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

Abstract

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.

Keywords: 5′-bispyrene; DNA detection; Exciplex; Fluorescence; Stokes shift; Trifluoroethanol (TFE).

Figure 1

Split probe system

Figure 2

Target strands and nomenclature used for exciplex formation with mismatched targets. Mismatches and inserts are shown in italic. The Exci-probes used were: CGGTTTGT-5′- bispyrene and naphthalene-3′-GTCTTAGC.

Figure 3

Fluorescence spectra of 5′-bispyrene alone (1), 5′- bispyrene and target (2) and 5′-bispyrene, target and 3′- naphthalene (3) in 80% TFE/ Tris buffer (0.01 M Tris, 0.1 M NaCl, pH 8.4) at 5°C. Component concentrations were 2.5 µM in a total volume of 100 µl. Excitation was at 350 nm, spectra are corrected for TFE and buffer.

Figure 4

Fluorescence spectra of 5′-bispyrene alone (1), 5′- bispyrene and target (2), and 5′-bispyren, target and 3′- naphthalene (3) in 0% TFE/ Tris buffer (0.01 M Tris, 0.1 M NaCl, pH 8.4) at 5°C. Component concentrations were 2.5 µM in a total volume of 100 µL. Excitation was at 350 nm, spectra are corrected for TFE and buffer.

Figure 5

Melting curve of the full system in 0% v/v TFE / Tris buffer (10 mM Tris, 0.1 NaCl, pH 8.4) showing the change in absorbance at 260 nm as temperature was ramped at 0.25°C/min.

Figure 6

Fluorescence spectra of 5′-bispyrene alone (1), 5′-bispyrene and target (2) and 5′-bispyrene, target and 3′-phosphate (3) in 0% TFE/ Tris buffer (0.01 M Tris, 0.1 M NaCl, pH 8.4) at 5°C. Component concentrations were 2.5 µM in a total volume of 100 µl. Excitation was at 350 nm, spectra are corrected for TFE and buffer.

Figure 7

Fluorescence spectra of (1) 5′-bispyrene+WT target (- buffer), (2) 5′-bispyrene+WT target+ 3′-naphthalene (-3′-naphthalene and buffer), (3) 5′-bispyrene+3′-DMM target+ 3′-naphthalene (-3′- naphthalene and buffer) and (4) 5′-bispyrene+2-insertions target +3′-naphthalene (-3′-naphthalene and buffer) in 0% TFE/ Tris buffer (0.01 M Tris, 0.1 M NaCl, pH 8.4) at 5°C. Component concentrations were 2.5 µM in a total volume of 100 µl. Excitation was at 350 nm, spectra are corrected (as indicated).

Figure 8

Fluorescence monitoring titration of 5′-bis-pyrene probe (1.7 µM) with different concentrations of complementary DNA target (A 0 µM, B 0.4 µM, C 0.8 µM, D 1.5 µM and E 2.0 µM) in Tris buffer (0.01 M Tris, 0.1 M NaCl, pH 8.4) at 5°C. The total volume was 100 µl. Excitation was at 350 nm, slit width 5 nm. The inset shows the fluorescence intensity at 480 nm of 5′-bis-pyrene probe (1.7 µM) versus the concentration of the 16-mer target.