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Nucleic acid detection in the near-infrared wavelength range

Akimitsu Okamoto 1
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INTRODUCTION
The use of near-infrared (NIR) photons is a promising approach for biomedical imaging in living tissue. The unique character of NIR dyes is directly reflected in improved image quality, and NIR fluorescence imaging has emerged as a powerful tool for cell and tissue imaging. It is important for the establishment of effective nucleic acid imaging in cells to design an NIR fluorescent probe that has the feature that the fluorescence is turned off when the probe does not recognize the target nucleic acid and turned on when the probe has hybridized the target nucleic acid. We focused on the system used in an exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probe. The fluorescence of the probe, in which two fluorescent dyes are attached to a thymine base, is well controlled by excitonic interaction. However, there is no ECHO probe in which the fluorescence is controlled in the range of NIR wavelengths. For this context, NIR nucleotides should be designed without loss of a high on–off performance as a hybridization-sensitive fluorescent probe based on excitonic interaction chemistry.

EXPERIMENTAL
A solution of the NIR probe in water (50 microM, 2 microL) was introduced into HeLa cells, using 2 microL of Lipofectamine 2000 (Invitrogen). After incubation for 1 h, the cells were washed with PBS and observed in phenol red-free DMEM. Images were acquired with a motorized inverted microscope equipped with a 20x objective.

RESULTS AND DISCUSSION
NIR fluorescent nucleotides, D644, D662, and D715, were synthesized and incorporated into oligonucleotides by DNA autosynthesizer to obtain NIR-ECHO probes. The fluorescence emission of Probe1(Dnnn) 5'-d(CGCAATDnnnTAACGC)-3' was suppressed in their unhybridized states, but emission appeared immediately after the probe was added to a solution of the complementary nucleic acid. The absorption band of the unhybridized probe appears at about 50, 58, and 70 nm shorter wavelengths for Probe1(D644), Probe1(D662), and Probe1(D715), respectively, than that of the hybrid. This blue shift suggested a splitting of the excited state because of the formation of a bichromophoric H-aggregate. The interdye excitonic interaction in a bichromophoric H-aggregate in the unhybridized state of the probes suppresses the fluorescence emission of the probes. This exciton-controlled fluorescence behavior was also observed for the probes with a polythymidine sequence, Probe2(Dnnn) 5'-d(TTTTTTDnnnTTTTTT)-3'.
Having established that the NIR-ECHO probes are effective for nucleic acid detection, the fluorescence behavior of the probe was investigated in living cells. The NIR-ECHO probe capable of binding to the polyA tail of mRNA, Probe2(D715), was transfected to HeLa cells using a transfection reagent, Lipofectamine 2000. The NIR fluorescence appeared clearly in transfected cells. The distribution of NIR fluorescence of Probe2(D715) in cells was very similar to that of Probe2(D514) bound to mRNA in cells.
NIR-ECHO probes are the first NIR nucleic acid probes possessing the function that the absorption and emission were controlled at various NIR wavelengths, and this function facilitates imaging of intracellular mRNA.