Abstract
CYP3A4*1B is a single nucleotide polymorphism of CYP3A4 and is associated with prostate cancer which exhibits higher nifedipine oxidase activity in liver. This research provides details of the effects of structural variation and medium effects for the recently reported split-oligonucleotide (tandem) probe system for excimers-based fluorescence detection of DNA. In this approach the detection system is split at a molecular level into signal-silent components, which must be assembled correctly into a specific 3-dimensional structure to ensure close proximity of the excimer partners and the consequent excimer fluorescence emission on excitation. The model system consists of two 11-mer oligonucleotides, complementary to adjacent sites of a 22-mer DNA target. Each oligonucleotide probeis equipped with functions able to form an excimer on correct, contiguous hybridization. The extremely rigorous structural demands for excimer formation and emission required careful structural design of partners for excimer formation, which are here described. This study demonstrates that the excimer formed emitted at ~480 nm with alarge Stokes shift (~130 - 140 nm).
Highlights
Reversible hybridisation of complementary polynucleotides is essential to the biological processes of replication, transcription, and translation
Fluorescence studies were made for solutions of ExciProbe (X1) and ExciProbe (X2) oligonucleotides with both probes complementary to each other (Figure 1)
Addition of the complementary target resulted in immediate quenching of the local excited state (LES) emission at 395 nm to less than onethird of its original value and the appearance of a new, broad emission band characteristic of pyrene excimer fluorescence after the full terminally located system had formed)[1, 2, 26]
Summary
Reversible hybridisation of complementary polynucleotides is essential to the biological processes of replication, transcription, and translation. Physical studies of nucleic acid hybridisation are required for understanding these biological processes on a molecular level. Fluorescence measurements present an improved sensitive measure of nucleic acid concentration compared to conventional solution-phase detection techniques. The sensitivity of fluorophores to their environments offers a means by which to differentiate hybridised from unhybridised nucleic acids without resorting to separation techniques. This was first demonstrated by attaching different fluorescent labels to the termini of oligonucleotides, which hybridise to adjacent regions on a complementary strand of DNA. Excimer emission from oligonucleotides containing 5-(1-pyrenylethynyl)uracil [6], trans-stilbene [7], and perylene [8] have been reported
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