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Conformational Dynamics of Cyanocobalamin and Its Conjugates with Peptide Nucleic Acids Tomasz Pie#ko, Aleksandra J. Wierzba, Monika Wojciechowska, Dorota Gryko, and Joanna Trylska J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b00649 • Publication Date (Web): 16 Mar 2017 Downloaded from http://pubs.acs.org on March 25, 2017

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The Journal of Physical Chemistry

Conformational Dynamics of Cyanocobalamin and Its Conjugates with Peptide Nucleic Acids Tomasz Pieńko,†,‡ Aleksandra J. Wierzba,¶ Monika Wojciechowska,† Dorota Gryko,∗,¶ and Joanna Trylska∗,† Centre of New Technologies, University of Warsaw, S. Banacha 2c, 02-097 Warsaw, Poland, Department of Drug Chemistry, Faculty of Pharmacy with the Laboratory Medicine Division, Medical University of Warsaw, S. Banacha 1a, 02-097 Warsaw, Poland, and Institute of Organic Chemistry, Polish Academy of Sciences, M. Kasprzaka 44/52, 01-224 Warsaw, Poland E-mail: [email protected]; [email protected]

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To whom correspondence should be addressed Centre of New Technologies, University of Warsaw, S. Banacha 2c, 02-097 Warsaw, Poland ‡ Department of Drug Chemistry, Faculty of Pharmacy with the Laboratory Medicine Division, Medical University of Warsaw, S. Banacha 1a, 02-097 Warsaw, Poland ¶ Institute of Organic Chemistry, Polish Academy of Sciences, M. Kasprzaka 44/52, 01-224 Warsaw, Poland †

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Abstract Vitamin B12 also called cobalamin (Cbl) is an important enzymatic co-factor taken up by mammalian and also by many bacterial cells. Peptide nucleic acid (PNA) is a synthetic DNA analogue that has the ability to bind in a complementary manner to natural nucleic acids. Provided that PNA is efficiently delivered to cells, it could act as a steric blocker of functional DNA or RNA and regulate gene expression at the level of transcription or translation. Recently, Cbl has been examined as a transporter of various molecules to cells. Also, PNA if covalently linked with Cbl, can be delivered to bacterial cells but it is crucial to verify that Cbl does not change the desired PNA biological properties. We have analyzed the structure and conformational dynamics of conjugates of Cbl with a PNA monomer and oligomer. We synthesized a cyanocobalamin derivative with a PNA monomer C connected via the triazole linker and determined its NMR spectra. Using microsecond-long molecular dynamics simulations, we examined the internal dynamics of cyanocobalamin-C, its conjugate with a 14-mer PNA, and free PNA. The results suggest that all compounds acquire rather compact structures but the PNA oligomer conformations vary. For the Cbl-C conjugate the cross-peaks from the ROESY spectrum corroborated with the clusters from molecular dynamics trajectories. Within PNA the dominant interaction is stacking but the stacking bases are not necessarily neighboring in the PNA sequence. More bases stack in free PNA than in PNA of the conjugate but stacking is less stable in free PNA. PNA in the conjugate is slightly more exposed to solvent. Overall, cyanocobalamin attached to a PNA oligomer increases the flexibility of PNA in a way that could be beneficial for its hybridization with natural nucleic acid oligomers.

