J Phys Chem B. 2016 Dec 8;120(48):12232-12248.

Photophysical Characterization of Enhanced 6-Methylisoxanthopterin Fluorescence in Duplex DNA 

Andrew Moreno†, J. L. Knee†, and Ishita Mukerji‡
†Departments of Chemistry and ‡Molecular Biology and Biochemistry, Molecular Biophysics Program, Wesleyan University, 52 Lawn Ave, Middletown, Connecticut 06459, United States.



Protein-DNA interactions govern many genetic regulatory mechanisms in the cell where the regulation results from the protein recognizing and binding to a specific section of DNA. In the recognition step, a key component of these interactions is the dynamics of the DNA duplexes encoded in the motions of the bases and backbone, which can be monitored with fluorescent base analogs (FBA). Incorporation of these analogs into duplex DNA is often accompanied by a reduction in quantum yield, a limiting factor for their potential application. In contrast, the fluorescent guanine analogue, 6-methylisoxanthopterin 6-MI, exhibits a 3- to 4-fold enhancement in fluorescence in the duplex form when incorporated into the following DNA sequences: ATFAA, AAFTA, or ATFTA (where F represents 6-MI). This enhancement allows the detection of protein-DNA complexes at picomolar concentrations. To identify the main factors underlying the 6-MI fluorescence increase upon duplex formation, we examined the effect of local sequence context and structural perturbations on 6-MI photophysics. We found that the duplex-enhanced fluorescence (DEF) depends on the composition of bases adjacent to 6-MI and the presence of adenines at locations n ± 2 from the probe. We have developed a model for DEF in which adenine residues located at n ± 2 from 6-MI constrains the probe in a geometry that prevents local motion, reduces dynamic quenching and produces an increase in 6-MI fluorescence. By examining a number of duplex sequences, we have developed some rules for designing sequences likely to exhibit DEF. Our results suggest that sequences with pyrimidine neighbors or purine/pyrimidine neighbors such as TFT or AFT coupled with the introduction of adenine residues two residues away from the probe should produce duplexes with moderate to high 6-MI enhanced fluorescence. These findings greatly increase the versatility and utility of the 6-MI base analog as a probe of protein-DNA interactions or to detect changes in DNA and RNA structure, as in nucleic acid aptamer sequences.

PMID: 27934220

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The structure and dynamic motions of bases in DNA underlie molecular interactions between proteins and DNA. Intrinsic fluorescent probes provide several advantages to the study of DNA conformational dynamics as the structural similarity between internal probes and native nucleosides enable detection of subtle features, such as local base perturbations and subtle changes in DNA structure. Although intrinsic fluorescent base analogs (FBA) are valuable for interrogating DNA conformation and function, they often exhibit low fluorescent quantum yields upon incorporation into DNA with similar properties in single and double-stranded DNA. While working with the fluorescent guanine analogue, 6-methylisoxanthopterin 6-MI, we identified a subset of sequences: ATFAA, AAFTA, or ATFTA that exhibit an unexpected 3- to 4-fold increase in relative quantum yield upon duplex formation.

This work investigates how sequence context and local DNA structure influence 6-MI photophysics. There are a few major benefits from this information. First, future development of new highly fluorescent nucleoside analogs will rely on this information for guided synthesis. Second, knowledge of the sequences and structure that lead to enhanced fluorescence extends the use of 6-MI to new and novel applications. Third, correct interpretation of protein-induced changes to FBA photophysics requires a detailed understanding of the relationship between conformational dynamics and fluorescence. By coupling photophysical techniques with molecular dynamic simulations we identified distinct conformations of 6-MI corresponding to “dark” and “bright” states.

