Hannah Elizabeth Savage

Target-assembled ExciProbes: Application to DNA Detection at the Level of PCR Product and Plasmid DNA

Abstract Recently, we introduced a novel exciplex-based approach for detection of nucleic acids using a model DNA-mounted exciplex system, consisting of two 8-mer ExciProbes hybridized to a complementary 16-mer DNA target. We now show, for the first time, that this approach can be used to detect DNA at the level of PCR product and plasmid, when the target sequence (5′-GCCAAACACAGAATCG-3′) was embedded in long DNA molecules (PCR products and ∼3 Kbp plasmid). A remarkably stringent demand is made of the solvent conditions for this exciplex emission to occur, viz., emission is optimal for DNA at 80% tri-fluoroethanol, even in the plasmid situations, raising the question of the molecular structural basis of this system. We show that a perfectly matched plasmid target can be differentiated from target containing single nucleotide substitutions; hence, ExciProbes could be applied to SNP analysis. The effect of counter cations (Na+, K+, and Mg2+) and PCR additives on exciplex emission has been also examined.

New concepts of fluorescent probes for specific detection of DNA sequences: bis-modified oligonucleotides in excimer and exciplex detection.

The detection of single base mismatches in DNA is important for diagnostics, treatment of genetic diseases, and identification of single nucleotide polymorphisms. Highly sensitive, specific assays are needed to investigate genetic samples from patients. The use of a simple fluorescent nucleoside analogue in detection of DNA sequence and point mutations by hybridisation in solution is described in this study. The 5′-bispyrene and 3′-naphthalene oligonucleotide probes form an exciplex on hybridisation to target in water and the 5′-bispyrene oligonucleotide alone is an adequate probe to determine concentration of target present. It was also indicated that this system has a potential to identify mismatches and insertions. The aim of this work was to investigate experimental structures and conditions that permit strong exciplex emission for nucleic acid detectors, and show how such exciplexes can register the presence of mismatches as required in SNP analysis. This study revealed that the hybridisation of 5′-bispyrenyl fluorophore to a DNA target results in formation of a fluorescent probe with high signal intensity change and specificity for detecting a complementary target in a homogeneous system. Detection of SNP mutations using this split-probe system is a highly specific, simple, and accessible method to meet the rigorous requirements of pharmacogenomic studies. Thus, it is possible for the system to act as SNP detectors and it shows promise for future applications in genetic testing.

Multipurpose indicator in cells using intramolecular FRET between GFP and chemical compounds

Specific, surface cysteine sites have been introduced into Green Fluorescent Protein (GFP) to allow site-specific chemical modification by thiol-directed reagents. These sites have been labelled using BODIPY/eosin/rhodamine reagents as chemical FRET partners for the native GFP chromophore. When they were excited at 488 nm these engineered GFP: conjugated-fluorophore constructs, showed quenching of the native GFP fluorophore emission at 511 nm. New emission bands appeared correponding to each chemical fluorophore emission. Thus the new GFP chimeras exhibited strong intramolecular FRET. GFP mutants were then engineered with trypsin-sensitive sequences located close to the chemical fluorophore-bearing cysteine site. Trypsinolysis caused major changes in the FRET fluorescence spectra. On trypsinolysis the FRET was destroyed, as the FRET partners were now on separate molecules in the cleaved products. T Consequently, the emission wavelength altered from that of the chemically conjugated FRET partner back to that of the native fluorophore of the GFP (511 nm). This provides the possibility of efficient, ratio-based detection. Thus, protein re-engineering has led to novel probes capable of enzymatic triggering based on intramolecular FRET between GFP and specifically sited chemical labels.