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Introduction Currently used antibiotics inhibit the synthesis of bacterial cell wall (β-lactams or glycopeptides), suppress bacterial protein synthesis (e.g. aminoglycosides, macrolides) or hinder nucleic acid synthesis (e.g. fluoroquinolones). 1 Another possible strategy to target pathogens is based on silencing the expression of essential genes with oligonucleotides complementary to bacterial DNA or RNA. 2–4 Since natural oligonucleotides are not biostable their synthetic analogues have to be used. Several modified oligonucleotides such as phosphorothioates, 2’O-methyl and 2’-O-methoxyethyl oligonucleotides, locked nucleic acids, phosphorodiamidate morpholino oligonucleotides, and peptide nucleic acids (PNAs) have been tested as antibacterials. 3 Their functionality is based on an antisense mechanism in which they bind to a specific mRNA fragment to prevent translation or an antigene mechanism in which they bind to DNA to preclude gene transcription. Peptide nucleic acids (PNAs) were first synthesized by Nielsen and co-workers in 1990s. 5 PNA is an analog of natural nucleic acids with the backbone composed of neutral N-(2aminoethyl)glycine units linked by peptide bonds. Nucleobases are connected to the backbone by methylene carbonyl groups. The notation for PNA sequences is thus the same as for peptides; from the N to C terminus. A similar pattern of spatial positioning of nucleobases in PNA as in natural nucleic acids makes PNA capable of pairing with DNA or RNA obeying the Watson-Crick rules. 6 Moreover, duplexes involving PNA are more thermally stable than duplexes of natural nucleic acids. Any single base mismatch between the bound strands in PNA-containing complexes decreases their stability. 7 Also, the neutral backbone of PNA allows it to efficiently hybridize with DNA or RNA at low salt concentrations. 8 In addition, PNA is stable in acidic environment. One of the drawbacks of PNA is that the uncharged backbone worsens its solubility in water (as compared to DNA or RNA), which causes sequence-dependent aggregation of PNA. PNA solubility in water is also affected by the length of an oligomer and the ratio of purines to pyrimidines. On the other hand PNA is not recognized by nucleases and proteases making it resistant to enzymatic cleavage and 3



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stable inside cells relative to natural nucleic acids. Whereas DNA or RNA based oligonucleotides can be delivered into eukaryotic cells through endocytosis, PNA is poorly transferred across the cell membrane. 9 Transport of PNA to prokaryotic cells is also negligible, however PNA membrane permeability may be augmented through incorporation of positively charged lysine or arginine. 10 Another way to deliver PNA to cells is its conjugation with cell penetrating peptides such as (KFF)3 K, 11 steroids 12 or antibodies. 13 The difficulty in efficient transport of PNA to bacterial cells remains the most limiting factor in the use of PNA in various applications pertaining to bacterial cells. To tackle this problem, we have recently employed vitamin B12 as a potential transporter of PNA to these cells. Vitamin B12 , also termed cobalamin (Cbl), is an important enzymatic cofactor for mammals and bacteria. Some microorganisms can synthesize cobalamin whereas delivery to mammals is through dietary intake. Vitamin B12 has been recognized as an efficient transporter of proteins, anticancer drugs, and radioisotopes to eukaryotic cells. 14–16 Therefore, it may be also successful in delivering PNA to some prokaryotic cells which need to uptake vitamin B12 from the environment. Indeed our experiments have shown that such uptake is possible (data not shown). To use cobalamin as a transporter of PNA to cells, a method of synthesis of cobalamin–PNA conjugates via click chemistry has been developed 17,18 but the structural dynamics of such conjugates has not been investigated. The solution structure of cyanocobalamin was determined by NMR restrained molecular dynamics. 19,20 More recently, a structural molecular dynamics study of vitamin B12 conjugated with an anorectic peptide PYY was reported. 21 The NMR and crystal structures of PNA-involving duplexes have been also determined but no structural data on PNA singlestranded oligomers has been reported. 22–24 MD simulations of PNA are scarce and were performed mainly for PNA–PNA and PNA–DNA duplexes 25,26 and for a PNA◦DNA◦PNA triplex. 27 Conformational dynamics of single-stranded PNA 28,29 and PNA with various modifications was also investigated. 30–33 However, the simulation time scale of these simulations

