Theoretical Studies Of The Electronic Spectra Of Some Newly Designed Fluorescent Base Analogues In DNA

Fluorescence spectroscopic techniques are extremely powerful tools for wide applications in biochemistry and biology due to their fascinating properties, including high speed, simplicity and convenience, exquisite sensitivity and selectivity. As we all know, natural nucleobases display extremely low fluorescent quantum yields and ultra-short excited-state lifetimes in both solution and gas phase. Thus, in order to more easily probe DNA/RNA strand conformational dynamics and the interactions between DNA/RNA and other molecules with spectroscopic techniques, the creation of fluorescent DNA base analogues is very important. In this work, the electronic spectroscopic properties of several kinds of newly designed fluorescent base analogues were investigated theoretically. Also considered were the effects of the Micro-Biologic environments, including the water solvent, linking to deoxyribose, base pairing, and base stacking on their photophysical characters. Understanding of the excited-state properties of these analogues and effects of perturbations induced by the biologic environment is helpful in finding ways for their direct usefulness of their fluorescence. In addition, the theoretical predictions are helpful in understanding the photophyscical properties of the newly designed DNA oligonucleotides and explaining experimental data in the future. Furthermore, it may help in the design and synthesize of new fluorescent base analogues. Some interesting phenomena and characters have been observed through the investigations, which can be described as follows:1. Firstly, the electronic spectroscopic properties of y-bases were investigated. Also examined were the effects of linking to deoxyribose and hydrogen bonding to their natural complementary bases. For the isolated bases, the calculated excitation and emission energies are in good agreement with the measured data where experimental results are available. The lowest singlet excited state of these bases is ofππ* character, which is mainly dominated by the configuration HOMO (H)→LUMO (L). The S1 geometries of yA, yT, and yT-m are found to be planar, while those for yG, yG-t2, and yC are nonplanar. In general, binding with deoxyriboses red-shifts the absorbance maxima and the fluorescence emission of the y-bases. Furthermore, the linking with deoxyriboses increases the fluorescence intensity of yA, but decreases those of yT and yC. The ground-state geometries of the WC base pairs (yAT, yTA, yGC, and yCG) are found to be planar, and the calculated interaction energies are very close to those of natural base pairs, indicating that the y-bases can pair with their natural complementary partners to generate stable base pairs. The S1 geometries of yAT and yTA base pairs are found to be quasi-planar, while those for yCG and yGC show propeller-twisted nonplanar structures resulting from the pyramidalization of the amino groups that belong to the y-base moieties. In general, base pairing does not have a significant effect on the fluorescence emissions of yA, yC, and yT, but blue shifts the fluorescence emission of yG by 22 nm.2. Based on the investigations of the y-bases, we extend our work to the yy-bases. Also examined were the effects of methanol solution and hydrogen bonding to their natural complementary bases, and the results were compared with those of y-bases. The nature of the lowest ten singlet transitions of the yy-bases were revealed in both gas phase and methanol solution. The lowest singlet excited state of these bases is ofππ* character, which is mainly dominated by the configuration H→L. The S1 geometries of yyC, yyG, and yyA are found to be nonplanar, while that for yyT is planar. The geometries of the lowest nn* states of yyC and yyT are highly nonplanar, involving the elongation of the associated carbonyl group by 0.07 and 0.075 A, respectively. The calculated excitation and emission maxima of yyC and yyT agree well with the measured data. In methanol solution, the fluorescence from yyA and yyG would be expected to occur around 539 and 562 nm, respectively, suggesting that yyA is a green-colored fluorophore, while yyG is a yellow-colored fluorophore. It was found that the methanol solution can red-shift both the absorption and emission maxima of yyA, yyT, and yyC, but blue-shift those for yyG. There is consensus that the intensity of the strongest absorption peak is greatly increased after solvation. Also it was found that the strategy of using the PCM model combined with the gas phase ground-state structures can well reproduce the absorption spectra of yy-bases in methanol solution. The ground-state geometries of the WC base pairs (yAT, yTA, yGC, and yCG) are found to be planar, and their lowest transitions are essentially identical to the first transition of free yy-base, and therefore can be classified as local excitations of the yy-bases. The S1 geometries of yyAT and yyTA base pairs are found to be quasi-planar, while those for yCG and yGC show propeller-twisted nonplanar structures resulting from the pyramidalization of the amino groups that belong to the yy-base moieties. Generally, though the base pairing has no significant