Locked Nucleic Acids and Intercalating Nucleic Acids in the Design of Easily Denaturing Nucleic Acids: Thermal Stability Studies

Filichev, Vyacheslav V.; Christensen, Ulf Bech; Pedersen, Erik B.; Babu, B. Ravindra; Wengel, Jesper

doi:10.1002/cbic.200400222

Cited by 30 publications

(18 citation statements)

References 37 publications

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“…As expected, we observed a large difference in Tm for all probes bound to fully complementary oligonucleotides vs. oligonucleotides containing one mismatch. The difference between the Tm values for each oligo-nucleotide was in agreement with the results published earlier (Filichev et al 2004;Kaur et al 2008;Pasternak and Wengel 2010). LNA/2′OMe and PNA showed high stability in all buffers, comparatively to LNA/DNA oligonucleotides.…”

Section: Discussionsupporting

confidence: 92%

Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids

Fontenete

Barros

Madureira

et al. 2015

Appl Microbiol Biotechnol

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In the past few years, several researchers have focused their attention on nucleic acid mimics due to the increasing necessity of developing a more robust recognition of DNA or RNA sequences. Fluorescence in situ hybridization (FISH) is an example of a method where the use of these novel nucleic acid monomers might be crucial to the success of the analysis. To achieve the expected accuracy in detection, FISH probes should have high binding affinity towards their complementary strands and discriminate effectively the noncomplementary strands. In this study, we investigate the effect of different chemical modifications in fluorescent probes on their ability to successfully detect the complementary target and discriminate the mismatched base pairs by FISH. To our knowledge, this paper presents the first study where this analysis is per-formed with different types of FISH probes directly in biological targets, Helicobacter pylori and Helicobacter acinonychis. This is also the first study where unlocked nucleic acids (UNA) were used as chemistry modification in oligonucleotides for FISH methodologies. The effectiveness in detecting the specific target and in mismatch discrimination appears to be improved using locked nucleic acids 2 (LNA)/2′-O-methyl RNA (2′OMe) or peptide nucleic acid (PNA) in comparison to LNA/DNA, LNA/UNA, or DNA probes. Further, the use of LNA modifications together with 2′OMe monomers allowed the use of shorter fluorescent probes and increased the range of hybridization temperatures at which FISH would work.

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Section: Discussionsupporting

confidence: 92%

Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids

Fontenete

Barros

Madureira

et al. 2015

Appl Microbiol Biotechnol

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“…Pseudo-complementary PNAs ( 7 ) have been used for targeting breathed plasmid DNA using non-physiological salt concentration ( 8 ). Recently, we have presented the design of easily denaturing nucleic acids constructed from DNA- and RNA-discriminating molecules ( 9 ). LNA binds better to ssRNA than to ssDNA ( 10 , 11 ).…”

Section: Introductionmentioning

confidence: 99%

Easily denaturing nucleic acids derived from intercalating nucleic acids: thermal stability studies, dual duplex invasion and inhibition of transcription start

Filichev

Vester²,

Hansen³

et al. 2005

Nucleic Acids Research

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The bulged insertions of (R)-1-O-(pyren-1-ylmethyl)glycerol (monomer P) in two complementary 8mer DNA strands (intercalating nucleic acids) opposite to each other resulted in the formation of an easily denaturing duplex, which had lower thermal stability (21.0°C) than the wild-type double-stranded DNA (dsDNA, 26.0°C), but both modified oligodeoxynucleotides had increased binding affinity toward complementary single-stranded DNA (ssDNA) (41.5 and 39.0°C). Zipping of pyrene moieties in an easily denaturing duplex gave formation of a strong excimer band at 480 nm upon excitation at 343 nm in the steady-state fluorescence spectra. The excimer band disappeared upon addition of a similar short dsDNA, but remained when adding a 128mer dsDNA containing the same sequence. When P was inserted into 2′-OMe-RNA strands, the duplex with zipping P was found to be more stable (42.0°C) than duplexes with the complementary ssDNAs (31.5 and 19.5°C). The excimer band observed in the ds2′-OMe-RNA with zipping P had marginal changes upon addition of both 8 and 128mer dsDNA. Synthesized oligonucleotides were tested in a transcriptional inhibition assay for targeting of the open complex formed by Escherichia coli RNA polymerase with the lac UV-5 promoter using the above mentioned 128mer dsDNA. Inhibition of transcription was observed for 8mer DNAs possessing pyrene intercalators and designed to target both template and non-template DNA strands within the open complex. The observed inhibition was partly a result of unspecific binding of the modified DNAs to the RNA polymerase. Furthermore, the addition of 8mer DNA with three bulged insertions of P designed to be complementary to the template strand at the +36 to +43 position downstream of the transcription start resulted in a specific halt of transcription producing a truncated RNA transcript. This is to our knowledge the first report of an RNA elongation stop mediated by a small DNA sequence possessing intercalators. The insertions of P opposite to each other in ds2′-OMe-RNA showed inhibition efficiency of 96% compared with 25% for unmodified ds2′-OMe-RNA.

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“…As previously, (S)-1aminopropane-2,3-diol serves as an acyclic linker between the phosphodiesters. Similar propanediol linkers have been used by other groups to prepare glycol nucleic acid (GNA), [25] twisted intercalating nucleic acids (TINA), [26] alkyne-modi-fied oligonucleotides for the click-type postsynthetic modification, [27] and by our group for fluorescent DNA base substitutions. [15,28] The linker has been attached to the thiazole ring of the TO dye (Scheme 1).…”

mentioning

confidence: 97%

Fluorescent Color Readout of DNA Hybridization with Thiazole Orange as an Artificial DNA Base

Berndl¹,

Wagenknecht²

2009

Angew Chem Int Ed

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Locked Nucleic Acids and Intercalating Nucleic Acids in the Design of Easily Denaturing Nucleic Acids: Thermal Stability Studies

Cited by 30 publications

References 37 publications

Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids

Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids

Easily denaturing nucleic acids derived from intercalating nucleic acids: thermal stability studies, dual duplex invasion and inhibition of transcription start

Fluorescent Color Readout of DNA Hybridization with Thiazole Orange as an Artificial DNA Base

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