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Theoretical methods System building and force field Cbl crystal structure was used as a starting point. 34 The hydrogen atoms were added by leap (AmberTools13). 35 Partial atomic charges were computed using Gaussian 09 36 and antechamber (AmberTools13) following the RESP procedure 37 of the AMBER force field. The bonded and Lennard-Jones non-bonded parameters were adopted from the work of Marques et al. 38 Next, a 20 Å thick layer of pre-equilibrated water molecules around Cbl was added. The net charge of Cbl is zero so no neutralizing ions were necessary. Four simulations types were performed; Cbl surrounded by TIP3P 39 or SPC/E 40 water molecules and either no ions or Na+ and Cl− ions were added to obtain 100 mM NaCl concentration. For each type three 500 ns production simulations were performed which differed in the assignment of starting velocities. The root-mean-square-deviation (RMSD) and radius of gyration of Cbl heavy atoms relative to the starting crystal structure show similar performance for each conditions (Figure S1). For Cbl in the production phases the highest RMSD does not exceed 1.5 Å. In all further simulations for Cbl conjugates the SPC/E water model with 100 mM NaCl was used. The structure of Cbl conjugated to a cytosine PNA monomer (C), via an alkyl-type linker, was prepared in leap (AmberTools13). The linker was attached to the 5’ hydroxyl group of Cbl via the carbamate bond and to the C monomer via the triazole (Figure 1). Partial charges of the linker atoms were computed in the same way as for Cbl. The missing parameters were generated using the GAFF force field and antechamber. For the C monomer, the AMBER parameters of Shields et al. 27 were used. The Cbl-PNA conjugate with a sequence of Cbl-linker-CATCTAGTATTTCT-Lys-NH2 (N→C) and the corresponding free 14-mer PNA were next prepared. Lys is typically added to the PNA terminus to improve its solubility. The starting conformation of the PNA oligomer was built in an extended form. For free PNA, the C monomer was protonated 6


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thus the atomic charges were recomputed according to the RESP procedure. Parameters for Lys were from AMBER ff12SB and for PNA from. 27 Free PNA and Cbl-PNA systems were neutralized with either two or one chlorine ions, respectively. Systems were surrounded by a 20 Å layer of SPC/E water molecules and ions to achieve 100 mM NaCl.

Molecular dynamics simulations protocol The MD simulation protocol included energy minimization, thermalization, equilibration and production. Initially, the systems were energy minimized under harmonic restraints of 50 kcal/mol set on non-hydrogen atoms with 5000 steps of the steepest descent followed by 3000 steps of conjugate gradient method using the sander program. 35 Subsequent phases were carried out using NAMD 2.8. 41 Thermalization, in the NVT ensemble, consisted of gradual increase of temperature from 30 to 310 K with an increment of 20 K every 20 ps with harmonic restraints of 50 kcal/mol imposed on solute heavy atoms. Equilibration was carried out in the NpT ensemble with a constant pressure of 1 atm controlled using Langevin piston method and a constant temperature of 310 K regulated by the Langevin thermostat. Energy restraints on solute heavy atoms were gradually decreased from 50 to 1.25 kcal/mol in 500 ps and then removed. Production simulations were repeated three times for 500 ns for free Cbl and 1000 ns for all other systems (each started from the same coordinates but with different randomly set atomic velocities). Data were saved every 5 ps. Where applicable the analyses were performed on three combined trajectories. Periodic boundary conditions and Particle Mesh Ewald method with a grid spacing of 1.0 Å were used. The SHAKE algorithm was applied and the integration time step of 2 fs. For non-bonded interactions, a short-range cutoff of 12 Å was applied.

Data analysis Trajectories were analyzed with the cpptraj program (AmberTools13) and VMD 1.9.2 was used for visualization. Plots were generated with xmgrace and Gnuplot(v4.6). Clustering 7



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was performed on combined trajectories using kmeans algorithm. The initial number of clusters was identified by trial and error (between 2 to 10). The quality of clustering was evaluated by comparing DBI (Davies-Bouldin Index), which is a measure of the separation of clusters, and pSF (pseudo-F statistic), which estimates the tightness of clusters. In general, low DBI and high pSF values are desired so based on this fact the number of clusters was set to 3 for Cbl-C and 5 for Cbl-PNA and PNA. The π − π stacking contacts between PNA nucleobases were assessed by measuring the distances between the centers of masses of the bases (considering the maximum threshold of 6.5 Å). The criteria for hydrogen bonds were 3.2 Å between the donor and acceptor and no less than 150 degrees angle between donor-hydrogen-acceptor. Solvent accessible surface area (SASA) was calculated with the maximal speed molecular surfaces algorithm 42 and a probe radius of 1.4 Å.

