Cy3 and Cy5 Dyes Terminally Attached to 5â²C End of DNA: Structure

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Cy3 and Cy5 Dyes Terminally Attached to 5’C End of DNA: Structure, Dynamics and Energetics Ondrej Kroutil, Ingrid Romancová, Miroslav Sip, and Zdenek Chval J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp509459y • Publication Date (Web): 03 Nov 2014 Downloaded from http://pubs.acs.org on November 10, 2014

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The Journal of Physical Chemistry

Cy3 and Cy5 Dyes Terminally Attached to 5’C End of DNA: Structure, Dynamics and Energetics

Ondřej Kroutil,a,b Ingrid Romancová,b Miroslav Šíp a and Zdeněk Chval a*

a

Department of Laboratory Methods and Information Systems, Faculty of Health and Social Studies, University of South Bohemia, J. Boreckého 27, 37011 České Budějovice, Czech Republic b

Institute of Physics and Biophysics, Faculty of Science, University of South Bohemia, Branišovská 31, 37005 České Budějovice, Czech Republic

Abstract:

Cy3 and Cy5 cyanine dyes terminally attached to the 5’C end (C1) of the DNA oligonucleotide were studied by metadynamics (MTD), molecular dynamics (MD), and density-functional methods with dispersion corrections (DFT-D). MTD simulations explored the free energy surface (FES) of the dye-DNA interactions which included stacking and major groove binding motifs and unstacked structures. Dynamics of the stacked structures was studied by the MD simulations. All possible combinations of stacking interactions between the two indole rings of the dyes and the neighbor guanine and cytosine rings were observed. The most probable interaction included the To whom correspondence should be addressed: e-mail: [email protected], tel.: +420-389-037-612 ACS Paragon Plus Environment


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stacking between the dye’s distal indole ring and the guanine base. In ~10% of the structures the delocalized π-electrons of the dyes’ polymethine linkers played a key role in the dye-DNA dispersion interactions. The stacked conformers of the Cy3 dye were confirmed as true minima by DFT-D full optimizations. The stacked dye decreased flexibility up to two neighbor base pairs.

Keywords: cyanine, fluorophore, base pair entropy, force field parametrization, thermal motions

Introduction: Cyanine Cy3 and Cy5 are fluorescent dyes with many applications in nonlinear optics, laser physics and particularly in biomedical imaging and single-molecule spectroscopy. Both dyes adopt an all-trans configuration in its ground state (Figure 1) and can be easily attached to nucleic acids and proteins. They are brighter than other fluorescent dyes, offer greater photostability and low pH sensitivity.1 Moreover being excitable in the visible and the near infrared region range they are preferable in biological applications since a much lower auto-fluorescence and a higher transmittance through the cell media are to be expected in this wavelength range.

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Figure 1: Structures of Cy3 and Cy5 cyanine dyes. For the purpose of structural analyses the structure of cyanine dyes was divided to three parts: the ‘distal’ indole ring (DIR) with a free hydroxypropyl side chain (C3OH), the polymethine linker (PML) and the ‘proximal’ indole ring (PIR) with a three-carbon linker attached covalently to the 5'-phosphate end of the DNA (cytidine C1 nucleotide).

Absorption and emission spectra of the cyanine dyes can be tuned by variation of the length of PML joining the two heads of the cyanine dye.2 Cy3 has the absorption and emission maxima at 550 nm and 570 nm, while Cy5 at 650 nm and 670 nm, respectively. Thus the Cy3 and Cy5 dyes are often used as the donor - acceptor pair in the fluorescence resonance energy-transfer (FRET) experiments to study the structure and dynamics of biomolecules.3 The FRET efficiency is dependent on the molecular environment within the biomolecule, and the distance and orientation of the two fluorophores. Recently, conformational dynamics of Cy3 and Cy5 dyes bound to 16base-pair RNA duplex was studied.4 It was shown that the dyes attached to the 3’ end of RNA explored much wider region of the configuration space than when bound to the 5’ end but even in the former case the free rotation approximation was not appropriate for calculations of the energy transfer in FRET.4 The same conclusion was achieved for tetramethylrhodamine and Cy5 dyes bound in the minor and major grooves of DNA, respectively.5 Cyanines linked to DNA internally are believed to partially bind to the minor or major grooves6 while cyanines linked to the DNA termini were shown to be mostly stacked at the end of the helix7–9 and their orientation depended on the length of a linker.10,11 Cyanine dyes were mostly studied with respect to their photophysical properties.1,6,12–18 The fluorescence quantum yield was strongly affected by the speed of cis-/trans- photoisomerization19



