Phenylethynylpyrene-labeled oligonucleotide probes for excimer fluorescence SNP analysis of 23S rRNA gene in clarithromycin-resistant Helicobacter pylori strains

Prokhorenko, Igor A.; Malakhov, Andrei D.; Козлова, А. А.; Momynaliev, K. Т.; Govorun, Vadim M.; Korshun, Vladimir A.

doi:10.1016/j.mrfmmm.2006.02.007

Cited by 16 publications

(6 citation statements)

References 33 publications

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“…This correlates with its short excited lifetime (7.72 ns for 1-PEPy in EtOH ). The absorbance and fluorescence of 1-PEPy derivatives on DNA are approximately 50 nm and 30 nm red-shifted, respectively, in comparison with pyrene derivatives; the first fluorescence maximum of 1-PEPy in oligonucleotides is within 400–405 nm – . These spectral features reduce 100-fold the 1-PEPy detection limit with respect to that of pyrene .…”

Section: Introductionmentioning

confidence: 89%

“…Recently, we – and others – introduced 1-PEPy fluorochrome as a label for nucleic acids. It is well-known that introduction of a phenylethynyl group in a fluorescent molecule leads to an increase in absorption together with a bathochromic shift in absorption and emission, a decrease in Stoke’s shift and excited lifetime, and an increase in emission quantum yield (fluorescence intensity) ( 48 and referecces cited therein).…”

Section: Introductionmentioning

confidence: 99%

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1-Phenylethynylpyrene (1-PEPy) as Refined Excimer Forming Alternative to Pyrene: Case of DNA Major Groove Excimer

et al. 2007

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1-Phenylethynylpyrene fluorochrome was studied as meta- and para-derivatives of arabino-uridine-2'-carbamates in ss and dsDNA. 1-PEPy showed red-shifted emission and increased fluorescence quantum yield compared to pyrene. Although 1-PEPy has very short excited lifetime (<2.5 ns), it is able to form inter- and intrastrand excimers on DNA, probably resulting from spatial preorganization of two dye molecules.

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Section: Introductionmentioning

confidence: 89%

Section: Introductionmentioning

confidence: 99%

1-Phenylethynylpyrene (1-PEPy) as Refined Excimer Forming Alternative to Pyrene: Case of DNA Major Groove Excimer

et al. 2007

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“…Resistance due to mutations at other positions has also been reported (Fontana et al, 2002;Khan et al, 2004;Toracchio et al, 2004). Several methods to detect CAM-resistant H. pylori from faecal samples have used RFLP (Alarcon et al, 2003), real-time PCR (Schabereiter-Gurtner et al, 2004) or other methods (Morris et al, 2005;Prokhorenko et al, 2006;van Doorn et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

Detection of mixed clarithromycin-resistant and -susceptible Helicobacter pylori using nested PCR and direct sequencing of DNA extracted from faeces

Noguchi

Rimbara

Kato

et al. 2007

Journal of Medical Microbiology

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The major cause of chemotherapy failure in patients with chronic gastritis and peptic ulcers caused by Helicobacter pylori is clarithromycin (CAM) resistance due to a mutation in the 23S rRNA gene. This study describes a non-invasive and accurate method for the detection of mixed CAM-resistant and -susceptible H. pylori by sequencing of the H. pylori 23S rRNA gene. Faeces were crushed with beads and the 23S rRNA gene was amplified using a nested PCR on the extracted DNA. Mutation analysis of this gene using this method showed that 20.4 % of patients carried mixed CAM-susceptible (wild type) and -resistant (A2142G or A2143G mutant) H. pylori. Furthermore, it was found that 66.6 % of patients who had been treated unsuccessfully carried one of these mutations in the 23S rRNA gene (including the mixed type), whilst standard culture detected CAM-resistant isolates in only 22.2 % of patients with unsuccessful treatment. These data suggest that, for successful therapy, the diagnosis method described here would more accurately detect CAM-resistant H. pylori, including mixed infections. INTRODUCTIONHelicobacter pylori is a Gram-negative, spiral bacterium found in the human stomach. This micro-organism causes chronic gastritis and peptic ulcers (Kuipers, 1997) and is linked to gastric cancer and other non-gastrointestinal diseases (Kusters et al., 2006;Leong & Sung, 2002). To treat H. pylori infections, a triple therapy is used comprising a combination of a proton pump inhibitor and two antimicrobial agents. This is reported to be the most successful method for eradication of infection (Malfertheiner et al., 2002). In Japan, a combination of lansoprazole, amoxicillin and clarithromycin (CAM) (the LAC regimen) is commonly used to eliminate H. pylori (Asaka et al., 2001). Although most patients are treated successfully with this triple therapy, some failure has necessitated the use of additional drugs such as levofloxacin (Asaka et al., 2001;Kato et al., 2000). The major cause of therapy failure in patients is resistance to CAM (Kato et al., 2000; Rimbara et al., 2005a). Therefore, a reliable diagnostic test to detect macrolide resistance would be useful in planning a therapeutic regimen. Standard culture is generally used to determine the susceptibility of H. pylori to antimicrobial agents; however, acquiring a test sample is invasive for the patient, as endoscopy is required to obtain gastric specimens. Faecal culture is not possible, as H. pylori coccoid forms are unculturable; however, there are some reliable non-invasive tests for the diagnosis of H. pylori infections (Graham et al., 1987;Leodolter et al., 2003;Shuber et al., 2002;Vaira et al., 1999).Resistance to CAM in H. pylori is due to a mutation in the 23S rRNA subunit in the 50S ribosome. The two most common mutations are an adenine-to-guanine transition at position 2142 or 2143, the latter leading to an adenineto-cytosine transversion at position 2142 (Alarcon et al., 2003;van Doorn et al., 2001). Resistance due to mutations at other positions has also been rep...

