ssDNA targets from densely packed fully modified ssLNA probes immobilized onto gold(111) surface by fluorescence-based measurement of the 'on-surface' melting temperatures. We find that mismatch discrimination can be clearly improved by applying the surface-tethered LNA probes, in comparison to the corresponding DNA probes. We show that concentration as well as type of cation (monovalent and polyvalent) can significantly influence thermal stability of the surface-confined LNA-DNA duplexes - the nature of concentration dependence contradicting the solution phase behavior. Since the ionic setting influenced the fully matched duplexes more strongly than the singly mismatched duplexes, the mismatch discrimination ability of the surface-confined LNA probes could be controlled by ionic modulations. To our knowledge, this is the first report on ionic regulation of melting behavior of surface-confined LNA-DNA duplexes.Analytical Chemistry 12/2012; 85(3). DOI:10.1021/ac3028382 · 5.83 Impact Factor
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ABSTRACT: A novel, highly sensitive technology for the detection, enrichment, and separation of trace amounts of target DNA was developed on the basis of amino-modified fluorescent magnetic composite nanoparticles (AFMN). In this study, the positively charged amino-modified composite nanoparticles conjugate with the negatively charged capture DNA through electrostatic binding. The optimal combination of AFMN and capture DNA was measured by dynamic light scattering (DLS) and UV-vis absorption spectroscopy. The highly sensitive detection of trace amounts of target DNA was achieved through enrichment by means of AFMN. The detection limit for target DNA is 0.4 pM, which could be further improved by using a more powerful magnet. Because of their different melting temperatures, single-base mismatched target DNA could be separated from perfectly complementary target DNA. In addition, the photoluminescence (PL) signals of perfectly complementary target DNA and single-base mismatched DNA as well as the hybridization kinetics of different concentrations of target DNA at different reaction times have also been studied. Most importantly, the detection, enrichment, and separation ability of AFMN was further verified with milk. Simple and satisfactory results were obtained, which show the great potential in the fields of mutation identification and clinical diagnosis.Analytical and Bioanalytical Chemistry 03/2010; 397(3):1251-8. DOI:10.1007/s00216-010-3625-8 · 3.58 Impact Factor
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ABSTRACT: DNA polymerases amplify DNA fragments through primer extension reactions. However, polymerization behavior of short primers in the primer extension process has not been systematically explored. In this study, we examined the minimal primer length required for primer extension, and the effect of primer length, mismatches and other conditions on DNA polymerization using a non-radioactive method. Under the condition we conducted, the shortest primers polymerized by Klenow fragment (KF) and Taq DNA polymerase in our experiments were respectively heptamer and octamer. The extension efficiency was also affected by the up-stream overhanging structure of the primer-template complex. We hypothesized a simple model to interpret these observations based on the polymerase structures. Furthermore, it was found that the longer the primer, the more efficient is the primer extension. These polymerization behavior of short primers lay foundation about DNA polymerization mechanism and development of novel nucleic acid detection assays.Acta Biochimica et Biophysica Sinica 10/2010; 42(10):722-8. DOI:10.1093/abbs/gmq082 · 2.09 Impact Factor