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Intrinsic DNA fluorescence

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The term intrinsic DNA fluorescence refers to the fluorescence emitted directly by DNA when it absorbs ultraviolet (UV) radiation. It contrasts to that stemming from labels that are attached to DNA strands, widely used in biological applications. The intrinsic DNA fluorescence was discovered in the 1960s by studying nucleic acids in frozen media.[1] Since the beginning of the 21st century, the much weaker emission of nucleic acids in fluid solutions is being studied in room temperature by means sophisticated spectroscopic techniques using as UV source femtosecond laser pulses and following the evolution of the emitted light from femtoseconds to nanoseconds. [2] [3][4][5] [6] Such studies contribute to understanding the very first steps of a complex series of events triggered by UV radiation, ultimately leading to DNA damage. Moreover, the knowledge of the fundamental processes underlying the DNA fluorescence paves the way for the development of label-free biosensors.

Conditions for measuring the intrinsic DNA fluorescence

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Due to the weak intensity of the intrinsic DNA fluorescence, specific cautions are necessary in order to perform correct measurements and obtain reliable results.[7] A first requirement concerns the purity of both the DNA samples and that of the chemicals and the water used to the preparation of the buffered solutions. A second requirement is associated with the DNA damage provoked by the exciting UV light. For example, a particular class of UV-induced reaction products, the pyrimidine (6–4) pyrimidone photoadducts, are much stronger emitters than the undamaged DNA.[8] Therefore, their generation during the experiment may alter the emission spectra. In order to overcome these difficulties, continuous stirring of the solution is needed. For measurements using laser excitation, the circulation of the DNA solution by means of a peristaltic pump is recommended.

Spectral shapes and quantum yields

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The fluorescence of all DNA systems in neutral aqueous solution peaks in the near ultraviolet (300-400 nm) when excited around 260 nm. In addition, a long tail, extending all over the visible domain is present in the fluorescence spectrum. The associated quantum yields Φ, that is the number of emitted photons over the number of absorbed photons, are typically in the range of 10-4-10-3. The highest values are encountered for G-quadruplexes.[9] A limited number of measurements were also performed upon UVA excitation (330 nm), where DNA single and double strands, but not their monomeric units, absorb weakly.[10] The UVA-induced fluorescence peaks at longer wavelengths (415-430 nm) and the corresponding Φ values are at least one order of magnitude higher compared to those determined with excitation around 260 nm.[11] Time-resolved studies, combined to theoretical calculations,[12],[13] showed that the fluorescence spectrum of DNA multimers (containing more than one nucleobase) is the envelope of multiple components, arising from the electronic coupling between the close-lying nucleobases.[14] Their relative importance depends on a series of factors, such as the base sequence, the secondary structure, the viscosity of the solution or, in the case of G-Quadruplexes, the metal ions in their central cavity.[15]

Applications

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The utilization of the intrinsic fluorescence of nucleic acids for sensing purposes started to be scrutinized just in 2019. The possibility of detecting target DNA[16] or Pb2+ ions,[17] the screening of a large number of sequences[18] or the authentication of COVID-19 vaccines[19] have been explored. Moreover, the possibility of detecting the DNA damage by probing its fluorescence at short wavelengths (300 nm) has been discussed.[20] Due to their modulable structure, G-quadruplexes, are particularly promising for the development of label-free and dye-free biosensors.[21]