Experimental methods Cbl-C synthesis The Cbl-C conjugate was synthesized via the azide-alkyne dipolar cycloaddition (AAC, "click" reaction) which is a powerful, bioorthogonal method for the preparation of complex molecular structures, polymers, peptide derivatives, conjugates etc. 43–46 The aromatic 1,4 triazole formed is a stable unit and due to its geometry and dipole moment can mimic a peptide bond. For this reason copper-catalyzed azide-alkyne cycloaddition (CuAAC) was chosen as a method for the conjugation of the PNA monomer 1 with Cbl at 5’ position (Scheme 1). The designed synthesis required preparation of the PNA alkyne and Cbl derived azide. To this end, suitably functionalized PNA monomer 1 on solid support was coupled with 4-pentynoic acid leading to compound 4 (Scheme 1). After cleavage from the resin alkyne 5 was obtained in a decent yield. The linker possessing the terminal azide functionality was attached to Cbl using the most 8


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and Varian 600 MHz spectrometers with TMS or the residual solvent peak used as an internal standard. High-resolution ESI mass spectra were recorded on Mariner and SYNAPT spectrometers. All reactions were monitored using RP-HPLC techniques. Preparative chromatography was performed using C18 reversed-phase silica gel 90 Å (Sigma-Aldrich) with redistilled water and HPLC grade MeCN as eluents. Product purity was determined using RP-HPLC and NMR spectroscopy techniques. HPLC measurement conditions were: column, Eurospher II 100-5, C18, 250 mm x 4.6 mm with a precolumn; detection, UV/vis; pressure, 10 MPa; temperature, 30 ◦ C; flow rate, 1 mL/min; wavelengths, and HPLC methods are given below.

Preparation of PNA monomer C-pentynoic acid derivative The C-pentynoic acid derivative (Scheme 1, 5) was synthesized manually by Fmoc chemistry on 0.12 mmol scale using 2.5 molar excess of the Fmoc/Bhoc protected monomer C, 3.0 molar excess of pentynoic acid and Tentagel resin (loading 0.024 mmol/g). Fmoc deprotection of the resin was carried with 20% piperidine in DMF (2 x for 5 min and 15 min). The monomer C and pentynoic acid were activated with the use of HATU (2.3 equiv.), NMM (2.5 equiv.) and lutidine (3.75 equiv.) mixture in DMF/NMP (1:1; v/v). Each coupling of the C monomer was performed twice for 30 min. Fmoc deprotection of the C monomer was performed with 20% of piperidine in DMF (2 x 2 min). After washing the resin (with DMF, DCM and again with DMF) pentynoic acid was coupled to the C monomer in the presence of coupling agents. Coupling was performed twice for 30 min. Deprotection and cleavage from the resin were carried out with the use of TFA/triisopropylsilane/m-cresol mixture (2.5 mL/31 µL/62 µL) in DCM (2.5 mL) for 30 min. The reaction mixture was then precipitated with Et2 O, centrifuged and the crude was subsequently lyophilized. Purification was performed with the use of semiprepartive RP-HPLC (Knauer C18 column 8 x 250 mm, 5 µm particle size; from 1% MeCN/H2O + 0.05% TFA to 70% MeCN/H2O + 0.05% TFA in 30 min). HRMS (ESI) m/z [M + Na]+ calcd for C15H20N6O4Na 371.1444, found 371.1457. tR (RP-HPLC, from 10


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1% MeCN/H2 O + 0.05 % TFA to 70% MeCN/H2O + 0.05% TFA in 15 min): 6.92 min (see Section S1).

