Intrinsic DNA fluorescence

Due to the weak intensity of the intrinsic DNA fluorescence, specific cautions are necessary in order to perform correct measurements and obtain reliable results. 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. The buffer emission must be systematically recorded and, in certain cases, subtracted in an appropriate way.{{cite journal |last1=Huix-Rotllant |first1=M. |date=2015 |title=Stabilization of mixed Frenkel-charge transfer excitons extended across both strands of guanine-cytosine DNA duplexes |url= |journal=J. Phys. Chem. Lett. |volume=6 |issue= 12|pages=3540–3593 |doi=10.1021/acs.jpclett.5b00813 |pmid=26266599 |access-date=}} A second requirement is associated with the DNA damage provoked by the exciting UV light which alters its fluorescence.{{cite journal |last1=Carroll |first1=G.T.|date=2023 |title=Intrinsic fluorescence of UV-irradiated DNA |url= |journal=J. Photochem. Photobiol. A |volume=437 |issue= |pages=114484 |doi=10.1016/j.jphotochem.2022.114484 |bibcode=2023JPPA..43714484C |access-date=}} 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; the reproducibility of successive fluorescence signal needs to be checked.

File:Nuceoside fluorescence spectra.tif

The fluorescence spectra of the DNA monomeric chromophores (nucleobases, nucleosides or nucleotides) in neutral aqueous solution, obtained with excitation around 260 nm, peak in the near ultraviolet (300-400 nm); and a long tail, extending all over the visible domain is present in their emission spectrum. The spectra of the DNA multimers (composed of more than one nucleobase) are not the sum of the spectra of their monomeric constituents. In some cases, in addition to the main peak located in the UV, a second band{{cite journal |last1=Ge |first1=G. |date=1991 |title=Excited-state properties of the alternating polynucleotide poly(dA-dT)poly(dA-dT) |url= |journal=Photochem. Photobiol. |volume=54 |issue= 2|pages=301–305 |doi=10.1111/j.1751-1097.1991.tb02020.x |pmid=1780364 |access-date=}}{{cite journal |last1=Stuhldreier |first1=M. C. |date=2013 |title=Ultrafast photo-initiated molecular quantum dynamics in the DNA dinucleotide d(ApG) revealed by broadband transient absorption spectroscopy |url= |journal=Faraday Disc. |volume=183 |issue= |pages=173–188 |doi=10.1039/c3fd00003f |pmid=24020202 |bibcode=2013FaDi..163..173S |access-date=}}{{cite journal |last1=Kwok |first1=W.-M. |date= 2006|title=Femtosecond time- and wavelength-resolved fluorescence and absorption study of the excited states of adenosine and an adenine oligomer |url= |journal=J. Am. Chem. Soc. |volume=128 |issue= 36|pages=11894–11905 |doi=10.1021/ja0622002 |pmid=16953630 |access-date=}} is present at longer wavelengths; it is attributed to excimer or exciplex formation.{{cite journal |last1=Improta |first1=R. |date=2011 |title=Interplay between "neutral" and "charge-transfer" excimers rules the excited state decay in adenine-rich polynucleotides |url= |journal=Angew. Chem. Int. Ed. |volume=20 |issue= 50|pages=12016–12019 |doi=10.1002/anie.201104382 |pmid=22012744 |access-date=}}{{cite journal |last1=Spata |first1=V. A |date=2015 |title=Photophysical deactivation pathways in adenine oligonucleotides |url= |journal=Phys. Chem. Chem. Phys. |volume=17 |issue= 46|pages=31073–31083 |doi=10.1039/c5cp04254b |pmid=26536353 |bibcode=2015PCCP...1731073S |access-date=}}