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which depended on the type of linkage used for attachment, DNA sequence and secondary structure.6 Except the A:T bp the activation energies of cis-trans isomerization were comparable to unstacking free energies for Cy3 covalently attached to DNA.20 Very recently it was shown by different fluorescence techniques that photoisomerization occurred with high efficiency even for doubly linked Cy3-DNA constructs.21 Cyanine dyes are also widely used for DNA labeling in many applications like DNA microarrays. The immobilized probes on one microarray should hybridize with the complementary target sequences in the sample simultaneously and under the same experimental conditions. They are designed to have similar melting temperatures and similar free energies of hybridization. The available software tools for the probe design rely on standard hybridization conditions.22,23 Cyanine dyes were shown to increase DNA duplex stability when attached to the duplex or strand ends24 but they have the destabilizing effect when attached internally.25 It may be the result of different thermodynamics since entropy plays a key role in the minor groove binding while intercalation (and stacking) are believed to be enthalpy driven.26 One of the aims of this study was to see how the bound dyes influence the DNA structure and the flexibility of the terminal base pairs. We will also explore in more detail the conformational behavior of Cy3 and Cy5 dyes when attached covalently to the 5'-C end of DNA via a threecarbon linker.

Computational Methods The systems. The double-stranded DNA with the palindromic CCACTAGTGG sequence was used in three atomic resolution explicit-solvent MD simulations. In two simulations the DNA strand was modified on one 5’C end by covalently bonded Cy3 and Cy5 dyes and the resulting


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systems were designated as Cy3-DNA10 and Cy5-DNA10, respectively (Figure 2). The third simulation was performed on the bare DNA as a reference. The shorter pentamer sequence CCACC with the dye attached at the 5’C end (C1) was used for MTD simulations. These systems were designated as Cy3-DNA5 and Cy5-DNA5. Despite only ten nucleotides are present their labeling is the same as for Cy3-DNA10 and Cy5-DNA10 systems (e.g. the neighbor bp to the dye is still designated as C1:G20). The starting structures of the DNA decamers and pentamers for the MD and MTD simulations, respectively, were built in the canonical B-DNA conformation using the NAB program (Amber Tools 13 program package). The dyes were covalently attached to the 5’ end of C1. The starting conformations of the dyes with respect to the neighboring C:G bp were based on the reference structures which were determined as the average structures from the constrained molecular dynamics calculations reproducing experimental NMR data.7,8 For DFT-D optimizations and single point energy calculations the smallest system was used. It consisted of the Cy3 dye, C1 cytidine and the guanine base of G20 and was designated as the Cy3-C:G system (Figure 2).

Figure 2: The top view of the Cy3-DNA10 system which is shown in the ribbon representation except the heavy atoms of the Cy3:GC system that are represented by the ball and stick model. The Cy5-DNA10 system is the same except the structure of the attached dye (cf. Figure 1). 5 ACS Paragon Plus Environment


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MD. All MD simulations were performed by the AMBER 10 program package.27 The parmbsc0 modification28 of the parm99 force field29 for DNA was adopted, together with the explicit TIP3P water model and standard parm99 parameterization for nineteen Na+ and one Clions which were added to neutralize the system electrostatically. The low ionic strength of ~ 0.1 M roughly corresponds to conditions in most experiments.8,10,11,13,15,19–21 For the Cy3 and Cy5 dyes standard gaff force field parameters were employed with exception of the torsional angles of PML’s which were parametrized by high level ab initio calculations (text in the Supporting Information, Figure S2 and Table S1). The RESP charges for the cyanine dyes were derived from the molecular electrostatic potential using the HF/6-31G* level of computation performed by the Gaussian 09 (G09) program,30 in connection with the Antechamber module of AMBER 10 (Tables S2 and S3). An equilibration protocol consisted of the series of energy minimizations and short constrained MD runs using an NVT ensemble. It was followed by the production run using an NPT ensemble at 298 K and the pressure of 101325 Pa to obtain volumetric mass density of 1g/cm3. Periodic boundary conditions, the Particle Mesh Ewald method to treat long-range interactions and SHAKE on hydrogen atoms (2 fs timestep) were used. Each trajectory was 150 ns long and 150 000 structures in 1 ps time intervals were saved for data analyses. Essential dynamics. The analyses of the motions of the cyanine dyes with respect to DNA were extracted by the g_covar and g_anaeig programs (parts of Gromacs program package31).