References

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  1. ^ Eisinger, J (1966). "EXCIMER FLUORESCENCE OF DINUCLEOTIDES POLYNUCLEOTIDES AND DNA". Proceedings of the National Academy of Sciences USA. 55 (5): 1015. doi:10.1073/pnas.55.5.1015.
  2. ^ Peon, J. (2001). "DNA/RNA nucleotides and nucleosides: direct measurement of excited-state lifetimes by femtosecond fluorescence up-conversion". Chem. Phys. Lett. 348 (3–4): 255. doi:10.1016/S0009-2614(01)01128-9.
  3. ^ Kwok, W-M. "Femtosecond time- and wavelength-resolved fluorescence and absorption study of the excited states of adenosine and an adenine oligomer". J. Am. Chem. Soc. 128: 11894. doi:10.1021/ja0622002.
  4. ^ Schwalb, N.K. (2008). "Base sequence and higher-order structure induce the complex excited-state dynamics in DNA". Science. 322. doi:10.1126/science.1161651.
  5. ^ Vaya, I. (2010). "Fluorescence of natural DNA: from the femtosecond to the nanosecond time-scales". J. Am. Chem. Soc. 132. doi:10.1021/ja102800r.
  6. ^ Gustavsson, T. (2023). "The Ubiquity of High-Energy Nanosecond Fluorescence in DNA Duplexes". J. Phys. Chem. Lett. 14: 2141. doi:10.1021/acs.jpclett.2c03884.
  7. ^ Markovitsi, D. "UVB/UVC induced processes in model DNA helices studied by time-resolved spectroscopy: pitfalls and tricks". J. Photochem. Photobiol. A-Chem. 183: 1.
  8. ^ Blais, J. "Fluorescence quantum yield determination of pyrymidine (6-4) pyrimidone photoadducts". Photochem. Photobiol. 59 (402). doi:10.1111/j.1751-1097.1994.tb05055.x.
  9. ^ Gustavsson; T. "Fundamentals of the Intrinsic DNA Fluorescence". Acc. Chem. Res. 54: 1226. doi:10.1021/acs.accounts.0c00603.
  10. ^ Mouret, S. "UVA-induced cyclobutane pyrimidine dimers in DNA: a direct photochemical mechanism?". Org. Biomol. Chem. 8: 1706. doi:10.1039/b924712b.
  11. ^ Banyasz, A. "Base-pairing enhances fluorescence and favors cyclobutane dimer formation induced upon absorption of UVA radiation by DNA". J. Am. Chem. Soc. 133: 5163. doi:10.1021/ja110879m.
  12. ^ Spata, V.A. (2016). "Excimers and Exciplexes in Photoinitiated Processes of Oligonucleotides". J. Chem. Phys. Lett. 6: 976. doi:10.1021/acs.jpclett.5b02756.
  13. ^ Martinez-Fernandez, L. (2022). "Nucleic Acids as a Playground for the Computational Study of the Photophysics and Photochemistry of Multichromophore Assemblies". Acc. Chem. Res. 55: 2077. doi:10.1021/acs.accounts.2c00256.
  14. ^ Gustavsson; T. "Fundamentals of the Intrinsic DNA Fluorescence". Acc. Chem. Res. 54: 1226. doi:10.1021/acs.accounts.0c00603.
  15. ^ Dao, N.T. "Following G-quadruplex formation by its intrinsic fluorescence". Febs Letters. 585: 3969. doi:10.1016/j.febslet.2011.11.004.
  16. ^ Xiang, X. "Label-free and dye-free detection of target DNA based on intrinsic fluorescence of the (3+1) interlocked bimolecular G-quadruplexes". Sens. Actuators B Chem. (290): 68. doi:10.1016/j.snb.2019.03.111.
  17. ^ Lopez, A. "Probing metal-dependent G-quadruplexes using the intrinsic fluorescence of DNA". Chem. Comm. 58: 10225. doi:10.1039/d2cc03967b.
  18. ^ Zuffo, M. "Harnessing intrinsic fluorescence for typing of secondary structures of DNA". Nucl. Ac. Res. 48: e61. doi:10.1093/nar/gkaa257.
  19. ^ Assi, S. "Authentication of Covid-19 Vaccines Using Synchronous Fluorescence Spectroscopy". J. Fluoresc. 33: 1165. doi:10.1007/s10895-022-03136-5.
  20. ^ Markovitsi, D. (2024). "10.1021/acsomega.4c02256". ACS Omega. doi:10.1021/acsomega.4c02256.
  21. ^ Markovitsi, D. (2024). "Processes triggered in guanine quadruplexes by direct absorption of UV radiation: From fundamental studies toward optoelectronic biosensors". Photochem. Photobiol. 100: 262. doi:10.1111/php.13826.
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