The duplex spectra are affected by their size{{cite journal |last1=Markovitsi |first1=D. |date=2010 |title=Fluorescence of DNA Duplexes: From Model Helices to Natural DNA |url= https://hal.archives-ouvertes.fr/hal-00538375/file/Markovitsi_Duplex_Fluorescence_revised_manuscript_2.pdf|journal=J. Phys. Chem. Lett. |volume=1 |issue= 22|pages=3271–3276 |doi=10.1021/jz101122t |access-date=}} and the viscosity of the solution,{{cite journal |last1=Georghiou |first1=S. |date=1998 |title=Environmental control of deformability of the DNA double helix |url= |journal=Photochem. Photobiol. |volume=67 |issue= 5|pages=526–531 |doi=10.1111/j.1751-1097.1998.tb09088.x |pmid=9613236 |access-date=}} while those of G-Quadruplexes by the metal cations present in their central cavity.{{cite journal |last1=Dao |first1=N.T.|date=2011 |title=Following G-quadruplex formation by its intrinsic fluorescence |url= |journal=FEBS Letters |volume=585 |issue= 24|pages=3969–3977 |doi=10.1016/j.febslet.2011.11.004 |pmid=22079665 |bibcode=2011FEBSL.585.3969D |hdl=10356/98618 |access-date=|hdl-access=free }}{{cite journal |last1=Hua |first1=Y. |date=2012|title=Cation Effect on the Electronic Excited States of Guanine Nanostructures Studied by Time-Resolved Fluorescence Spectroscopy |url= |journal=J. Phys. Chem. C |volume=116 |issue= 27|pages=14682–14689 |doi=10.1021/jp303651e |access-date=}}{{cite journal |last1=Ma |first1=M.S. |date=2019 |title=Real-time Monitoring Excitation Dynamics of Human Telomeric Guanine Quadruplexes: Effect of Folding Topology, Metal Cation, and Confinement by Nanocavity Water Pool |url= |journal=J. Phys. Chem. Lett. |volume=10 |issue= 24|pages=7577–7585 |doi=10.1021/acs.jpclett.9b02932 |pmid=31769690 |access-date=}} Due to the fluorescence dependence on the secondary structure, it is possible to follow the formation{{cite journal |last1=Dao |first1=N.T.|date=2011 |title=Following G-quadruplex formation by its intrinsic fluorescence |url= |journal=FEBS Letters |volume=585 |issue= 24|pages=3969–3977 |doi=10.1016/j.febslet.2011.11.004 |pmid=22079665 |bibcode=2011FEBSL.585.3969D |hdl=10356/98618 |access-date=|hdl-access=free }} and the melting{{cite journal |last1=Markovitsi |first1=D.|date=2004 |title=Cooperative effects in the photophysical properties of self-associated triguanosine diphosphates |url= |journal=Photochem. Photobiol. |volume=79 |issue= 6|pages=526–530 |doi=10.1562/2003-12-12-RA.1|doi-broken-date=28 December 2024 |pmid=15291304 | access-date=}} of G-Quadruplexes by monitoring their emission; and also to detect the occurrence of hairpin loops in these systems.{{cite journal |last1=Kwok |first1=C. K. |date=2013 |title=Effect of Loop Sequence and Loop Length on the Intrinsic Fluorescence of G-Quadruplexes |url= |journal=Biochemistry |volume=52|issue= 18|pages=3019–3021 |doi=10.1021/bi400139e |pmid=23621657 |access-date=}}{{cite journal |last1=Feng |first1= H. |date=2022 |title=Spectroscopic analysis reveals the effect of hairpin loop formation on G-quadruplex structures |url= |journal=RSC Chem. Biol. |volume=3 |issue= 4|pages=431–435 |doi=10.1039/d2cb00045h |pmid= 35441140 |access-date=|pmc=8984947 }}

The fluorescence 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.{{cite journal |last1=Gepshtein |first1=R. |date=2008 |title=Radiationless transitions of G4 wires and dGMP |url= |journal=J. Phys. Chem. C |volume=112 |issue= 32|pages=12249–12258 |doi=10.1021/jp803301r |access-date=}}{{cite journal |last1=Sherlock |first1=M. E. |date=2016|title=Steady-State and Time-Resolved Studies into the Origin of the Intrinsic Fluorescence of G-Quadruplexes |url= |journal=J. Phys. Chem. B |volume=120 |issue= 23|pages=5146–5158 |doi=10.1021/acs.jpcb.6b03790 |pmid=27267433 |access-date=}}{{cite journal |last1=Gustavsson |last2=T. |date=2021 |title=Fundamentals of the Intrinsic DNA Fluorescence |url= https://hal.science/hal-03911926/file/DNA%20fluorescence%20revised%20manuscript_2.pdf|journal=Acc. Chem. Res. |volume=54 |issue= 5|pages=1226–1235 |doi=10.1021/acs.accounts.0c00603 |pmid= 33587613|access-date=}} The DNA nucleoside thymidine (dT) was proposed as a reference for the determination of small fluorescence quantum yields.{{cite journal |last1=Morshedi |first1=M. |date=2024 |title=References for Small Fluorescence Quantum Yields |url= |journal=Journal of Fluorescence |volume= |issue= |pages= |doi=10.1007/s10895-024-03729-2 |pmid=38748338 |access-date=|doi-access=free }}

A limited number of measurements were also performed with UVA excitation (330 nm), where DNA single and double strands, but not their monomeric units, absorb weakly.{{cite journal |last1=Sutherland |first1=J. C. |date=1980 |title=Absorption spectrum of DNA for wavelengths greater than 300 nm |url= |journal=Radiation Res. |volume=86 |issue= 3|pages=399–410 |doi=10.2307/3575456 |jstor=3575456 |pmid=6264537 |access-date=}} The UVA-induced fluorescence peaks between 415 and 430 nm; the corresponding Φ values are at least one order of magnitude higher compared to those determined with excitation around 260 nm.{{cite journal |last1=Banyasz |first1=A. |date=2011 |title=Base-pairing enhances fluorescence and favors cyclobutane dimer formation induced upon absorption of UVA radiation by DNA |url= https://hal.archives-ouvertes.fr/hal-00591797/file/UVA_base_pairing_effect_revised_manuscript.pdf|journal=J. Am. Chem. Soc. |volume=133 |issue= 14|pages=5163–5165 |doi=10.1021/ja110879m |pmid=21417388 |bibcode=2011JAChS.133.5163B |access-date=}}