Synthesis of the conjugate of Cbl with C-pentynoic acid derivative CuI (4 mg, 0.021 mmol) and TBTA (20 mg, 0.038) were stirred in DMF (2 mL) for 20 min and subsequently transferred into a solution of Cbl-5’-C6-N3 (115.5 mg, 0.076 mmol) and PNA monomer C-pentynoic acid derivative (26.4 mg, 0.076 mmol) in DMF (2 mL). Reaction progress was monitored by the RP-HPLC. When the substrate was consumed (approx. 16 h) the reaction mixture was poured into AcOEt (approx. 50 mL) and centrifuged. The precipitate was then washed twice with Et2 O and after drying, dissolved back in water (approx. 5 mL) and centrifuged. The supernatant was purified by RP column chromatography gradually with MeCN/H2 O (from 10 to 15% v/v). Fractions containing desired product (determined by RP-HPLC) were evaporated and characterized by NMR and MS techniques. HRMS (ESI) m/z [M + 2Na]2 + calcd for C85 H120 N24 O19 PCoNa 947.40981, found 947.40960. tR (RP-HPLC, from 1% MeCN/H2O + 0.05% TFA to 70% MeCN/H2 O + 0.05% TFA in 15 min): 9.68 min (see Section S1).

Results and Discussion Global measures from molecular dynamics simulations of Cbl conjugates and PNA To monitor the overall stability of the Cbl conjugates in MD trajectories, we calculated RMSD and radii of gyration of the solutes with respect to their starting structures (Figures S2 and S3). RMSD fluctuations suggest that the systems are internally flexible. The radius of gyration decreases in the solute equilibration phase, suggesting substantial conformational changes from the extended starting structure of PNA, but it levels after 30 ns. This decrease

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implies that especially PNA and Cbl-PNA prefer globular and compact structures in aqueous solution which corroborates polyacrylamide gel electrophoresis studies showing that singlestranded PNA form compact structures in water. 48,49 To compare solvent accessibility of the PNA oligomer in its free and conjugated forms, we calculated SASA of the PNA moiety in Cbl-PNA and PNA (Figure S4.) The first PNA unit was not considered in the calculations because it has a different number of atoms in Cbl-PNA and PNA (due to either its connection with the linker or PNA terminus, respectively). PNA in the conjugated form demonstrates a slightly larger SASA signifying that on average it is more exposed to solvent than free PNA. However, for the entire oligomer on average the difference is only about 60-70 Å2 .

Distributions of end-to-end distances of PNA and the linker To determine the flexibility of the PNA backbone and linker, we measured the distances between terminal atoms within the PNA and linker. Figure 2 shows the fraction of occurrence of these distances. The termini in free PNA sample a broader range of distances than the same terminal atoms in the conjugate with Cbl. For Cbl-PNA the profile is shifted toward larger distances in comparison to free PNA, and ranges from 3 to 45 Å. The most occupied, for almost 20% of simulation time, is the end-to-end distance around the 19 Å peak in the conjugated Cbl-PNA. The histograms of the distances between the linker endings in the Cbl-C and Cbl-PNA are similar, ranging from to 3 to 15 Å. Peaks showing maximal occupation (of about 30%) are at the distance of 10 Å for the linker in Cbl-C and 11 Å for the linker in Cbl-PNA. Endto-end distances show that both conjugating PNA to Cbl and extending the PNA sequence make the PNA and linker more extended in comparison with free PNA.

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Figure 3: Occurrence of dihedral angles in the free PNA and Cbl-PNA calculated from MD simulations. C1, A2, T3 etc. denote the type and order of the nucleobase in the PNA sequence. Four sequential N* atoms were selected to define each dihedral pseudo-angle in PNA (for the terminal lysine Cα (C*) was also included, Figure 1). Values for each angle (in each row) were normalized to 1. PNA. The occurrence of these angles is shown in Figure 4. A profile of occurrence of the R4 –OR8 –N64 –P3 pseudo-angle differs between Cbl-C and Cbl-PNA (Figure 1). In Cbl-C

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NMR assignments for Cbl-C and comparison with molecular dynamics simulations Based on various mono- and two dimensional NMR spectra (1 H,