MTD. The MTD simulations were performed with the same force field as in the unbiased MD calculations but using the Gromacs 4.6.3 program package31 with the Plumed 2.0.2 plug-in.32 The Cy3-DNA5 and Cy5-DNA5 systems were solvated by explicit TIP3P water molecules and 1 6 ACS Paragon Plus Environment

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chloride and 9 sodium ions were added. The equilibration protocol was exactly the same as for MD (see above). The 680 ns long production run employed well-tempered MTD with the bias potential acting on two collective variables representing stacking and Debye-Hückel electrostatic interaction energy (DHEIE). Stacking was defined by the coordination number (CN): N C::( G ) N Cy ( x )

S stack =

1 − (rij / r0 ) 6

∑ ∑ 1 − (r i

j

ij

(1)

/ r0 )12

where the reference distance ‫ݎ‬଴ for stacking was set to 4 Å,33 N C :( G ) ran over the heavy atoms of the C1 and G20 bases, N Cy ( x ) ran over the heavy atoms of either Cy3 or Cy5 dye excluding the atoms of the linker between the dye and DNA. rij denoted the distance between the interacting atoms i and j. DHEIE34 between the dye and the DNA pentamer was calculated as:

S DH

1 = k B Tε w

N 5 DNA N Cy ( x )

∑ ∑ qq i

i

j

e j

−κrij

rij

(2)

where κ was the Debye-Hückel parameter, ε w relative permittivity of water (80) and qi , q j were partial atomic charges. Both summations went over all atoms of the interacting systems. The hills of the bias potential were added every 1 ps, giving 680,000 hills in total for every simulation. With LINCS algorithm used to constrain covalent bonds with hydrogen atoms, simulation step was set to 2 fs and temperature of the system to 298 K. Results were analyzed using VMD graphic interface35 and Gromacs analysis tools.

DFT-D. The geometries of the Cy3-C:G residues (Figure 2) were extracted from the representative structures of the conformers found by the MD simulations (see below). The structures were fully optimized by the RI-TPSS-D3/def2-TZVP/COSMO method with a tight



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converge maximum norm of cartesian gradient of 10-4 a.u. and the energy convergence criterion of 10-8 hartree. The method comprised the TPSS functional36 including the resolution of identity (RI) approximation of the Coulomb integrals, the empirical dispersion term for the main group elements,37 and the COSMO continuum solvation model with the cavities constructed based on the Klamt’s atomic radii38 and water as the medium. The Coulomb potentials of all elements were approximated by auxiliary basis sets developed by Weigend.39 The nature of the obtained stationary points was always checked by a numeric evaluation of the Hessian matrix. Calculated frequencies were corrected by a default scaling factor of 0.9914. Thermal contributions to the energetic properties were calculated using the canonical ensemble at standard gas-phase conditions (T = 298 K, p = 101.325 kPa). These calculations were performed by the Turbomole 6.4 program package.40 Relative energies of the optimized structures were obtained by the TPSS-D3/ccpVQZ/IEFPCM/UFF single point calculations. These calculations were carried out by the G09 program30 with the Integral Equation Formalism-PCM solvent model (IEFPCM and UFF scaled radii for cavity construction). Wavefunction .wfx files were used for calculations of reduced density gradients (RDGs) and analyzed by the program Multiwfn.41

Results: MD. Clustering of the structures. We were interested mainly in the conformational changes of the dyes with respect to the neighboring C1:G20 bp. All attempts to group the structures by standard clustering techniques available in the AmberTools 13 software failed. It may be the consequence of smooth transitions between different conformers (see below). Thus, at first the structures were grouped on the base of the distances between the mass-weighted centers of the G20, C1 rings and 8 ACS Paragon Plus Environment

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the three parts of the cyanine dyes molecules (as depicted in Figure 1): distal and proximal indole rings (DIR and PIR, respectively) and the polymethine linker (PML). For some structures a more detailed analysis of distances was performed splitting the condensed rings of G20, DIR and PIR into two 6- and 5-membered rings. All unstacked structures were excluded from the next analyses. The cyanine dye was considered as unstacked from the C1:G20 bp when all distances between the mass weighted centers of DIR, PML, PIR of the dye and the ones of G20, C1 of the bp were longer than 5.0 Å. Only 0.3% and 1.0% of the structures fulfilled this condition for the Cy3-DNA10 and Cy5-DNA10 complexes, respectively. The longest time intervals when the Cy3 and Cy5 dyes remained unstacked were 60 ps and 160 ps long, respectively. In the next step the two indole rings of the Cy3 and Cy5 dyes were transformed into a pair of purine rings and the dye molecule was treated by the X3DNA program42 as an additional A:A bp (Figure S3). The relative orientation of the dyes with respect to the C1:G20 bp was described by six base-pair step parameters. Mainly Slide, Shift, Twist and Tilt were found to be important structural parameters while the values of Rise and Roll were much less dependent on the conformer structure. Taking together the distances between mass-weighted centers and the base-pair step parameters we could define nine and seven clusters of the structures (conformers) for Cy3-DNA10 and Cy5DNA10 complexes, respectively, with occurrence higher than 0.1%. They cover more than 98% of the analyzed structures for both complexes. The means of the base-pair step parameters of these clusters are summarized in Tables S4 and S5 (Supporting information) for Cy3- and Cy5-DNA10 complexes, respectively. The slide, shift and twist values are shown graphically in Figure 3.