The fluorescence of some minor, naturally occurring nucleobases, such as 5-methyl cytosine, N7-methylated guanosine or N6-methyladenine, has been studied both in monomeric form and in multimers.{{cite journal |last1=Daniels |first1=M. |date=2007 |title=Intrinsic fluorescence of B and Z forms of poly d(G-m(5)C).poly d(G-m(5)C), a synthetic double-stranded DNA: spectra and lifetimes by the maximum entropy method |url= |journal=Photochem. & Photobiol. Sci. |volume=6 |issue= 8|pages=883–893 |doi=10.1039/b615670c |pmid=17668119 |access-date=}}{{cite journal |last1=Wang |first1=D. H. |date=2022 |title=Excited State Dynamics of Methylated Guanosine Derivatives Revealed by Femtosecond Time-resolved Spectroscopy |url= |journal=Photochemistry and Photobiology |volume=98|issue= 5|pages=1008–1016 |doi=10.1111/php.13612 |pmid=35203108 |access-date=}}{{cite journal |last1=Wang |first1=D. H. |date=2024 |title=Methylation Induces a Low-energy Emissive State in N6-methyladenine Containing Dinucleotides |url= |journal=ChemPhotoChem |volume=8 |issue= 7|pages= |doi=10.1002/cptc.202300235 |access-date=}} The emission spectra of these systems are red-shifted compared to those of the major nucleobases and give rise to exciplexes.

The emission spectra described in this section are derived from fundamental studies; they may differ from those reported in application-oriented studies, which are shifted to longer wavelengths. The reason is that the latter are usually recorded for solutions with higher concentration. As a result, photons emitted at short wavelengths are reabsorbed by the DNA solution (inner filter effect) and the blue part of the spectrum is truncated.

The specificity of the intrinsic DNA fluorescence is that, contrary to most fluorescent molecules, its time-evolution cannot be described by a constant decay rate (described by a mono-exponential function). For the monomeric units, the fluorescence lasts at most a few picoseconds. In the case of multimers, the fluorescence continues over much longer times, lasting in some cases, for several tens of nanoseconds. The time constants derived from fittings with multi-exponential functions depend of the probed time window.

In order to obtain a complete picture of this complex time evolution, a femtosecond laser is needed as excitation source. Time-resolved techniques employed to this end are fluorescence upconversion,{{cite journal |last1=Peon |first1=J. |date=2001 |title=DNA/RNA nucleotides and nucleosides: direct measurement of excited-state lifetimes by femtosecond fluorescence up-conversion |url= |journal=Chem. Phys. Lett. |volume=348 |issue=3–4 |pages=255–262 |doi=10.1016/S0009-2614(01)01128-9 |bibcode=2001CPL...348..255P |access-date=}}{{cite journal |last1=Schwalb |first1=N.K. |date=2008 |title=Base sequence and higher-order structure induce the complex excited-state dynamics in DNA |url= |journal=Science |volume=322 |issue= 5899|pages=243–245 |doi=10.1126/science.1161651 |pmid=18845751 |bibcode=2008Sci...322..243S |access-date=}}{{cite journal |last1=Vaya |first1=I. |date=2010 |title=Fluorescence of natural DNA: from the femtosecond to the nanosecond time-scales |url= https://hal.archives-ouvertes.fr/hal-00516393/document|journal=J. Am. Chem. Soc. |volume=132 |issue= 34|pages=11834–11835 |doi=10.1021/ja102800r |pmid=20698570 |bibcode=2010JAChS.13211834V |access-date=}}{{cite journal |last1=Wang |first1=D. H. |date=2022 |title=Excited State Dynamics of Methylated Guanosine Derivatives Revealed by Femtosecond Time-resolved Spectroscopy |url= |journal=Photochemistry and Photobiology |volume=98|issue= 5|pages=1008–1016 |doi=10.1111/php.13612 |pmid=35203108 |access-date=}} Kerr-gated fluorescence spectroscopy{{cite journal |last1=Kwok |first1=W.-M. |date= 2006|title=Femtosecond time- and wavelength-resolved fluorescence and absorption study of the excited states of adenosine and an adenine oligomer |url= |journal=J. Am. Chem. Soc. |volume=128 |issue= 36|pages=11894–11905 |doi=10.1021/ja0622002 |pmid=16953630 |access-date=}} and time-correlated single photon counting.{{cite journal |last1=Vaya |first1= I.|date=2016 |title=High energy long-lived mixed Frenkel – charge transfer excitons: from double-stranded (AT)_n to natural DNA |url= |journal=Chem. Eur. J. |volume=22 |issue= 14|pages=4904–4914 |doi=10.1002/chem.201504007 |pmid= 26928984|access-date=}} In addition to the changes in the fluorescence intensity, all of them allow the recording of time-resolved fluorescent spectra{{cite journal |last1=Onidas |first1=D. |date=2007 |title=Fluorescence of the DNA double helix (dA)20.(dT)20 studied by femtosecond spectroscopy – effect of the duplex size on the properties of the excited states |url= https://hal.archives-ouvertes.fr/hal-00170399/file/_dA_20_dT_20_revised_manuscript.pdf|journal=J. Phys. Chem. B |volume=111 |issue= 32|pages=9644–9650 |doi=10.1021/jp072508v |pmid=17658793 |access-date=}}{{cite journal |last1=Ma |first1=C. |date=2015 |title=Remarkable effects of solvent and substitution on the photo-dynamics of cytosine: a femtosecond broadband time-resolved fluorescence and transient absorption study |url= |journal=Phys. Chem. Chem. Phys. |volume=17 |issue= 29|pages=19045–19057 |doi=10.1039/c5cp02624e |pmid=26126728 |bibcode=2015PCCP...1719045M |access-date=}} and fluorescence anisotropies,{{cite journal |last1=Hua |first1=Y |date=2012|title=Cation Effect on the Electronic Excited States of Guanine Nanostructures Studied by Time-Resolved Fluorescence Spectroscopy |url= |journal=J. Phys. Chem. C |volume=116 |issue= 27|pages=14682–14689 |doi=10.1021/jp303651e |access-date=}}{{cite journal |last1=Ma |first1=M.S. |date=2019 |title=Real-time Monitoring Excitation Dynamics of Human Telomeric Guanine Quadruplexes: Effect of Folding Topology, Metal Cation, and Confinement by Nanocavity Water Pool |url= |journal=J. Phys. Chem. Lett. |volume=10 |issue= 24|pages=7577–7585 |doi=10.1021/acs.jpclett.9b02932 |pmid=31769690 |access-date=}} which provide information about the relaxation of the excited electronic states and the type of the emitting excited states.