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“…An excimer band was also observed upon insertion of more than one dye into the DNA strand in close proximity to each other. For 4‐(pyren‐1‐ylethynyl)phenyl derivatives, a strong pyrene excimer at approximately 505 nm was observed for ssONs with two intercalators inserted as neighbors58 and as next‐nearest neighbors 15. A relatively higher excimer band was observed for ssON with p TINA monomers as next‐nearest neighbors in comparison with the o TINA analogues ( ON5 and ON9 , Figure 4 A and B).…”

Section: Resultsmentioning

confidence: 96%

1‐, 2‐, and 4‐Ethynylpyrenes in the Structure of Twisted Intercalating Nucleic Acids: Structure, Thermal Stability, and Fluorescence Relationship

Filichev

Astakhova

Malakhov

et al. 2008

Chemistry A European J

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A postsynthetic, on-column Sonogashira reaction was applied on DNA molecules modified by 2- or 4-iodophenylmethylglycerol in the middle of the sequence, to give the corresponding ortho- and para-twisted intercalating nucleic acids (TINA) with 1-, 2-, and 4-ethynylpyrene residues. The convenient synthesis of 2- and 4-ethynylpyrenes started from the hydrogenolysis of pyrene that has had the sulfur removed and separation of 4,5,9,10-tetrahydropyrene and 1,2,3,6,7,8-hexahydropyrene, which were later converted to the final compounds by successive Friedel-Crafts acetylation, aromatization by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, and a Vilsmeier-Haack-Arnold transformation followed by a Bodendorf fragmentation. Significant alterations in thermal stability of parallel triplexes and antiparallel duplexes were observed upon changing the attachment of ethynylpyrenes from para to ortho in homopyrimidine TINAs. Thus, for para-TINAs the bulge insertion of an intercalator led to high thermal stability of Hoogsteen-type parallel triplexes and duplexes, whereas Watson-Crick-type duplexes were destabilized. In the case of ortho-TINA, both Hoogsteen and Watson-Crick-type complexes were stabilized. Alterations in the thermal stability were highly influenced by the ethynylpyrene isomers used. This also led to TINAs with different changes in fluorescence spectra depending on the secondary structures formed. Stokes shift of approximately 100 nm was detected for pyren-2-ylethynylphenyl derivatives, whereas values for 1- and 4-ethynylpyrenylphenyl conjugates were 10 and 40 nm, respectively. In contrast with para-TINAs, insertion of two ortho-TINAs opposite each other in the duplex as a pseudo-pair resulted in formation of an excimer band at 505 nm for both 1- and 4-ethynylpyrene analogues, which was also accompanied with higher thermal stability.

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Phenylethynylpyrene-labeled oligonucleotide probes for excimer fluorescence SNP analysis of 23S rRNA gene in clarithromycin-resistant Helicobacter pylori strains

Cited by 16 publications

References 33 publications

1-Phenylethynylpyrene (1-PEPy) as Refined Excimer Forming Alternative to Pyrene: Case of DNA Major Groove Excimer

1-Phenylethynylpyrene (1-PEPy) as Refined Excimer Forming Alternative to Pyrene: Case of DNA Major Groove Excimer

Detection of mixed clarithromycin-resistant and -susceptible Helicobacter pylori using nested PCR and direct sequencing of DNA extracted from faeces

1‐, 2‐, and 4‐Ethynylpyrenes in the Structure of Twisted Intercalating Nucleic Acids: Structure, Thermal Stability, and Fluorescence Relationship

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