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Figure 3: Slide and Shift values (A & C) and fluctuations of the Twist values during the MD run (B & D) for Cy3-DNA10 (A & B) and Cy5-DNA10 (C & D). Coloring of points is done according the conformer: A & B: blue 3A, red 3B; black 3C; green 3D, orange 3E, magenta 3F, cyan 3G, brown 3H, yellow 3I. C & D: blue 5A, red 5B; green 5C; brown 5D, cyan 5E, yellow 5F, black 5G.

Cy3-DNA10. In agreement with previous experimental evidence4,7,9 the position of the Cy3 dye is rather flexible with respect to DNA. All possible combinations of the stacking interactions between DIR and PIR of Cy3 on one side and G20 and C1 rings of DNA on the other side were observed. DIR preferably interacted with G20 while PIR with C1. Interactions of the C1:G20 bp with DIR and PIR were involved in 81.0% and 2.6% of structures, respectively. All nine conformers are shown in Figure 4.


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Figure 4: The structural alignment of the Cy3-DNA conformers 3A-J done with respect to the bases of the C1:G20 bp (black licorice). The ribose, the phosphate and the linker are represented by a wire model while the rest of the Cy3 dye residue by a licorice model. The hydrogen atoms are not shown for clarity. The conformers are distinguished by different coloring. 3A conformer (blue color: probability 80.2%) is shown in all three parts of the Figure as a reference structure. A: 3B (red: 12.9%); 3F (pink: 0.8%); 3E (orange: 0.8%). B: 3C (grey: 2.0%); 3G (cyan: 0.6%); 3H (brown: 0.2%). C: 3D (green: 1.8%); 3I (yellow: 0.2%); 3J (tan: not sampled in MD but existed as a minimum of FES in MTD- see below).

In the most abundant conformer 3A the Cy3 dye is placed in parallel above the C1:G20 bp and is stabilized by the stacking interactions between G20 and DIR. These structures correspond to previously derived structures based on the NMR experiments and subsequent restrained molecular dynamics calculations.7,8 In the second most abundant binding motif representing 15.0% of the structures the two indole rings of Cy3 are not stacked and only the delocalized electrons of PML are involved in the dispersion interactions with the C1:G20 bp (see below). Cy3 interacts only through PML mostly with C1 (conformer 3B) and to lesser extent with G20 (3C). 3B is the most flexible conformer



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and can be considered as a transient structure from which transitions to all other conformers as well as to unstacked structures were observed. Considering 3A as the starting structure the analysis of the transitions between the conformers revealed that the movements of the Cy3 dye with respect to the C1:G20 bp can be summarized in following four movements: i) The anti-clockwise rotational- translational motion of the Cy3 dye in the direction 3A → 3B → 3F → 3E (Figure 4A) connected with a steep increase of the slide, shift and twist values (Figure 3 and Table S4). It leads to changes of the stacking interactions: in 3A DIR is stacked over G20; in 3B both indole rings of the Cy3 dye are fully unstacked; in 3F and 3E PIR is stacked with C1 and G20, respectively. ii) The sliding motion of the Cy3 dye along the long axis of the C1:G20 bp which forms the 3D structure with DIR-C1 interaction. In the opposite direction this motion leads to the 3I structure (Figure 4C) mainly decreasing the value of slide. If this motion continues with larger amplitude it would probably lead to the 3J conformer which was not sampled by MD but it was found as a minimum on FES in the MTD simulation (see below). iii) The translational motion of the Cy3 dye along the short axis of the C1:G20 bp which leads to conformation 3G. It shows the same interactions as 3A but G20-DIR stacking is weakened and 3G has the higher value of shift (Figure 4B). iv) The complex translational motion along the long axis of Cy3 connected with the large increase of slide and moderate increase of shift and twist. In resulting 3C PML is placed roughly along the short axis of C1:G20 (Figure 4B). In the next step 3H may be formed from 3C by a slight increase of shift but this time twist has to be decreased back to the value of 3A. Thus, 3H lies lengthwise to 3A but is stabilized by C1-PIR stacking.


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The essential dynamics analysis was performed to see the movements of Cy3 in the context of the whole Cy3-DNA10 system. The main conformational changes of the cyanine dyes were coupled with the thermal motions of the DNA chain, mainly with modes 4, 5, 6, 8, 10, 12. All these modes were connected with the changes of the widths of the major and minor grooves by the global twisting movements.43 On the other hand global tilting movements (modes 7, 9, 11) did not change significantly the position of the Cy3 dye with respect to the neighbor C1:G20 bp. These movements could be documented by casual close contacts between the C3OH group of Cy3 and 5’CH2 group of the C2 nucleotide. The normal mode analysis of the truncated Cy3-C:G system (Figure 1) confirmed the existence of the motions discussed above. Here, the third mode represented the rotational motion of Cy3 over C1:G20 which led to the unstacking of the indole rings (equivalent to the motion i). The fourth mode showed the sliding motion of DIR over C1:G20 (motion ii). The fifth mode reflected deformations of the sugar-phosphate backbone of both strands which led to a shifting motion of DIR over G20 (motion iii). The next three modes (6-8) represented motions of G20, the last one being connected with the changes of tilt values between Cy3 and C1:G20. The modes 9 and 10 showed motions of the free C3OH side chain.