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clear COSY, HMBC, HSQC) the complete assignment of 1 H and

C, homo- and heteronu13

C spectra of Cbl-C in

DMSO-d6 was performed (see whole Section S2). With these in hands, we attempted to identify the relative position of the PNA monomer with respect to the Cbl moiety. The ROESY spectrum provided valuable data though due to its high complexity, only well-separated signals served as a source of information. We were able to observe resonances correlating to Cbl and PNA fragments, respectively. There are clear cross peaks between the proton from P11 in cytosine aromatic ring and protons N63 , N52 and C46 in Cbl moiety suggesting interaction of Cbl and PNA moieties (Figure 5). This group (P11 ) has also NOE correlation to L2 , L4 and L5 . The alkyl spacer L3 resonances have NOE cross-peaks with R1 , Pr2 and N52 , and a weak correlation with a signal corresponding to P10 aromatic cytosine proton was also present, which in turn correlates to L2 , L4 and L5 in the alkyl spacer. The P9 group was found to interact with the same set of protons as the group P10 , moreover, the NOE cross-peak with P2 was also present in the spectrum. The L5 protons were found to couple with the ribose proton R1 and B7 from dimethylbenzimidazole. Correlations present in the 1 H-1 H ROESY spectrum imply that in DMSO solution the PNA monomer is in proximity to and e- and g-amide groups in Cbl. Moreover, the presence of correlations within the PNA moiety and alkyl spacer suggests rather compact arrangement of this part of the molecule. Two set of signals were observed for groups P1 -P13 and N65 -N67 in 13 C and 1 H NMR spectra (approx. ratio of 6:5 based on the ratio of signals coming from proton N65 ). These suggest the presence of two main conformers resulting from the restricted rotation around the alkyl spacer. As a result in the ROESY spectrum cross-peaks for both interactions P11 -N63 and P11 -N52 are observed (Figure 5). In MD simulations of the Cbl-C conjugate we also observed fluctuations of the positions of atoms corresponding to P11 -N63 and P11 -N52 proton interactions. Figure 6 shows that these 16


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Figure 6: The distances between the P11 -N63 and P11 -N52 atom positions derived from an MD trajectory as a function of the simulation time. Data are shown as a running average over 10000 points. The distances correspond to correlations marked by black lines in Figure 5. either the triazole ring (cluster 2) or flat carbamate moiety (cluster 3). For an all-atom representation and different view of these clusters see Figure S5. The NMR data were compared with representative Cbl-C conformations shown in Figure 7. N63 -P11 , R1 -L3 , R1 -L5 and B7 -L5 interactions evidenced by cross-peaks in the ROESY spectrum were found in cluster 1 while N52 -P11 , L2 -P10 , L2 -P11 , L4 -P10 , L5 -P10 were present in cluster 2. Two different representations from molecular dynamics (cluster 1 and 2) are consistent with the 1 H and

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C NMR spectra that show two sets of signals corresponding

to the cytosine moiety. Moreover, both N63 and N52 signals have NOE correlations with the P11 protons in the ROESY spectrum. This is only possible if two conformers are present in the solution. Also among interactions present in MD representations and ROESY NMR one can mention L2 -P10 in clusters 2 and 3, while L5 -P9 and L2 -P9 are present in cluster 3.

