ME Hawkins and FM Balis. Use of pteridine nucleoside analogs as hybridization probes (2004) NAR 32 e62
ME Hawkins. Fluorescent Pteridine Nucleoside Analogs: A Window on DNA Interactions (2001) CBB 34 257 (review)
ME Hawkins et al. Synthesis and fluorescence characterization of pteridine adenosine nucleoside analogs for DNA incorporation (2001) Anal. Biochem 298 231
EL Rachofsky et al. Emission Kinetics (1998) SPIE 3256 68
ME Hawkins et al. Fluorescence Properties of Pteridine Analogs as Monomers and Incorporated into Oligonucleotides (1997) Analytical Biochem 244 86
SL Driscoll et al. Fluorescence Properties of a Guanosine Analog Incorporated into Small Oligonucleotides (1997) Biophys J 73 3277
ME Hawkins et al. Incorporation of a Fluorescent Guanosine Analog into Oligonucleotides and Its Application to a Real Time Assay for the HIV-1 Integrase 3'-processing Reaction. (1995) NAR 23 2872
FOR RESEARCH USE ONLY
The highly fluorescent pteridine-based nucleoside analog probes share some unique structural features causing them to behave quite differently from standard linker arm-attached fluorophores. The properties defined below render them exquisitely sensitive to changes in their immediate environment. As a result of this sensitivity these probes can be used in a variety of applications for the study of DNA interactions.
A major feature of these probes is that they are designed as structural analogs to guanosine or adenosine and they exhibit very high fluorescence quantum yields. Like native nucleosides, they attach to the oligonucleotide through a deoxyribose linkage and, since they are produced as phosphoramidites, they can be site-specifically incorporated into DNA using automated DNA synthesis.
The deoxyribose attachment positions the probe within the oligonucleotide and dramatically increases fluorescence sensitivity to effects from adjacent bases as compared to linker-attached fluorophores.
Changes in base stacking or base pairing in the vicinity of the probe are directly reflected through measurable changes in fluorescence. In addition to increased sensitivity, a major advantage of this intimate association with the DNA is that changes in fluorescence can be observed in real time without the need for separation of products. Figure 1 displays the extremes in fluorescence intensity between equimolar amounts of the monomer form of the probe, 6MAP and the same probe contained within an oligonucleotide. The rate of P1 nuclease cleavage of 6MAP from the oligonucleotide (Figure 2) demonstrates how this reaction can be observed in real time. Relative quantum yields of the probes within oligonucleotides are dependent on the immediate nucleoside environment and range from below quantifiable limits to 0.28. The quantum yields of the monomer forms range from 0.39 to 0.88 so the potential for increase in signal upon enzymatic (or chemical) removal of a probe from an oligonucleotide single or double strand is substantial.
Most of the quenching (loss of fluorescence signal) of the nucleoside analogs is associated with incorporation into a single stranded oligonucleotide. Since this phenomenon is apparently associated with base stacking interactions, we investigated the fluorescence of these probes after deliberate disruption of base stacking through the formation of a bulge or hairpin. In this application the probe is positioned as an insertion in a sequence that is complementary to a sequence of interest. Since the fluorophore does not have a base-pairing partner in the target sequence, when annealing occurs, it is squeezed out of the strand to form a single base hairpin and this results in a strong increase in fluorescence intensity. The fluorescence change associated with annealing of the two strands is dependent on the identity of bases neighboring the probe and produces an increase of up to 27 times the background signal (equimolar single strand containing the probe). The associated depression of melting temperature for the resulting hairpin or bulge in a 21-mer is 1 deg C, an indication that there is little perturbation in annealing caused by the probe. Figure 3 shows the type of increase in intensity observed in this hairpin hybridization application. This approach allows one to probe for a specific sequence within a mixture without separation of products.
These probes can be incorporated into oligonucleotides site-specifically and since they are more heavily quenched by neighboring purines, and much less quenched by neighboring pyrimidines, they can be selectively placed within an oligonucleotide to get a brighter or quieter response. Oligonucleotides containing the probe in a terminal position or containing multiple probes per strand are extremely bright.
Although we have focused primarily on fluorescence intensity changes possible with these probes, changes in fluorescence decay and anisotropy can also be monitored to explore structure and kinetics of oligonucleotides. Since the probes have an absorption maximum near 350 nm, which overlaps the emission maximum of tryptophan, they could also function as a donor in an energy transfer between probe-containing DNA and tryptophan. This feature could be exploited to study interactions between DNA and tryptophan-containing DNA binding proteins.
The attractive features of these fluorophores are that they:
Covered by U.S. Patents 5,525,711, 5,612,468, 6,716,971. Patents pending. Licensed from the National Institutes of Health.