Cy5-DNA10. In the prevailing 5A conformer the interacting DIR and G20 rings were placed on exactly the same relative positions with respect to each other as in 3A. Similarly to Cy3-DNA10, the movements of the Cy5 dye could be described as the thermal motions starting from 5A. Calculated entropies with respect to atoms of DIR showed a similar flexibility of Cy5 compared to Cy3. Thus, the lower number of the conformers of the Cy5-DNA10 complex found by MD can be explained only by mechanical and geometrical reasons which come from the longer PML of Cy5 compared to Cy3. Starting from 5A a larger displacement was required to establish PIR-G:C 13 ACS Paragon Plus Environment


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interactions compared to 3A and structures with these binding motifs were less frequent (1.92% and 0.01% for Cy3 and Cy5, respectively). The MTD simulation showed the conformers with PIR-G:C interactions existed as local minima on the FES (see below and 5H and 5I in Figure 5).

Figure 5: The structural alignment of the Cy5-DNA conformers 5A-J done with respect to the bases of the C1:G20 bp (black licorice). The ribose, the phosphate and the linker are represented by a wire model while the rest of the Cy5 dye by a licorice model. The hydrogen atoms are not shown for clarity. The conformers are distinguished by different coloring. 5A conformer is shown in all three parts of the Figure as a reference structure (blue color: probability 76.4%). A: 5B (red: 9.7%); 5D (tan: 1.8%). B: 5C (green: 6.0%); 5E (cyan: 1.8%); 5F (yellow: 1.3%); 5G (iceblue: 1.1%). C: conformers which were not sampled in MD but are local minima on FES (cf. Figure 7): 5H (orange); 5I (magenta)

Influence of the dyes on the DNA structure and flexibility. At first we compared the means of the base-pair and step base-pair parameters of the DNA decamer in the three systems, Cy3DNA10, Cy5-DNA10 and bare DNA. The influence of both dyes on the DNA structure was very similar. The base pair parameters of the first C1:G20 bp were changed at most with the 14 ACS Paragon Plus Environment

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differences up to 0.6 Å (Shear for Cy3-DNA10 vs. bare DNA) and 14.4° (Buckle for Cy5-DNA10 vs. bare DNA). The other base-pair parameters of the C1:G20 bp were affected less: up to 0.3 Å (Cy5-DNA vs. DNA), 9° (Cy3-DNA vs. DNA) and -4° (Cy3-DNA & Cy5-DNA vs. DNA) for stagger, propeller and opening, respectively. Only the parameters of the first base pair step were influenced significantly by the presence of the dyes decreasing values of shift, rise, tilt, roll and twist by up to 0.3 Å (Cy5-DNA vs. DNA), 0.2 Å (Cy5-DNA vs. DNA), 3° (Cy3-DNA vs. DNA), 4° (Cy3-DNA vs. DNA) and 3° (Cy3DNA vs. DNA), respectively, and increasing the value of slide up to 0.2 Å (Cy3-DNA vs. DNA). There were no systematic changes of parameters which would span deeper to the helix except the decreases of roll20 and tilt values for the first three base-pair steps. However, starting from the second base-pair step the differences were smaller than 1°. The same was true for random fluctuations of the other helical parameters whose changes did not mostly exceed 0.1 Å and 1° in agreement with previous simulations of the QSY 21-DNA and Rhodamine 6G-DNA systems.44 In fact in our simulations this was true only up to T8:A13 bp since the parameters of the last two bps on the free unlabeled end were strongly affected by the opening events of the G10-C11 bp in the Cy5-DNA10 and bare DNA systems. Entropies were calculated by the Schlitter formula45 which works with fluctuations in Cartesian coordinates and provides a quantitative measure of the conformational freedom of the system moving in a quasi-harmonic regime. It also enables to evaluate the components of total configurational entropy related to the defined parts of the molecule or the system.46 To see the influence of the dyes on the flexibility of DNA we compared the entropies of individual bps along both dye-labeled DNA chains. Note that the same reference system had to be defined for all bps to perform a direct numerical comparison of calculated entropies. 32 heavy atoms were chosen for both A:T and C:G bps (Figure 6). The side chain atoms of the purine and pyrimidine 15 ACS Paragon Plus Environment