The early studies were performed using time-correlated single photon counting combined with nanosecond sources (synchrotron radiation or lasers).{{cite journal |last1= Ballini |first1=J. P. |date=1982 |title=Wavelength-resolved lifetime measurements of emissions from DNA components and poly rA at room temperature excited with synchrotron radiation |url= |journal=Journal of Luminescence |volume=27 |issue= 4|pages=389–400 |doi=10.1016/0022-2313(82)90039-4 |bibcode=1982JLum...27..389B |access-date=}}{{cite journal |last1= Ballini |first1=J. P. |date=1983|title=Synchrotron excitation of DNA fluorescence: decay time evidence for excimer emission at room temperature |url= |journal=Biophys. Chem. |volume=18 |issue= 1|pages=61–65 |doi=10.1016/0301-4622(83)80027-1 |pmid=17005122 |access-date=}}{{cite journal |last1=Ballini |first1=J. P. |date=1991 |title=Time-resolved fluorescence emission and excitation spectroscopy of d(TA) and d(AT) using synchrotron radiation |url= |journal=Biophys. Chem. |volume=91 |issue= 3|pages=253–265 |doi=10.1016/0301-4622(91)80003-A |pmid=1863687 |access-date=}} Although they discovered the existence of nanosecond components exclusively for multimeric nucleic acids, they failed to obtain a full picture of the fluorescence dynamics.

File:Cartoon conical intersection.jpg

Emission from the monomeric DNA chromophores arises from their lower in energy electronic excited states, that is the ππ* states of the nucleobases. These are bright states, in the sense that they are also responsible for photon absorption.{{cite journal |last1=Improta |first1=R. |date=2016 |title=Quantum Mechanical Studies on the Photophysics and the Photochemistry of Nucleic Acids and Nucleobases |url= |journal=Chem. Rev. |volume=116 |issue= 6|pages=3540–3593 |doi=10.1021/acs.chemrev.5b00444 |pmid=26928320 |access-date=}}

