ABSTRACT
Coronary artery aneurysmal dilation is a rare finding with poorly understood mechanism of action that is found in small population of patients undergoing coronary angiography. Mycotic coronary aneurysm is an even rarer cause of coronary aneurysmal dilatation that develops as a potentially fatal complication of bacteremia. We present a case of mycotic right coronary artery aneurysm in a nonsurgical candidate with complex medical comorbidities treated with percutaneous coronary intervention via covered stents.
Subject(s)
Aneurysm, Infected , Coronary Aneurysm , Coronary Vessels , Percutaneous Coronary Intervention , Stents , Humans , Coronary Aneurysm/etiology , Coronary Aneurysm/surgery , Coronary Angiography/adverse effects , Coronary Vessels/diagnostic imaging , Percutaneous Coronary Intervention/adverse effects , Stents/adverse effects , Treatment Outcome , Aneurysm, Infected/etiology , Aneurysm, Infected/surgeryABSTRACT
Quadruplex priming amplification (QPA) allows isothermal amplification of nucleic acids with improved yield and simplified detection. This assay is based on a DNA quadruplex, GGGTGGGTGGGTGGG (G3T), which in the presence of specific cations possesses unusually high thermal stability. QPA employs truncated G3T sequences as primers, which upon polymerase elongation, self-dissociate from the binding site and allow the next round of priming without thermal unfolding of amplicons. The rate of amplification strongly depends on the thermal stability of the primer/primer binding site (PBS) complex and to date QPA has been demonstrated to work over a narrow temperature range. To expand the capabilities of QPA, in the present study, we studied the fold and thermodynamic properties of the wild-type G3T and variants containing sequence modifications or extensions at the 5'-end. Circular dichroism studies demonstrate that the substitution of thymidines by other nucleotides or GC addition at the 5'-end does not change the parallel fold of G3T. Thermal unfolding experiments revealed that purine bases incorporated at loop positions and 5'-end dinucleotide extension significantly destabilize the quadruplex, while loop pyrimidines have almost no effect. Overall, the results of these studies suggest that linear isothermal QPA can be performed over a wide temperature range to accommodate both thermophilic and mesophilic DNA polymerases.
Subject(s)
DNA Primers/chemistry , DNA/genetics , G-Quadruplexes , Nucleic Acid Amplification Techniques/methods , Base Sequence , DNA/chemistry , DNA Primers/genetics , Nucleic Acid Conformation , Temperature , ThermodynamicsABSTRACT
We previously developed a method, known as quadruplex priming amplification (QPA), which permits isothermal amplification of DNA. The assay is based on a DNA quadruplex formed by the GGGTGGGTGGGTGGG (G3T) sequence. G3T has three unique properties that are fundamental for QPA; (i) G3T forms a quadruplex with significantly more favorable thermodynamics than the corresponding DNA duplexes; (ii) removal of guanines at the 3'-end inhibits quadruplex formation; and (iii) incorporated fluorescent nucleotides, such as 2-aminopurine (2AP) or 6-methylisoxanthopterin (6MI), which are quenched by neighboring nucleotides, regain maximum emission upon quadruplex formation. New model studies carried out here with primers missing one, two and three guanines reveal that the driving force for QPA comes from the difference in thermal stability between the primer/template and the product complexes. Primers missing one and two guanines are able to self-dissociate from the template upon elongation, whereas QPA is not observed when the primer lacks three 3'-nucleotides. QPA reaches its maximum rate at temperatures slightly higher than the T(m) of the primer/template complex and is more efficient in the presence of only dGTP. QPA-based assays also revealed that Taq is able to incorporate thymidines opposite template 2AP, while no significant incorporation was observed opposite template 6MI.
Subject(s)
DNA Primers/genetics , DNA/genetics , G-Quadruplexes , Nucleic Acid Amplification Techniques/methods , 2-Aminopurine/chemistry , Base Sequence , DNA/chemistry , DNA Primers/chemistry , Fluorescent Dyes/chemistry , Guanine/chemistry , Spectrometry, Fluorescence , Temperature , Templates, Genetic , Thermodynamics , Xanthopterin/analogs & derivatives , Xanthopterin/chemistryABSTRACT
Quadruplexes are involved in the regulation of gene expression and are part of telomeres at the ends of chromosomes. In addition, they are useful in therapeutic and biotechnological applications, including nucleic acid diagnostics. In the presence of K(+) ions, two 15-mer sequences d(GGTTGGTGTGGTTGG) (thrombin binding aptamer) and d(GGGTGGGTGGGTGGG) (G3T) fold into antiparallel and parallel quadruplexes, respectively. In the present study, we measured the fluorescence intensity of one or more 2-aminopurine or 6-methylisoxanthopterin base analogs incorporated at loop-positions of quadruplex forming sequences to develop a detection method for DNA sequences in solution. Before quadruplex formation, the fluorescence is efficiently quenched in all cases. Remarkably, G3T quadruplex formation results in emission of fluorescence equal to that of a free base in all three positions. In the case of thrombin binding aptamer, the emission intensity depends on the location of the fluorescent nucleotides. Circular dichroism studies demonstrate that the modifications do not change the overall secondary structure, whereas thermal unfolding experiments revealed that fluorescent analogs significantly destabilize the quadruplexes. Overall, these studies suggest that quadruplexes containing fluorescent nucleotide analogs are useful tools in the development of novel DNA detection methodologies.
