Enhanced Discrimination Of Single LNA Modified Oligonucleotide Probes Against Non-Complementary Hybridization

Enhanced Discrimination of Single-LNA-Modified Oligonucleotide Probes against Non-Complementary HybridizationObjectiveComplementary hybridization has laid a solid foundation for nucleic acid analysis such as SNP genotyping,gene expression profiling,and mini-sequencing.To achieve the expected high detection accuracy,oligonucleotide probes should form relatively stable duplexes with its DNA or RNA target strands through complementary hybridization,and discriminate effectively those non-complementary strands containing mismatches or insertions.To ensure the detection specificity,the melting temperature differences between PM and MMs are expected to be as large as possible so that at a given hybridization temperature,all target strands form stable complementary duplexes with probes and all the non-complementary nucleotide strands are left single-stranded.In practical applications,there are many DNA and RNA fragments in the sample solutions whose sequences differ from the target strands by only one or two nucleotides,and their melting temperatures vary from that of complementary duplexes by a small margin as little as 0.5℃or even less.Therefore, effective discrimination of the target and non-targets needs to widen the melting temperature gap between the complementary duplexes and mismatched hybrids.LNA is a bicyclic furanose unit containing 2'-O,4'-C-methylene D-ribofuranosyl nucleotide.It is the bridged structure that effectively 'locks' the ribose in the N-type (3'-endo) conformation that is dominant in A-form DNA and RNA.This conformation enhances base stacking and phosphate backbone pre-organization and leads to the improved binding affinity toward complementary DNA or RNA strands. It has been reported that LNA-modified probes improved the complementary duplex stability by elevating T_m,normally in the range of 3.0 to 9.6℃per LNA modification. However,there is very limited study regarding the question how LNA-modifications improve the discrimination capability against the non-complementary DNA or RNA strands,that is,how LNA-modifications destabilize the duplexes containing mismatches.In this article,we report a study on the duplex stability affected by mismatches and insertions.The effects of the chemical nature,the positions,the numbers,and the cooperative behavior of mismatches as well as insertions on the 20-mer and 30-mer duplexes were examined.We investigated the ability of the LNA-modified probe to improve the hybridization stability of the complementary duplexes and to discriminate the non-complementary nucleotides.We designed a series of oligonucleotide probes containing single-LNA-modification and a group of DNA oligonucleotides containing different mismatches as well as insertions,and examined the effect of chemical nature and the cooperative behavior of mismatches and insertions on the stability of duplexes. We also utilized real-time PCR to further confirm the discrimination capability of the single-LNA modified oligonucleotide probes.The current results provide valuable information for designing LNA-modified oligonucleotide probes for mutation identification,medical diagnosis,forensic determination and other the hybridization-based DNA or RNA detections.Methods1.E-coil cultureTake out of Escherichia coli which freezed in -80℃refrigerator,and put it in LB medium then shaking culture it in 37℃incubator above 12 hour.2.extraction of E-coil genomic DNAE-coil genomic DNA was extracted using bacterial genomic DNA extraction kit.3.Melting temperature measurementThe melting process of oligonucleotide duplexes were monitored by measuring the absorbance at 260 nm on an UV-VIS spectrophotometer equipped with a temperature control accessory.The melting temperatures were determined in terms of the peak position of the first-derivative of melting curves by the software.4.Temperature gradient PCR experimentThe effect of mismatch on the duplex stabilities was further investigated using temperature gradient PCR experiment.5.Real-Time PCRThe discrimination capability of LNA-modified probes was further investigated using real-time PCR experiment.6.Common PCR experimentThe discrimination capability of LNA-modified probes was further investigated using common PCR experiment.Results1.Duplex thermal stability affected by single mismatchWhen 1-MM are included in the duplex hybridization,the melting curves are shifted leftward with respect to the PM curve,reducing the melting temperature.2.Duplex thermal stability affected by double mismatchThe second MM shifts the melting curve leftward further,leading to a big decrease of melting temperature.The FWHH(full width of half height) for the 2-MM duplex was broader than 1-MM and PM.3.Duplex thermal stability affected by mismatched positionsWhen cytosine substitute was made from the 5'-end to the 3'-end in a 20-mer oligonucleotide,the relationship of T_m with respect to the substitution position showed a U-like profile,indicating that the mismatches at the central portion can generate the most profound destructive effect.4.Duplex thermal stability affected by insertionsInsertions,just like mismatches,will reduce the duplex stability.5.Temperature gradient PCR experimentDuring the PCR amplification cycles,primers can hybridized with the PM,1-MM and 2-MM DNA template,the production of 2-MM<1-MM G:G > G:A> A:A > or≈T:T > or≈A:C > T:C > C:C.2.The mismatches at the center of the duplexes create more profound destructive (?) the thermal stability than at the edge.3.Just like mismatches,insertions also show a similar effect on the hybridization stability.4.LNA-modification in the oligonucleotide probes did not only increase the duplex stability significantly,but also destabilized the hybrids containing N:m mismatches.5.Particularly,the LNA-purines offer a greater potential to recognize the mismatches than LNA-pyrimidines and DNA-purines.6.The duplex becomes less unstable when the mismatch moves toward the ends of the duplex,and the duplex stability is primarily dependent upon the nature of mismatches and less dependent upon the position of complementary LNA:DNA hasapairs.7.The insertions on the target DNA strand of LNA-DNA duplexes create a larger destructive effect on the duplex stability than that on DNA-DNA duplexes,the insertion discrimination is greater when LNA modified purine bases than it modified pyrimidine bases.8.When being incorporated at the 3'-end of the PCR primers,LNA demonstrate a high discrimination potential to ensure the amplification success.