File:Relaxation via conformational motions.jpg

Their lifetimes are extremely short: they fully decay within, at most, a few ps.{{cite journal |last1=Peon |first1=J. |date=2001 |title=DNA/RNA nucleotides and nucleosides: direct measurement of excited-state lifetimes by femtosecond fluorescence up-conversion |url= |journal=Chem. Phys. Lett. |volume=348 |issue=3–4 |pages=255–262 |doi=10.1016/S0009-2614(01)01128-9 |bibcode=2001CPL...348..255P |access-date=}}{{cite journal |last1=Onidas |first1=D. |date=2002 |title=Fluorescence properties of DNA nucleosides and nucleotides: a refined steady-state and femtosecond investigation |url= |journal=J. Phys. Chem. B |volume=106 |issue= 43|pages=11367–11374 |doi=10.1021/jp026063g}}{{cite journal |last1=Pancur |first1=T. |date=2005 |title=Femtosecond fluorescence up-conversion spectroscopy of adenine and adenosine: experimental evidence for the ps* state? |url= |journal=Chem. Phys. |volume=313 |issue= |pages=199–212 |doi=10.1016/j.chemphys.2004.12.019 |access-date=}}{{cite journal |last1=Kwok |first1=M.-W. |date=2008 |title=A doorway state leads to photostability or triplet photodamage in thymine DNA |url= |journal=J. Am. Chem. Soc. |volume=130 |issue= 15|pages=5131–5139 |doi=10.1021/ja077831q |pmid=18335986 |bibcode=2008JAChS.130.5131K |access-date=}} Such ultrafast decays are due to the existence of conical intersections connecting the excited state with the ground state.{{cite journal |last1=Matsika |first1=S. |date=2005|title=Three-state conical intersections in nucleic acid bases |url= |journal=J. Phys. Chem. A |volume=109 |issue= 33|pages=7538–7545 |doi=10.1021/jp0513622 |pmid=16834123 |bibcode=2005JPCA..109.7538M |access-date=}}{{cite journal |last1=Giussani |first1=A. |date=2015 |title=Excitation of nucleobases from a computational perspective I: reaction paths |url= |journal=Top. Curr. Chem. |series=Topics in Current Chemistry |volume=355|issue= |pages=57–97 |doi=10.1007/128_2013_501 |pmid=24264958 |isbn=978-3-319-13370-6 |access-date=}}{{cite journal |last1=Improta |first1=R. |date=2016 |title=Quantum Mechanical Studies on the Photophysics and the Photochemistry of Nucleic Acids and Nucleobases |url= |journal=Chem. Rev. |volume=116 |issue= 6|pages=3540–3593 |doi=10.1021/acs.chemrev.5b00444 |pmid=26928320 |access-date=}} Therefore, the dominant deactivation pathway is non-radiative,{{cite journal |last1=Welborn |first1=V. V. |date=2018 |title=Non-radiative deactivation of cytosine derivatives at elevated temperature |url= https://figshare.com/articles/journal_contribution/6159728|journal=Molecular Physics |volume=116 |issue= 19–20|pages=2591–2598 |doi=10.1080/00268976.2018.1457806 |bibcode=2018MolPh.116.2591W |access-date=}} leading to very low fluorescence quantum yields.

The evolution toward the conical intersection is accompanied by conformational movements. An important part of the photons is emitted while the system is moving along the potential energy surface of the excited state, before reaching a point of minimum energy. As motions on a low-dimensional surfaces do not follow exponential patterns,{{cite journal |last1=Drake |first1=J. M. |date=1990 |title=Dynamics of Confined Molecular-Systems |url= |journal=Physics Today |volume=43 |issue= 5|pages=46–55 |doi=10.1063/1.881244 |bibcode=1990PhT....43e..46D |access-date=}}{{cite journal |last1=Benichou |first1=O. |date=2010 |title=Geometry-controlled kinetics |url= |journal=Nat. Chem. |volume=2 |issue= 6|pages=472–477 |doi=10.1038/nchem.622 |pmid=20489716 |arxiv=1006.3477 |bibcode=2010NatCh...2..472B |access-date=}} the fluorescence decays are not characterized by constant decay rates.{{cite journal |last1=Gustavsson |first1=T. |date= 2010|title=DNA/RNA: Building Blocks of Life Under UV Irradiation |url= https://hal.archives-ouvertes.fr/hal-00496298/file/Gustavsson_JPCL_Perspective_rev1.pdf|journal=J. Phys. Chem. Lett. |volume=1 |issue= 13|pages=2025–2030 |doi=10.1021/jz1004973 |access-date=}}

