• dna
• energy transfer
• fluorescence
• guanine
• lanthanide
• lanthanide probe
• luminescence
• metadynamics
• molecular dynamics
• terbium

The interest of scientists towards lanthanides, a group of elements that belongs to the f-block of the periodic table, has grown considerably in the last decades. This is due to their unique chemical, magnetic, and optical properties, which ultimately arise from their partially filled 4f orbitals, buried deep below the full 5s and 5p shells.
Lanthanide-based analytical probes are complexes made by lanthanide ions and organic ligands, and are designed to show fluorescence variations upon interaction with a specific analyte. These probes are very popular thanks to their selectivity and efficiency, which is due to their working principle, i.e. the antenna effect. In fact, organic ligands are required to excite the lanthanide ion, as free lanthanide ions have a small molar extinction factor and therefore cannot be efficiently excited by light absorption. This is because f-f electric dipole transitions are parity forbidden. Even in the presence of a less symmetric external ligand field, where this selection rule is partially lifted, the lanthanide ion transitions remain virtually dark. The ligands near the ion are excited by light absorption, and then transfer their electronic energy to the lanthanide ion via a process called Excitation Energy Transfer (EET). The excited lanthanide ion may get rid of the excess energy by emitting light. Through the antenna effect, small geometric and/or chemical variations induced by the interaction of the ligand result in large variations in the emitted intensity, which make these probes very sensitive to the surroundings.
In the present work, we performed an integrated experimental and computational study about a probe that is selective towards guanine bases in DNA. Instead of using a complex whose ligands are designed to interact with guanine bases, we suggest a novel approach where free terbium ions are used as a probe. This works because it is directly the analyte, i.e. guanine, that acts as an antenna to sensitise the emission of terbium ions upon interaction. Skipping the whole need to design a suitable ligand and the cost of organic synthesis would make such a probe inexpensive and readily accessible.
The purpose of this work is to assess whether conditions are right to use free terbium ions as a probe for the G-C to A-T ratio in DNA sequences and to provide an atomistic interpretation of the interaction.
Spectrophotometric experiments were performed to check the stability of DNA in the presence of terbium ions. This was done through time-drive acquisitions, DNA denaturation tests, and circular dichroism spectrophotometry. The selectivity of the probe towards guanine residues was proven by performing spectrofluorimetric titrations of a terbium solution using different polynucleotides (natural calf-thymus DNA and synthetic poly(dA)poly(dT)) and mononucleotides (GMP and AMP). Fluorescence enhancement only happens in the presence of guanines. Equilibrium constants were inferred by fitting the resulting binding isotherms. The dependence on temperature of equilibrium constants was analysed.
Molecular dynamics simulations were performed to study the effect of terbium ions on the conformation of two different DNA dodecamers, i.e. poly(dG)poly(dC) and poly(dAdT), and to investigate the ion distribution around them. In the case of poly(dG)poly(dC), the presence of terbium ions results in increased mobility of terminal base pairs and a slight conformational change and reduced mobility of central base pairs. Through the analysis of ion distribution, it was evident that the usual 12-6 Lennard-Jones parametrization of non-bonded interaction for terbium ions is faulty. Indeed, no interactions with guanine residues are present in the dynamics, which contradicts our experimental observations. Conversely, the 12-6-4 Lennard-Jones potential includes charge-induced dipole interactions and was able to explain the reason why guanine residues sensitise the fluorescence of terbium ions while adenine cannot: our simulations show that the first one bonds with terbium, while the latter does not. Transient ion density displacement until equilibration was discussed. To quantify the affinity of terbium ions towards guanine residues, the average number of terbium ions within the grooves, near the phosphate backbone, and in the bulk was studied and an estimate of the free energy difference between these positions was provided.
Two well-tempered metadynamics simulations were performed on a simplified model in order to explore the free energy surface (FES) and characterise the interaction between a terbium ion and guanine. The model consisted in a single terbium ion and a trimer made of guanines, whose conformation is forced to be the same that they would have if they were embedded in poly(dG)poly(dC). Well-tempered metadynamics is a type of enhanced sampling technique, used to overcome high energy barriers along one or more collective variables representing the slow degrees of freedom of the system. Particular attention is paid to the difference between first shell and outer shell bonding. The two runs differ in the number (one or two) of the biased collective variables. We found that using two collective variables, i.e. the distance between the terbium ion and the carbonyl oxygen of the central guanine and the number of water molecules in the first terbium solvation shell, was more effective and allowed to estimate the free energy difference.
In conclusion, our investigation about the feasibility of using terbium ions as a selective probe for DNA gave promising results. This work succeeded in providing a qualitative atomistic explanation of the reason why terbium fluorescence enhancement is selective towards guanine. The observed selectivity was explained in terms of the incapability of adenine at binding terbium ions. The effects of terbium ions on the conformation of DNA were also studied.