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rings (except O2) and atoms of the phosphate groups were not considered for entropy calculations. The bps from C1:G20 to T8:A13 showed rather similar entropies for both Cy3-DNA10 and Cy5-DNA10 systems (Figure 6). The two simulations differ for the last two G9:C12 and G10:C11 bps on the unlabeled free end. The Cy5-DNA10 system showed much higher values due the opening of the last G10:C11 bp in the last 5 ns of the simulation. For the Cy3-DNA10 system in which no opening events occurred the increase of the flexibility of two terminal bps was 5% and 24% for the next-to-last G9:C12 and the last G10:C11 bps, respectively, compared to the internal C4:G17, G7:C14 bps. Entropy of internal A:T and T:A bps was by ~6% higher compared to internal G:C and C:G bps and reflected the difference in the base pairs rigidity. The bound Cy3 and Cy5 dyes prevented fraying of the DNA end. For a palindromic sequence the influence of the dye on the DNA flexibility can be most easily resolved as the difference between the entropy values of the bps on the labeled and unlabeled ends. For Cy3-DNA10 the dye decreases flexibility only of the neighbor bp C1:G20 by ~ 6% (with respect to the unlabeled G10:C11). In case the opening event(s) occurred (as in the Cy5-DNA10 simulation) then the stabilization effect of the dye was much larger: the last G10:C11 and the next-to-last G9:C12 bps were affected showing the entropy decrease by ~ 33% and ~ 15%, respectively, compared to the unlabeled end.


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Figure 6: Entropies of the bps calculated by the Schlitter formula.45 Calculations were performed on 32 atoms of each bp (A/G: N1, C2, N3, C4, C5, C6, N7, C8, N9; T/C: N1, C2, O2, N3, C4, C5, C6; ribose: C1’, C2’, C3’, O3’, C4’, O4’, C5’, O5’)

MTD. The attached cyanine dyes stabilized the double helix by stacking and also by electrostatic interactions as the outcome of the +1 charge of the dyes. Two-dimensional MTD simulations were performed for each dye with the bias potential acting on the stacking and electrostatic potential as collective variables. The FESs interactions between the dyes and DNA were explored and showed an enhanced sampling of the binding motifs compared to the MD simulations. MTD covered stacking, unstacking structures as well as for Cy3-DNA5 complex also the structures with major groove binding of the dye. Figure 7A shows FES of the Cy3 dye’s interactions with DNA. The absolute minimum corresponds to the 3A conformer and has free energy of -8.3 kcal/mol. The second most stable minimum with the same CN but larger DHEIE belongs also to the 3A conformer but shows closer C3OH-phosphate contacts (Figure S4) and here it is designated as 3Ab. The conformer 3J was not sampled in the MD run. It shows the G20-PIR stacking interactions but unlike the 3E conformer the dye lies almost in parallel with the long axis of the C1:G20 bp (Figure 4).



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Figure 7: Free energy surfaces (in kcal/mol) of MTD simulations for interactions of the Cy3 (A) and Cy5 (B) dyes with DNA. The proposed minimum free energy paths for unstacking are shown by the black discontinuous lines. Positions of the local minima which are discussed in the text are designated by the labels. Contour lines represent the free energy difference of 1 kcal/mol.

Starting from the stacked structures the minimum free energy paths for unstacking is connected with a large change of CN representing the stacking interactions (Figure 7). In the group of structures designated as 3U (Figures 7 and 8) the dye was already unstacked with free energy ~ -2.1 kcal/mol. It included different structures, some of them still preserved interactions between the dye and the C1:G20 bp (mainly CH(PIR) - π(C1) interaction), the other showed initial binding of the dye to the major groove (Figure 8). The unstacking energy of ~ 6.2 kcal/mol corresponds to the free energy barrier between the stacked structures and structures with the major grove binding. It is lower compared to the value of 8.4 kcal/mol which was determined by Spiriti et al. using umbrella sampling simulations.20 In agreement with the cited study electrostatics play a minor role in the unstacking since DHEIE shows a smooth decrease by ~ 1.3 kcal/mol. However, it can be even less since FES is very flat in this region (Figure 7).


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Figure 8: A: The group of structures corresponding to the region 3U on FES of the Cy3-DNA5 complex (Figure 7A). It represents the transition between the structures with the CH(PIR)-π(C1) interactions and the structures with Cy3 bound in the major groove. B: The lowest energy structures with Cy3 bound in the major groove (minimum 3V in Figure 7) which show C3OHphosphate (phosphate groups of C1, C2 and A3 nucleotides) H-bond interactions. Geometries of the Cy3 dye are represented by the wire model and structures are aligned with respect to the average structure of the CCACC duplex which is shown by the licorice model.

Starting from 3U the electrostatic interactions might be strengthened substantially while keeping a low CN value which lead to the structures with major groove binding of the dye to DNA. The most stable structures (designated as the minimum 3V in Figure 7) have free energy ~ -4.7 kcal/mol and show tight contacts between C3OH of the dye and the sugar-phosphate backbone of the CCACC strand (Figure 8). It was shown that a high salt content (~1.0 M) influenced binding energies of the cationic methylene blue dye with DNA compared to the low ionic strength solution.47 In our case a salt addition should lead to a weakening of the major groove binding with the dominant contribution of electrostatic interactions while the stability of the stacked structures should be much less affected.