Due to their close proximity, nucleobases in DNA multimers may be electronically coupled. This leads to delocalization of the excited states responsible for photon absorption (Franck-Condon states) over more than one nucleobase (collective states).{{cite journal |last1=Bouvier |first1=B. |date=2002 |title=Dipolar coupling between electronic transitions of the DNA bases and its relevance to exciton states in double helices |url= |journal=Chem. Phys. |volume=275 |issue= 1–3|pages=75–92 |doi=10.1016/S0301-0104(01)00523-7 |bibcode=2002CP....275...75B |access-date=}}{{cite journal |last1=Czader |first1=A. |date= 2008|title=Calculations of the exciton coupling elements between the DNA bases using the transition density cube method |url= |journal=J. Chem. Phys. |volume=128 |issue= 3|pages=035101 |doi=10.1063/1.2821384 |pmid=18205523 |arxiv=0708.1128 |bibcode=2008JChPh.128c5101C |access-date=}}{{cite journal |last1=Plasser |first1=F. |date=2015 |title=Electronic Excitation Processes in Single-Strand and Double-Strand DNA: A Computational Approach |url= |journal=Top. Curr. Chem. |series=Topics in Current Chemistry |volume=356 |issue= |pages=1–38 |doi=10.1007/128_2013_517 |pmid=24549841 |isbn=978-3-319-13271-6 |access-date=}}{{cite journal |last1=Blancafort |first1=L. |date=2014 |title=Exciton delocalization, charge transfer, and electronic coupling for singlet excitation energy transfer between stacked nucleobases in DNA: An MS-CASPT2 study |url= |journal=J. Chem. Phys. |volume=140 |issue= 9|pages= |doi=10.1063/1.4867118 |pmid=24606381 |bibcode=2014JChPh.140i5102B |hdl=10256/11471 |access-date=|hdl-access=free }}{{cite journal | url=https://pubs.acs.org/doi/10.1021/acs.accounts.2c00256 | doi=10.1021/acs.accounts.2c00256 | title=Nucleic Acids as a Playground for the Computational Study of the Photophysics and Photochemistry of Multichromophore Assemblies | date=2022 | last1=Martínez Fernández | first1=Lara | last2=Santoro | first2=Fabrizio | last3=Improta | first3=Roberto | journal=Accounts of Chemical Research | volume=55 | issue=15 | pages=2077–2087 | pmid=35833758 | url-access=subscription }} The electronic coupling depends on the geometrical arrangement of the chromophores. Therefore, the properties of the collective states are affected by factors that determine the relative position of the nucleobases.{{cite journal |last1=Aarabi |first1=M. |date= 2025|title=Effect of A-DNA and B-DNA Conformation on the Interplay between Local Excitations and Charge-Transfer States in the Ultrafast Decay of Guanine–Cytosine Stacked Dimers: A Quantum Dynamical Investigation |url=https://doi.org/10.1021/acs.jpca.4c06672 |journal=J. Phys. Chem. A |volume= 129|issue= 4|pages= 985–996|doi= 10.1021/acs.jpca.4c06672|pmid=39828990 |access-date=|url-access=subscription}} Among others, the conformational disorder characterizing the nucleic acids modulates the coupling values,{{cite journal |last1=Bouvier |first1=B. |date=2003 |title=Influence of conformational dynamics on the exciton states of DNA oligomers |url= |journal=J. Phys. Chem. B |volume=107 |issue= 48|pages=13512–13522 |doi=10.1021/jp036164u |access-date=}}{{cite journal |last1=Nogueira |first1=J. J. |last2=Plasser |first2=Felix |last3=González |first3=Leticia |date=2017 |title=Electronic delocalization, charge transfer and hypochromism in the UV absorption spectrum of polyadenine unravelled by multiscale computations and quantitative wavefunction analysis |url= |journal=Chem. Sci. |volume=8 |issue= 8|pages=5682–5691 |doi=10.1039/c7sc01600j |pmid=28989607 |pmc=5621053 |access-date=}} giving rise to a large number of Franck-Condon states. Each one of them evolves along a specific energy surface.

One can distinguish two limiting types of emitting states in DNA. On the one hand, ππ* states, localized on single nucleobases or delocalized over several of them. And on the other, excited charge transfer states in which an important fraction of an atomic charge has been transferred from one nucleobase to another. The latter are weakly emissive. And between these two types, there is a multitude of emitting states, more or less delocalized, with different amounts of charge transfer. The properties of the emitting states may be modified during their lifetime under the effect of conformational motions of the nucleic acid, occurring on the same time-scale.{{cite journal |last1=Young |first1=M. A. |date=1997 |title=A 5-nanosecond molecular dynamics trajectory for B-DNA: Analysis of structure, motions, and solvation |url= |journal=Biophysical Journal |volume=73 |issue=2313–2336 |pages=2313–2336 |doi= 10.1016/S0006-3495(97)78263-8|pmid=9370428 |pmc=1181136 |bibcode=1997BpJ....73.2313Y |access-date=}}{{cite journal |last1=Giudice |first1=E. |date=2002 |title=Simulations of nucleic acids and their complexes |url= |journal=Accounts of Chemical Research |volume=36 |issue= 6|pages=350–357 |doi=10.1021/ar010023y |pmid=12069619 |access-date=}}{{cite journal |last1=Sponer |first1=J. |date=2007 |title=Molecular dynamics simulations and their application to four-stranded DNA |url= |journal=Methods |volume=43 |issue= 4|pages=278–290 |doi=10.1016/j.ymeth.2007.02.004 |pmid=17967698 |pmc=2431124 |access-date=}}{{cite journal |last1=Rinnenthal |first1=J. |date=2011 |title=Mapping the Landscape of RNA Dynamics with NMR Spectroscopy |url= |journal=Accounts of Chemical Research |volume=44 |issue= 12|pages=1292–1301 |doi=10.1021/ar200137d |pmid=21894962 |access-date=}} Because of this complexity, the description of the fluorescence decays by multiexponential functions is only phenomenological.{{cite journal |last1=Markovitsi |first1=D. |date=2006 |title=Complexity of excited state dynamics in DNA |url= |journal=Nature |volume=441 |issue= 7094|pages=E7 |doi=10.1038/nature04903 |pmid=16760929 |access-date=}}