We evaluated 21 different sequences to examine how subtle changes in the sequence context surrounding 6-MI influence this enhanced fluorescence. We determined that the duplex-enhanced fluorescence (DEF) depends on the composition of bases adjacent to 6-MI, where pyrimidine or pyrimidine/purine neighbors are more likely to lead to enhanced fluorescence. The presence of adenines at locations n ± 2 from the probe reduces dynamic quenching and further increases the DEF. The excited state decay of 6-MI in the DEF reveals a sharp reduction in dynamic quenching compared to sequences with low quantum yield, which could be due to an extrahelical conformation. However, longer rotational correlation times for 6-MI and reduced solvent accessibility instead indicates a constrained geometry within the duplex. This was a surprising result; as FBA quenching is generally thought to arise from stable Ï€-Ï€ stacking interactions between the FBA and adjacent bases. To further investigate the relationship between FBA rigidity and fluorescence we introduced local DNA structural deformations.

To monitor the effect of dynamics on the fluorescence in both enhanced and quenched sequences we systematically altered the local DNA structure using either an unpaired base or base-bulge near to the probe or a mismatch opposite to the 6-MI. These modified duplexes allowed us to probe how different degrees of conformational flexibility perturbs the fluorescence. Consistent with our findings, greater 6-MI flexibility results in a decrease in local duplex stability and an increase in solvent accessibility, leading to a reduction in fluorescence. Interestingly, increasing 6-MI flexibility in both DEF and the quenched state results in a reduction in hypochromicity, an indication of base stacking, but not increasing fluorescence. This result indicates 6-MI enhanced fluorescence does not arise from a reduction in ground state base stacking. We observed that reducing the steric 6-MI constraints in the DEF results in increased dynamic quenching of the excited state. We had hypothesized the bulge or mismatch in sequences with low quantum yield reduce 6-MI/base interactions. However, even in these sequences, greater conformational flexibility correlates with increasing dynamic quenching of 6-MI. Taken together the results indicate that multiple structural components influence 6-MI fluorescence.

To better understand the factors leading to this fluorescence increase, we used molecular dynamic simulations to examine in greater detail the structural differences between DEF and quenched sequences and identify key structural components influencing 6-MI quantum yield. The simulations revealed that under twisting of the X-F step (F = 6-MI) relative to B-DNA was a common feature of the quenched sequences. In fact, changing the identity of the base at the n ± 2 position from adenine to cytosine results in a similar degree of under twisting at the X-F step as in the quenched sequences. Significantly, we find that introduction of a mismatch in the DEF sequence leads to untwisting of the X-F step.

In summary, evidence from photophysical experiments and structural details from molecular dynamic simulations support a model in which the DEF arises from a constrained geometry of 6-MI in the duplex. In these duplexes, 6-MI remains H-bonded to cytosine, stacked with adjacent bases and inaccessible to quenchers. Also, when the base identity at the n ± 2 position is an adenine there a greater degree of twist and, consequently, an increase in the overall fluorescence. These results point to a model where adenine residues located at n ± 2 from 6-MI induce a structural geometry with greater twist in the duplex that hinders local motion reducing dynamic quenching and producing an increase in 6-MI fluorescence. Thus, placement of adenines two residues away from the 6-MI and neighboring pyrimidine or pyrimidine/purine bases is likely to lead to sequences that exhibit enhanced fluorescence upon duplex formation. This new insight into the design of sequences that can lead to 6-MI enhanced fluorescence broadens the potential application of 6-MI in the study of protein-DNA interactions and nucleic acid structure. This work is crucial for the rational design of 6-MI containing sequences exhibiting DEF and the design of other analogs with DEF, and for understanding the relationship between protein-induced changes in FBA fluorescence and DNA conformational dynamics.



Figure 1: Schematic of the 6-MI Duplex-Enhanced Fluorescence observed in the AAFTA, ATFAA and ATFTA sequences. A 3- to 4-fold enhancement in fluorescence is observed upon duplex formation.




Figure 2: Relative quantum yield of single-stranded DNA, double-stranded DNA and 6-MI monomer. In the sequences exhibiting duplex-enhanced fluorescence, the relative quantum yield in the duplex is significantly higher than the single strand and is close to that of the monomer.