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Representative conformations of PNA oligomers from molecular dynamics simulations The PNA and Cbl-PNA conjugates are internally flexible so, similar as in the case of CblC, we clustered the MD-derived trajectory frames. The representative conformations from clustering of all trajectories and key interactions for free PNA are shown in Figure 8 and for Cbl-PNA in Figure 9. Again the PNA structures are rather compact but the PNA backbone geometries vary among clusters. Nevertheless, key interactions in both cases involve stacking between bases. Only in two cluster representatives hydrogen bonding was detected as an additional characteristic interaction. In the PNA oligomer, in most cases, two PNA bases are stacked but also triples and even four bases in a row are stacked. Interestingly, the stacked bases are not always the neighbouring ones in the sequence (contrary to typical A-form RNA oligomers). 50 The occurrence of the PNA and Cbl–PNA clusters presented in Figures 8 and 9 but in individual trajectories is shown in Figure S6. For both systems there are up to three clusters identified in one trajectory. The transitions between clusters occur up to 250–300 ns of the simulation. Thus, overall each trajectory is dominated by a different kind of cluster, which suggests that simulating PNA-containing systems multiple times with a different set of initial atomic velocities for at least 300 ns should suffice. However, to observe major conformational changes of PNA within one trajectory using classical MD, the one microsecond simulation time is not enough and should be extended. On the other hand, much longer simulations might result in error accumulation.

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Intramolecular non-bonded interactions in PNA Clustering of MD conformations revealed that stacking interactions between the PNA bases determine the conformational shape of the PNA structure. To further quantify the stacking contacts we used the geometric criterion (see Methods). Figure 10 shows the normalized occurrence of the distances between the centers of masses of the bases grouped into four distance ranges. For the distance higher than 3.5 Å, free PNA is more frequently stacked than the conjugated PNA. However, the closest stacking contacts (up to 3.5 Å) are more frequent in Cbl-PNA than in free PNA.

Figure 10: The normalized occurrence of the distances between the centers of masses of PNA bases and hydrogen bond distances between any donor and acceptor in PNA (H-bonding) from all trajectories of PNA and Cbl-PNA. The occurrence of each kind of interaction was normalized to the total occurrence of stacking and H-bonding in PNA. Figure 11 presents the stacking occurrence between the bases with respect to their relative position in the PNA sequence. In both PNA forms, stacking of bases neighboring in the PNA sequence is preferred (n+1 type where n numbers the order of the base in the PNA sequence). 23



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In free PNA, other favorable stacked motifs are n+2, n+3, n+4, n+5, and n+11. In CblPNA n+1 and n+2 are the dominant patterns. Among the long-range contacts, n+11 type is apparently the most favorable.

Figure 11: The occurrence of distances between the centers of masses (below 5.5 Å) of PNA bases as a function of the position of the base in the PNA sequence. The occurrence of the distances was normalized to the total stacking occurrence in PNA. Stacking dependence on the base type is according to the following order: TT>AT>CT>GT>GA>CA>AA>CG>CC in the free PNA and AT>TT>CT>AG>CA>GT>GC>CC>AA in Cbl-PNA. This order suggests that rather the relative position of the base in the PNA sequence not its type determines if the bases stack. The lowest stacking occurrence is observed for AA, CC, and GC; in the simulated PNA sequence (CATCTAGTATTTCT) these bases are not close in the sequence. In contrast, close positioning of the C, A and T bases in the PNA sequence increases the occurrence of TT, AT, and CT pairs in accord with Figure 11. We also examined intramolecular hydrogen bonds. The maximal time that a single 24


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hydrogen bond is present does not exceed 24% for Cbl-PNA and 20% for free PNA. However, the total number of detected intramolecular hydrogen bonds in all trajectories within PNA is 1209 in Cbl-PNA and 1573 in free PNA. Total occurrence of hydrogen bonds is thus higher in free PNA than in the conjugate (Figure 10). Larger number of hydrogen bonds in free PNA may result in less stable stacking but overall the hydrogen bond-stacking balance makes the PNA oligomer tertiary structure highly compact.