Experimentally, the different types of emitting states can be differentiated through their fluorescence anisotropy.{{cite journal |last1=Wilson |first1=R. W. |date= 1976|title=Excitons, energy transfer, and charge resonance in excited dinucleotides and polynucleotides. A photoselection study |url= |journal=Journal of Physical Chemistry |volume=80 |issue= 20|pages=2280–2288 |doi=10.1021/j100561a029 |access-date=}} The charge transfer character of an excited state lowers the fluorescence anisotropy.{{cite journal |last1=Spata |first1=V. A. |date= 2015|title=Photophysical deactivation pathways in adenine oligonucleotides |url= |journal=Phys. Chem. Chem. Phys. |volume=17 |issue= 46|pages=31073–31083 |doi= 10.1039/c5cp04254b|pmid=26536353 |bibcode=2015PCCP...1731073S |access-date=}} The decrease of fluorescence anisotropy observed for all the DNA multimers on the femtosecond time-scale was explained by an ultrafast transfer of the excitation energy among the nucleobases.{{cite journal |last1=Markovitsi |first1=D. | title=Collective behavior of Franck-Condon excited states and energy transfer in DNA double helices | date=2005 |journal=J. Am. Chem. Soc. | volume=167 | issue= 49| pages=17130–17131 |doi= 10.1021/ja054955z|pmid=16332029 |access-date=}}{{cite journal |last1=Bittner |first1=E. R. |date=2006 |title=Lattice theory of ultrafast excitonic and charge transfer dynamics in DNA |url= |journal=J. Chem. Phys. |volume=125 |issue= 9|pages=094909 |doi=10.1063/1.2335452 |pmid=16965121 |bibcode=2006JChPh.125i4909B |access-date=}}{{cite journal |last1=Bittner |first1=E. R. |date=2007 |title=Frenkel exciton model of ultrafast excited state dynamics in AT DNA double helices |url= |journal=J. Photochem. Photobiol. A |volume=190 |issue= 2–3|pages=328–334 |doi=10.1016/j.jphotochem.2006.12.007 |arxiv=cond-mat/0606333 |bibcode=2007JPPA..190..328B |access-date=}}{{cite journal |last1=Vaya |first1=I. |date= 2012 |title=Electronic Excitation Energy Transfer between Nucleobases of Natural DNA |url= |journal=American Chemical Society |volume=134 |issue= 28|pages=11366−11368 |doi=10.1021/ja304328g |pmid=22765050 |bibcode=2012JAChS.13411366V |access-date=}}{{cite journal |last1=Ma |first1=C. S. |date=2023 |title=Excited-State Charge Transfer, Proton Transfer, and Exciplex Formation Revealed by Ultrafast Time-Resolved Spectroscopy in Human Telomeric Ribonucleic Acid Quadruplex |url= |journal=Journal of Physical Chemistry Letters |volume=14 |issue= 22|pages=5085–5094 |doi=10.1021/acs.jpclett.3c00806 |pmid=37232555 |access-date=}}

File:Duplex fluorescence.png

A particular class of emitting excitons with weak charge transfer character{{cite journal |last1=Huix-Rotllant |first1=M. |date=2015 |title=Stabilization of mixed Frenkel-charge transfer excitons extended across both strands of guanine-cytosine DNA duplexes |url= |journal=J. Phys. Chem. Lett. |volume=6 |issue= 12|pages=3540–3593 |doi=10.1021/acs.jpclett.5b00813 |pmid=26266599 |access-date=}}{{cite journal |last1=Vaya |first1= I.|date=2016 |title=High energy long-lived mixed Frenkel – charge transfer excitons: from double-stranded (AT)_n to natural DNA |url= |journal=Chem. Eur. J. |volume=22 |issue= 14|pages=4904–4914 |doi=10.1002/chem.201504007 |pmid= 26928984|access-date=}} was detected in all types of duplexes, including genomic DNA.{{cite journal |last1=Gustavsson |first1=T. |date=2023 |title=The Ubiquity of High-Energy Nanosecond Fluorescence in DNA Duplexes |url= https://cnrs.hal.science/hal-04002412/file/HENE_HAL.pdf|journal=J. Phys. Chem. Lett. |volume=14 |issue= 8|pages=2141–2147 |doi=10.1021/acs.jpclett.2c03884 |pmid=36802626 |access-date=}} Their specificity is that their emission appears at short wavelengths (λ<330 nm) and represents the longest-living components of the overall duplex fluorescence, decaying on the nanosecond timescale. It contrasts with the excimer/exciplex emission, characterized by a pronounced charge transfer character, appearing at long wavelengths and decaying on the sub-nanosecond time-scale. The contribution of the high energy emitting states to the total fluorescence increases with the local rigidity of the duplex (depending on the number of the Watson-Crick hydrogen bonds or the size of the system) and the excitation wavelength. The latter point, associated with the very weak spectral width observed for the most representative example (polymeric duplex with alternating guanine-cytosine sequence) is reminiscent of the emission stemming from J-aggregates.