The absolute minimum on FES of the Cy5-DNA5 complex has energy of -7.8 kcal/mol and corresponds to the 5A conformer. The FES shows two other local minima of the stacked structures which were not sampled by MD: the conformers 5H and 5I (Figures 5C and 7B) are



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stabilized by the PIR-C1:G20 stacking interactions and can be considered as the structural analogs of 3E and 3F Cy3-DNA conformers (cf. Figure 4A), respectively. A structural analog to the 3Ab conformer (see above and Figure S4) was not found for the Cy5 dye probably due to decreased flexibility of the linker when DIR is stacked over G20. Unstacking proceeds similarly to Cy3 decreasing sharply CN while DHEIE is decreased moderately. Since the structures with major groove binding of the dye were not found on FES we proposed a straightforward minimum free energy path for the unstacking which supposed a gradual decrease of CN down to zero (Figure 7B). The maximum free energy along this path is ~ -1.6 kcal/mol and the resulting unstacking free energy is ~ 6.2 kcal/mol. The two local minima 5X and 5Y correspond to structures with disrupted C1:G20 bp and G20 ring is flipped away towards the minor groove. The dye is either stacked over C1 and C2 which lie almost in one plane (Figure S5A) or it is intercalated between C1 and C2 base rings with possible stacking with G19 (Figure S5B and S5C).

DFT-D. The stacking interactions can be properly described only by the quantum chemical methods with correct description of dispersion interactions. The DFT-D methods were chosen for our calculations since they represent a reasonable compromise between the cost and the accuracy. Due to computational cost we focused only on the Cy3-C:G system (Figure 1). We performed 33 optimizations which started from the representative MD structures and covered all nine conformers 3A - 3I observed in the MD run and also the 3J conformer which was found as the FES minimum by MTD. The free valences on the N9 and O3’ atoms of G20 and C1 nucleotides, respectively, were filled by the hydrogen atoms. The resulting O3’-H and N9-H groups might form H-bonds which 20 ACS Paragon Plus Environment

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could not be possible in the maternal Cy3-DNA10 complex. This happened during optimizations of five complexes and these were not considered for further analyses. Three optimizations led to the lowest DFT energy structure which is representative of the 3A conformer (see below) and two pairs of optimizations led to the same local minima. Thus, 25 different structures were optimized. Since the starting structures for particular conformers were not geometrically distinct and still might lead to different minima we suggest the existence of the high number of local minima on the potential energy surface separated by low energy barriers.48 The optimized structures may represent only local minima of the conformers. However, our goal was to confirm the existence of the conformers described by MD and to give more insight into the nature of the binding between the Cy3 dye and the neighbor C1:G20 bp. We were able to find a local minimum for all conformers except 3D for which the DIR-C1 interaction was always replaced by PML-C1 and DIR-G20 interactions characteristic for 3G. The decrease of importance of stacking interaction with C1 at the expense of G20 could be also observed in 3B -> 3C transitions during a few optimizations and by a higher stability of 3C conformers compared to 3B (see below). Relative DFT energies of optimized structures of the MD 3A - 3I conformers were within 7.4 kcal/mol. The most stable 3A conformer is stabilized by G20-DIR interactions and by the internal C3OH-O4’ H-bond (Figure 9). The second most stable 3C conformer shows PML-C1, CHπ(G20) dispersion interactions and C3OH-O4’ H-bond (Figure 9) and have relative DFT energy 1.8 kcal/mol. The other conformers are stabilized only by stacking/dispersion interactions. Conformers 3B, 3F and 3I are almost equi-energetic with relative energies in the range of 4.4 4.6 kcal/mol. After inclusion of thermal contributions to the DFT energies the maximum difference between the conformers was smaller (~ 6.6 kcal/mol) and the order of the most stable conformers 3C, 3A 21 ACS Paragon Plus Environment


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and 3B changed with relative free energies -0.4; 0.0 and +1.3 kcal/mol, respectively. Note that these numbers should be taken with some caution since use of ideal gas-phase approximation is not completely consistent for the solvent-optimized structures.

Figure 9: Reduced density gradient isosurface maps with isovalue of 0.5 of the most stable RITPSS-D optimized structures of conformers 3A, 3B and 3C. The value of sign(λ2(r))ρ(r) in isosurfaces is represented by filling color. Stabilization of the system by weak dispersion interactions, by internal H-bonds and destabilization by steric ring repulsion are represented by green, blue and red isosurfaces, respectively. Calculations of the isosurface maps were performed by the MultiWFN program41 and results visualized by VMD.

