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, 8 (1), 16874

A 5'-BODIPY End-label for Monitoring DNA Duplex-Quadruplex Exchange

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A 5'-BODIPY End-label for Monitoring DNA Duplex-Quadruplex Exchange

Prashant S Deore et al. Sci Rep.

Abstract

Fluorescent probes that can distinguish different DNA topologies through changes in optical readout are sought after for DNA-based diagnostics. In this work, the 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene (BODIPY) chromophore attached to cyanophenyl substituents (BODIPY-CN) has been tethered to the 5'-end of the 15-mer thrombin binding aptamer (TBA) that contains the guanine (G) nucleobase. TBA folds into a unimolecular antiparallel G-quadruplex (GQ) upon binding thrombin and certain metal ions. The 5'-BODIPY-CN-TBA sample possesses a Stokes shift of ~40 nm with wavelengths of excitation/emission at 550/590 nm and exhibits a 2-fold increase in emission intensity compared to the free BODIPY-CN in aqueous buffer that possesses a brightness (εΦfl) of ~16,956 M-1. cm-1. However, when 5'-BODIPY-CN-TBA is base-paired to a complementary strand in the B-form duplex, the emission of the BODIPY-CN end-label increases 7-fold, 14-fold compared to the free-dye. This signal-on response enables the BODIPY-CN end-label to serve as a quencher-free fluorescent probe for monitoring duplex-GQ exchange. The visible end-label minimally perturbs GQ stability and thrombin binding affinity, and the modified TBA can act as a combinatorial logic circuit having INHIBIT logic functions. These attributes make BODIPY-CN a highly useful end-label for creating nanomolecular devices derived from G-rich oligonucleotides.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Synthesis of BODIPY-CN alcohol 4 and its phosphoramidite 8.
Figure 2
Figure 2
Perspective drawing of the BODIPY-CN alcohol 4 in the crystal with the three phenyl rings attached to the BODIPY core labeled A–C in the schematic.
Figure 3
Figure 3
Photophysical properties of the BODIPY-CN alcohol 4: (a) fluorescence response (λex = 550 nm, λem = 586 nm) of 4 (1 μM) in water with increasing percentages of methanol and glycerol; inset, fluorescence images of 4 in methanol versus water, (b) fluorescence response of 4 (1 μM) as a function of temperature in methanol:glycerol (1:4).
Figure 4
Figure 4
Overlay of excitation and emission spectra of BODIPY-CN alcohol 4 (Free-dye (1.75 μM), dashed blue trace) versus BODIPY-CN-TBA (1.75 μM) in the absence (GQ, dashed red trace) and presence of CS-10 (1.5 equiv., Duplex, solid green trace).
Figure 5
Figure 5
(a) Fluorescence titration of BODIPY-CN-TBA (1.75 μM) with CS-10 at 21 °C; initial trace of BODIPY-CN-TBA depicted by the solid red line, while dashed traces depict duplex formation upon successive addition of CS-10; insert plot of the fluorescence intensity versus [CS-10]. (b) Fluorescence thermal melting analysis of BODIPY-CN-TBA (1.75 μM) in the absence (green traces) and presence (red traces) of 1.5 equiv. CS-10; heating ramps are solid lines, cooling ramps are dashed.
Figure 6
Figure 6
(a) Fluorescence overlay spectra of BODIPY-CN-TBA:CS-10 (1.75 μM) in the absence (solid green trace) and presence of 2 equiv. thrombin (dashed red trace). (b) Fluorescence emission (λex = 550 nm; λem = 590 nm) intensity trace as a function of time for BODIPY-CN-TBA:CS-10 (1.75 μM) in the presence of 2 equiv. thrombin.
Figure 7
Figure 7
(a) Fluorescence emission spectra of BODIPY-CN-TBA at different input conditions, namely GQ, single strand and duplex DNA; (b) the truth table for sequential logic circuit, where ‘0’ = ‘Off’ and ‘1’ = ‘On’ signals; (c) fluorescence emission spectra of BODIPY-CN-TBA at different inputs according to the truth table (b). Fluorescence intensities higher and lower than the threshold value (50) at 590 nm are assigned as ‘1’ and ‘0’ respectively; and (d) INHIBIT logic gate has been constructed based on results obtained in (a,b) and (c).

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References

    1. Seeman NC. From genes to machines: DNA nanomechanical devices. Trends Biochem. Sci. 2005;30:119–125. doi: 10.1016/j.tibs.2005.01.007. - DOI - PMC - PubMed
    1. Liu J, Cao Z, Lu Y. Functional nucleic acid sensors. Chem. Rev. 2009;109:1948–1998. doi: 10.1021/cr030183i. - DOI - PMC - PubMed
    1. Krishnan Y, Simmel FC. Nucleic acid based molecular devices. Angew. Chem. Int. Ed. 2011;50:3124–3156. doi: 10.1002/anie.200907223. - DOI - PubMed
    1. Guo Y, et al. Multiple types of logic gates based on a single G-quadruplex DNA strand. Sci. Rep. 2014;4:7315. doi: 10.1038/srep07315. - DOI - PMC - PubMed
    1. Alberti P, Mergny J-L. DNA duplex-quadruplex exchange as the basis for a nanomolecular machine. Proc. Natl. Acad. Sci. USA. 2003;100:1569–1573. doi: 10.1073/pnas.0335459100. - DOI - PMC - PubMed

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