effects on the absorption and fluorescence maxima of yyA, yyC, and yyT, it blue-shifts those for yyG. In addition, it was found that the methanol solution has a similar effect on the absorption spectra of the y-bases compared with those for yy-bases, that is to say, the methanol solution can red-shift the absorption of yA, yT, and yC, but blue-shift that for yG. Compared with y-bases, both the absorption and emission maxima of yy-bases are red-shifted. Furthermore, the intensity of the excitation maxima corresponding to yy-bases are lower compared with those for y-bases, while the intensity of the strongest absorption bands are much higher than those of y-bases.3. Thirdly, in order to understand the electronic spectroscopic properties of the guanine (G) analogues designed by our group,4 analogues named A1-A4 are explored using ab initio calculations, and the results were compared with those of natural G and xG. The pairing effect with their complementary base, cytosine, on both the absorption and emission processes were examined as well. For the isolated bases, the onset absorption peaks of these newly designed analogues are red-shifted compared with that of natural guanine, while the fluorescence wavelengths are blue-shifted. The calculated excitation energies are in good qualitative agreement with measured data where experimental results are available. In general, the S1 singlet excited states are nonplanar for these newly designed base analogues, and the Stokes-Shifts are much smaller than that for guanine, suggesting they have stronger rigidity of molecule than guanine. Therefore, the fluorescence quantum yields of these analogues are expected to be higher than that of natural guanine. When the effect of the pairing with C is taken into account, these bases can be divided into two groups. The first group includes G, Al, and A2 (one-bond-intercalated at the C5 site), and their parings with C could reduce the oscillator strengths of both the firstππ* transitions by 27%-60% and the fluorescence emissions by 19%-23%. In addition, the energy gaps between the first local excitedππ* state and the charge transfer state (from the H of purine to the L of cytosine) are close to each other for their corresponding base pairs. The other three bases (A3, A4, and xG) considered here make up the second group, which are two-bonds-intercalated at the C5 sites. The pairing with C increases the oscillator strengths of both the firstππ* transitions by 11%-15% and the fluorescence emissions by 3%-20%, and the corresponding energy gaps are much larger than that of the GC pair. In general, if the pairing with C does not affect the nonradiative process, it can reduce the fluorescence quantum yield for G, A1, and A2, but enhance it for A3, A4, and xG.4. Finally, the excited-state properties of the size-expanded cytosine analogue xC were investigated. The cytosine analogue xC by benzo-homologation is fluorescent with a fluorescent quantum yield of 0.52. Most importantly, it was demonstrated that a DNA polymerase is able to read the chemical information stored in xC and that the full replication machinery of E. coli is able to recognize the sequence encoded by xC correctly and efficiently, suggesting that it is a promising candidate for direct usefulness. Clearly, an in-depth understanding of the excited-state properties and the effects of perturbations induced by the biologic environments, the water solvent, linking to deoxyribose, base pairing, and base stacking is very important and helpful in finding ways for its direct usefulness of its fluorescence. Therefore, a detailed and systemic computational study on the properties of xC with the aim of gaining more insight into the properties of the excited states by investigating the dependence of absorption and emission spectra on the biologic environments mentioned above. For isolated xC, the nature of the low-lying singlet transitions are discussed and the calculated electronic absorption peaks and emission maximum are in good agreement with reported experimental values. The experimental observed absorption band for dxC below 250 nm in methanol solution must be related to the calculated 223 nm in the gas phase. Water hydration and hydrogen bonding to G are demonstrated to have hyperchromic effect on the excitation maximum of xC. It was found that hydration blue-shifts the excitation maximum and fluorescence by 12 and 8 nm, respectively. Overall, linking to deoxyribose has a hyperchromic effect in the low energy region and a hypochromic effect in the high energy region on the absorption of xC. Similarly, base paring with G blue shifts the fluorescence of xC by 0.09 eV (9 nm), and hydration of xCG blue shift the fluorescence of xC further (by 0.19 eV,17 nm). Furthermore, the fluorescent quantum yield would be increased after hydration, linking to deoxyribose, and base paring with G. In the case of the stacked configurations, a significant decrease of the oscillator strength as well as a red shift of the dipole-allowed transition with respec(?)to free xC is observed in all cases. Because of the strong mixing of the molecular orbitals, the fluorescence quantum yield of xC are expected to be lowered in the stacked complexes due to a static quenching mechanism.