A fiber optic biosensor for fluorimetric detection of triple-helical DNA

Abstract

A fiber optic biosensor was used for the fluorimetric detection of T/AT triple-helical DNA formation. The surfaces of two sets of fused silica optical fibers were functionalized with hexaethylene oxide linkers from which decaadenylic acid oligonucleotides were grown in the 3'to 5'and 5'to 3'direction, respectively, using a DNA synthesizer. Fluorescence studies of hybridization showed unequivocal hybridization between oligomers immobilized on the fibers and complementary oligonucleotides from the solution phase, as detected by fluorescence from intercalated ethidium bromide. The complementary oligonucleotide, dT10, which was expected to Watson-Crick hybridize upon cooling the system below the duplex melting temperature ( T m), provided a fluorescence intensity with a negative temperature coefficient. Upon further cooling, to the point where the pyrimidine motif T*AT triple-helix formation occurred, a fluorescence intensity change with a positive temperature coefficient was observed. The reverse-Hoogsteen T.AT triplex, which is known to form with branched nucleic acids, provided a corresponding decrease in fluorescence intensity with decreasing temperature. Full analytical signal evolution was attainable in minutes.

Design of a Molecular Beacon DNA Probe with Two Fluorophores

“…The ratio of I coumarin / I FAM for MB 1 is 0.164±0.002. Based on a signal/noise ratio of 3/1, the detection limit for DNA 1 was determined to be 0.17 n M , which is one order of magnitude lower than that for many other DNA probes 11, 12. It is worth noting that this detection limit is achieved with a conventional spectrometer with a mercury lamp and can be further improved with a more efficient optical detection system and a better optical excitation by using lasers.…”

Drosophila melanogaster kl-3 and kl-5 Y-loops harbor triple-stranded nucleic acids

“…Finally, a number of proteins that are able to bind triplexes have been identified (Kiyama and Camerini-Otero, 1991;Musso et al, 1998;Musso et al, 2000;Nelson et al, 2000), one of which has been isolated in D. melanogaster (Jimenez-Garcia et al, 1998). Several techniques have been developed to detect triplexes in vitro (Uddin et al, 1997;Cherny et al, 1998;Rutigliano et al, 1998;Mariappan et al, 1999), but most of these techniques can artificially induce their formation. To date, triplexes can be most reliably detected by using either DNA intercalary substances (Lee et al, 1993;Fang et al, 1994;Moraru-Allen et al, 1997;Shchyolkina and Borisova, 1997) or triplex-directed antibodies.…”

MicroCantilever (MC) based nanomechanical sensor for detection of molecular interactions

“…The initial peak and subsequent decay may be due to different rates of physisorption between silicon or gold surface, charge transduction, or reversed-Hoogsteen T·AT triplex formation on the reference cantilever. It could also be sampling interactions involving reverse Hoogsteen or other configurations between the poly A chains on sensing cantilever (Uddin, Piunno et al 1997;Wu, Ji et al 2001;Cheng, Cuda et al 2006).…”