Discussion: We present a combined theoretical study of the conformational behavior of the cyanine Cy3 and Cy5 dyes with respect to DNA when bound covalently to the terminal C:G bp. The 150 ns MD runs explored dynamics of the stacked structures. They were followed by the 680 ns MTD runs to sample rare minima, major groove binding and unstacked structures. In agreement with previous studies4,20 the position of both dyes is rather flexible with respect to DNA. The most probable (MD) and stable (MTD) 3A and 5A conformers correspond to the 22 ACS Paragon Plus Environment

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previously published structures (rmsd ~ 1.0 Å).7,8 For the Cy3-DNA10 complex the existence of most of the conformers were confirmed by full DFT-D optimizations. Thus, the combination of modified gaff and Amber force fields should provide reasonable geometries of the cyanine-DNA complexes. The calculated unstacking energy for Cy3-DNA of ~ 6.2 kcal/mol is lower than previously published value of 8.4 kcal/mol.20 It may reflect differences between the Amber-gaff and Charmm force fields. Our value corresponds to the barrier between the stacked structures and the ones with Cy3 bound in the major groove. If a simple straightforward free energy path is considered as for Cy5-DNA (cf. the two free energy paths in Figure 7) then the unstacking energy for Cy3-DNA would be ~6.6 kcal/mol. Thus, the unstacking energy for Cy3-DNA could be higher by up to 0.4 kcal/mol than for Cy5-DNA since 3U has higher value of CN compared to the reference unstacked structure for the Cy5-DNA free energy path. The DFT-D calculations showed the maximum difference of 6.6 kcal/mol in Gibbs free energies between the stacked conformers of the Cy3-G:C complex. However, they suffered by a poor sampling and problems with the exact evaluation of thermal contributions. Entropy seems to play an important role in cyanine-DNA stacking interactions. On one hand it decreases flexibility of up to two neighbor bps, on the other the entropy term may counterbalance the enthalpy loss connected with the disruption of the DIR:G20 stacking interaction. In the 3A → 3B transition the stacking is replaced by weaker but more flexible PML-C1:G20 dispersion interactions and excellent agreement between MD and DFT-D methods was achieved calculating the 1-2 kcal/mol free energy increase. Discrepancy between the two methods still exists for 3A and 3C conformers since MD predicted that 3A is more stable than 3C by ~ 2.2 kcal/mol (cf. probabilities in Figure 4) while DFT-D showed an opposite trend: 3C was by 0.4 kcal/mol more stable. In our opinion considering the precision of the current force fields and the problems with 23 ACS Paragon Plus Environment


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DFT-D calculations which are discussed above, the difference of 2.6 kcal/mol is within the experimental error. Some improvements of our results could be reached only by quantum mechanical MD simulations at least tens of nanoseconds long which are currently not feasible.

Conclusions: The MD and MTD calculations were performed to study behavior of the Cy3 and Cy5 dyes covalently attached via the three carbon linker to the 5’C end of the DNA oligonucleotide. Torsional force field parameters of PMLs were derived for both dyes using the highly correlated ab initio MP4 method. The prevailing binding motif included DIR-G interaction but frequent transitions between the conformers connected with the changes of the nature of stacking interactions were observed. For Cy3-DNA10 the MD simulation sampled also the conformers with PIR-G:C interactions while it was not the case for Cy5-DNA10 due to longer PML of the dye although these conformers existed as the local minima on FES in MTD. With ~10% probability the interactions between the dye and the G:C base pair were mediated only through the dispersion interactions of PML. For Cy3 the existence of eight from nine MD conformers was confirmed by full RI-DFT-D/COSMO optimizations. The calculated unstacking energies were ~6.2 kcal/mol for both the Cy3-DNA5 and Cy5-DNA5 complexes. For Cy3-DNA complex the structures with major groove binding of the dye were also sampled and were by 3.6 kcal/mol less stable than the stacked ones. Our results show a positive effect of the Cy3 and Cy5 dyes attached to the 5`C end on the stability of the resulting duplex, which should be taken into account in accurate calculations of hybridization probes labeled with these fluorescent dyes.


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Supporting Information Available: Description of the force field parameterization for the torsions of the dyes’ PMLs, Figures S1-S5, Tables S1-S5, full references 27 and 30. This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgements: This project was supported by the grant from the Czech Science Foundation (project No. 208/12/0622). The access to the MetaCentrum (grant LM2010005) and CERIT-SC (grant CZ. 1.05/3.2.00/08.0144) computing and storage facilities is greatly appreciated. We are grateful to David G. Norman from The University of Dundee for providing us the Cartesian coordinates of the Cy3-DNA-Cy5 complex. We also thank to Martin Zacharias and Mahmut Kara from TU Munich and to Vojtěch Spiwok from ICT Prague for valuable discussions about free energy calculations.

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Cy3 and Cy5 Dyes Terminally Attached to 5â²C End of DNA: Structure

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Cy3 and Cy5 Dyes Terminally Attached to 5â²C End of DNA: Structure