Interaction energy of PNA and Cbl-PNA with solvent We have also analyzed the MD-derived internal energy of PNA and its interactions with solvent in the free form and in the Cbl-PNA conjugate. To make the calculated energies of PNA in both forms comparable, the first PNA monomer was excluded from the calculations because it had a different number of atoms in free PNA and Cbl-PNA. Thus the internal potential energy of 13 PNA nucleobases in the free and conjugated forms was computed as the sum of all bonded and non-bonded terms considered in the force field. The internal potential energy of PNA is higher in the free PNA than in the conjugate, which is mainly due to about 45-50 kcal/mol less favorable dihedral angle term in free PNA. PNA dihedral energy term is more favorable in a more extended geometry as it occurs in the Cbl-PNA conjugate. Contrary, the non-bonded energy is more favorable in the free PNA than in the conjugate (on average by about 25-30 kcal/mol). Further, the interaction energy of PNA with solvent was estimated as the sum of nonbonded interactions between PNA and water molecules (including Na+ and Cl− ions). The interaction energy of PNA with solvent is more favorable for free PNA (on average by about 50 kcal/mol) than for PNA within the conjugate, even though SASA of free PNA is slightly lower. It seems that free PNA optimizes its interactions with solvent by lowering SASA and compacting its structure. The next question is why the same PNA sequence upon conjugation with Cbl does not follow the same conformational and energetic pattern as free PNA. Figure 12 shows 25




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occludes the Cbl corrin ring (see the geometry of the 7% occupancy cluster in Figure 9 corresponding to cluster no. 5 in Figure S6). In this cluster the PNA geometry is extended so the interaction of PNA with solvent is less favorable. At the same time, the interaction energy of Cbl with solvent is highly negative (see the top plot in Figure 12). This occurs because upon attaching PNA to Cbl corrin ring, the hydrophilic parts of Cbl (such as amide bonds, hydroxyl or phosphate groups) become more accessible to solvent and form hydrogen bonds with water molecules (data not shown). Naturally, the interaction of PNA with Cbl becomes then less favorable.

Conclusions We presented a structural and dynamics study of conjugates of cyanocobalamin with peptide nucleic acids. With microsecond MD simulations and NMR we determined the internal mobility of Cbl connected via a triazole linker to a PNA mono- and 14-mer. We also compared the dynamics of free PNA oligomer with its conjugate with Cbl. Both PNA and Cbl-PNA acquire compact forms with stacking as a prevalent interaction but the stacked bases are not necessarily the ones that are neighbors in PNA sequence. The PNA oligomer once conjugated with Cbl is on average slightly more exposed to solvent and has a larger end-to-end distance than free PNA. Overall, we propose that the dynamics of Cbl-PNA in aqueous environment arises from two types of contacts between PNA and Cbl. In one type PNA is maximally contracted and diminishes Cbl interactions with water molecules. The second type of contact favors Cbl interaction with solvent but at the same time requires PNA to occupy a more extended structure. Such decrease in conformational stability of PNA through its conjugation with Cbl may be advantageous for biological activity of vitamin B12 -PNA conjugates. Solubility of PNA constructs might be thus enhanced not only due to a hydrophilic carrier but also via increased

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internal dynamics of the PNA strand. Hybridization of nucleic acids with PNA embedded in a conjugate may be also more effective because in the conjugate PNA would be prone to unfold more easily than free PNA. Therefore, the influence of Cbl on the physicochemical properties of PNA might be important for recognizing the final molecular target and Cbl, using its own protein transporters, may also help PNA penetrate cells.

Abbreviations cobalamin Cbl; MD, molecular dynamics; PNA, peptide nucleic acid; RMSD, root-meansquare deviation; SASA, solvent accessible surface area; NMR, nuclear magnetic resonance.

Acknowledgement These studies were supported by National Science Centre (DEC 2014/12/W/ST5/00589, SYMFONIA). Simulations were performed at the Centre of New Technologies and Interdisciplinary Centre for Mathematical and Computational Modelling (G31-4 and GA65-16), University of Warsaw.

Supporting Information Available Supporting Information contains RMSD and radius of gyration versus simulation time (Figures S1–S3), solvent accessibility of PNA (Figure S4), representative conformations from clustering of Cbl-C (Figure S5), occurrence of clusters as a function of the simulation time (Figure S6), copies of HPLC chromatograms (Section S1: Table S1–S2, Figures S7–S8), NMR spectra and assignments in Cbl-C (Section S2: Figures S9–S16).

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Conformational Dynamics of Cyanocobalamin and ... - ACS Publications

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