{{cite journal |last1= Bricks |first1=J. L. |date=2018 |title=Fluorescent J-aggregates of cyanine dyes: basic research and applications review |url= |journal=Methods Appl. Fluoresc. |volume=6 |issue= 1|pages=012001 |doi=10.1088/2050-6120/aa8d0d |pmid=28914610 |bibcode=2018MApFl...6a2001B |access-date=|doi-access=free }}{{cite journal |last1=Hecht |first1=M. |date=2021 |title=Supramolecularly Engineered J-Aggregates Based on Perylene Bisimide Dyes |url= |journal=Accounts of Chemical Research |volume=54 |issue= 3|pages=642–653 |doi=10.1021/acs.accounts.0c00590 |pmid=33289387 |access-date=}}

The utilization of the intrinsic fluorescence of nucleic acids for various applications has been under scrutiny since 2019. Several approaches have been explored, primarily focusing on the variation of its intensity upon binding of different molecular species to nucleic acids. Thus, target DNA in human serum,{{cite journal |last1=Xiang |first1=X|date=2019 |title=Label-free and dye-free detection of target DNA based on intrinsic fluorescence of the (3+1) interlocked bimolecular G-quadruplexes |url= |journal=Sens. Actuators B Chem. |volume= 290|issue=290 |pages=68–72|doi=10.1016/j.snb.2019.03.111 |bibcode=2019SeAcB.290...68X |access-date=}} Pb²⁺ ions in water,{{cite journal |last1=Lopez |first1=

A.|date=2022 |title=Probing metal-dependent G-quadruplexes using the intrinsic fluorescence of DNA |url= |journal=Chem. Comm. |volume=58 |issue= 73|pages=10225–10228 |doi=10.1039/d2cc03967b |pmid= 36001027|access-date=}} aptamer binding,{{cite journal |last1=Lu |first1=C. |date= 2022|title=Using the Intrinsic Fluorescence of DNA to Characterize Aptamer Binding |url= |journal= Molecules|volume=27 |issue= 22|pages= 7809|doi=10.3390/molecules27227809 |doi-access=free |pmid=36431910 |pmc=9692703 |access-date=}} as well as the interaction of quinoline dyes (commonly used in the food and pharmaceutical industries){{cite journal |last1=Gu |first1=Y. |date=2025 |title=Chitosan as a fluorescent probe for the detection of the AIE-active food colorant quinoline yellow |url= |journal=Analytical Methods |volume=17 |issue= 4|pages= 671–676|doi=10.1039/d4ay02087a |pmid=39711316 |access-date=}} were detected.

In parallel, the screening of a large number of sequences was explored by multivariate analysis.{{cite journal |last1=Zuffo |first1=M. |date=2020 |title=Harnessing intrinsic fluorescence for typing of secondary structures of DNA |url= |journal=Nucl. AC. Res. |volume=48 |issue= 11|pages=e61 |doi=10.1093/nar/gkaa257 |pmid=32313962 |pmc=7293009 |access-date=}} The technique of synchronous fluorescence scanning was employed for the authentication of COVID 19 vaccines.{{cite journal |last1=Assi |first1=S. |date=2023 |title=Authentication of Covid-19 Vaccines Using Synchronous Fluorescence Spectroscopy |url= |journal=J. Fluoresc. |volume=33 |issue= 3|pages=1165–1174 |doi=10.1007/s10895-022-03136-5 |pmid=36609659 |pmc=9825072 |access-date=}} And the assessment of the intrinsic fluorescence was included in a multi-attribute analysis of adeno-associated virus.

{{cite journal |last1=Xie |first1=Y. J. |date=2023 |title=Multi-attribute analysis of adeno-associated virus by size exclusion chromatography with fluorescence and triple-wavelength UV detection |url= |journal=Analytical Biochemistry |volume=680 |issue= |pages=115311 |doi=10.1016/j.ab.2023.115311 |pmid=37666384 |access-date=|doi-access=free }} Along the same line, an optical assay has been developed in order to assess the binding to G-Quadruplexes small molecules with potential anticancer properties.{{cite journal |last1=Bednarz |first1=A. |date= 2024|title=Probing G-quadruplex-ligand binding using DNA intrinsic fluorescence |url= |journal=Biochimie |volume=227 |issue= Pt A|pages=61–67 |doi=10.1016/j.biochi.2024.06.009 |pmid=38936685 |access-date=|doi-access=free }}

The prospect of probing DNA damage by monitoring the intrinsic fluorescence has been also discussed.{{cite journal |last1= Markovitsi|first1=D. |date=2024 |title=10.1021/acsomega.4c02256 |url= |journal=ACS Omega |volume=9 |issue=25 |pages= 26826–26837|doi=10.1021/acsomega.4c02256 |pmid=38947837 |pmc=11209687 |access-date=}} This potential application could leverage the short wavelength emission of duplexes, associated with collective excited states whose properties are highly sensitive to the geometrical arrangement of the nucleobases. And the generation of various lesions are known to induce structural distortions.

{{cite journal |last1=Wang |first1=C.-I. |date=1991 |title=Site specific effect of thymine dimer formation on dAn.dTn track bending and its biological implications |url= |journal=Proc. Natl. Acad. Sci. USA |volume=88 |issue= 20|pages=9072–9076 |doi=10.1073/pnas.88.20.9072 |doi-access=free |pmid=1924370 |pmc=52654 |access-date=}}{{cite journal |last1=Lukin |first1=M. |date=2006 |title=NMR structures of damaged DNA |url= |journal=Chem. Rev. |volume=106 |issue= 2|pages=607–686 |doi=10.1021/cr0404646 |pmid=16464019 |access-date=}}

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Category:Biochemistry

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Category:Fluorescence

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