Application of nucleobase-induced fluorescence enhancement in Cy3–DNA conjugates to sequence identification errors and mismatch quantification

Abstract

In Cy3–DNA conjugates, cyanine dye–nucleobase interactions can alter Cy3 fluorescence intensity, potentially causing significant errors in fluorescence-based bioanalytical assays. We quantify the error due to Cy3 fluorescence intensity enhancement with reference to free Cy3, when bonded to hybridized and single stranded DNA oligonucleotides, with the help of a normalised parameter Δ_H. Homo-nucleotide probes with adenine repeats having purine bases adjacent to Cy3 showed high accuracy in hybridization (p-value ≤ 0.0005) and mismatch (p-value ≤ 0.0001) detection. The Δ_H was 100% for Cy3–adenine (A), 70% for Cy3–thymine (T), 80% for Cy3–guanine (G) and 60% for Cy3–cytosine (C) at 488 nm excitation. Compared to free Cy3 having single exponential decay, Cy3–DNA conjugation resulted in slower, excitation-dependent double exponential decay of Cy3 fluorescence, indicating non-radiative kinetic steps. The fluorescence lifetime of Cy3 in single stranded and hybridized Cy3–A increased by an excitation-dependent factor of ∼3, in comparison with the single stranded and hybridized counterparts of the other probes. The sensitivity to detect single nucleotide mismatches, quantified by the accuracy parameter α, was highest for Cy3–A and Cy3–G probes, with maximum α = 0.66 (66%) at 488 nm. In GC-rich hetero-nucleotide sequences, an adjacent purine in the complementary strand yielded maximum fluorescence enhancement, with a systematic decrease in fluorescence intensity upon shifting the position of G from Cy3, illustrating the single base distance sensitivity of fluorescence enhancement.