Data for isolation and properties analysis of diastereomers of a mono-substituted phosphoryl guanidine trideoxyribonucleotide

This article presents new data on the properties of the diastereomers of a mono-substituted phosphoryl guanidine trideoxyribonucleotides d(TpCp*A) [1,2]. The data include information on isolation, identification, treatment with snake venom phosphodiesterase and structural analysis by 1D and 2D NMR spectroscopy and restrained molecular dynamics analysis. The data can be used for preparation, analysis, application of phosphoryl guanidine oligonucleotide and for development of new nucleic acids derivatives. This data article is associated with the manuscript titled “Diastereomers of a mono-substituted phosphoryl guanidine trideoxyribonucleotide: isolation and properties” [1].


Data
Data reported here describe the features of diastereomers of a trideoxynucleotide 5 0 -TpCpA-3 0 modified at the phosphate group near the 3 0 -end with a single 1,1,3,3-tetramethyl guanidine group revealed from studies by Revese phase HPLC (RP-HPLC) separation and analysis, SVPDE digestion, circular dichroism spectroscopy, 1D and 2D NMR analysis and restrained molecular dynamics simulation.

RP-HPLC analysis of oligonucleotides reaction mixture after synthesis
Revese phase HPLC analysis of oligonucleotides were performed for reaction mixture of native d(TpCp*A) and mono-substituted phosphoryl guanidine (PG) oligonucleotides d(TpCp*A) after synthesis. Аnalytical and preparative chomatograms are shown in Fig. 1.
MALDI-TOF MS spectra of oligonucleotides. Matrix-assisted laser desorption ionization e time of flight mass spectroscopy (MALDI-TOF MS) was conducted for the isolated by RP-HPLC samples of d(TpCpA) and diasteremers of d(TpCp*A) (Fig. 2).
Specifications Table   Subject area Chemistry, Physical Chemistry, Biology

More specific subject area
Biochemistry and physical chemistry of nucleic acids Native trinucleotide d(TpCpA) and individual diastereomers of mono-substituted phosphoryl guanidine oligonucleotide d(TpCp*A) were purified and analyzed Experimental features

Type of data
Individual diastereomers of mono-substituted phosphoryl guanidine oligonucleotide d(TpCp*A) were purified by RP-HPLC using C18 sorbent and gradient of acetonitrile. MALDI-TOF MS analysis was conducted on Reflex III, Autoflex Speed with 3-hydroxypicolinic acid as a matrix. Temperature series of CD spectra were measured on a J-600 spectropolarimeter. NMR spectra were acquired on a Bruker Avance 600 MHz spectrometer. MD simulation was performed using AMBER 14 MD modeling software with GPU accelerated code Data source location Value of the data Data on the isolation, SVPDE digestion and identification of the mono-substituted phosphoryl guanidine oligonucleotide d(TpCp*A) diastereomers can be helpful for other researchers to analyze phosphate modified nucleic acids derivatives The data can be used by other researchers with an interest in synthesis, purification and application of nucleic acid derivative and analogues Our data contribute to the properties of phosphate-substituted oligonucleotides This data could be useful for the researchers with an interest in biosensor development and biomedical application of nucleic acids

RP-HPLC profiles of oligonucleotides after SVPDE digestion
We treated native and mono-substituted oligonucleotides with snake venom phosphodiesterase (SVPDE) for 150 h. Three oligomers after digestion by SVPDE were analyzed by RP-HPLC (Fig. 3).

Circular dichroism spectra of oligonucleotides at high and low temperatures
Circular dichroism spectra were used for chracterisation structure of native and modyfied oligonucleotides at low (25 C) and high (95 C) temperatures (Fig. 4).

NMR spectroscopy analysis of oligonucleotides
1D and 2D NMR spectroscopy experiments were performed for isolated mono-substituted phosphoryl guanidine oligonucleotides d(TpCp*A) and their mixture (Figs. 5e9, Tables 1e10).
Assignment of the NMR signals.

Molecular dynamics simulation data analysis
Molecular dynamics simulation with the NOESY NMR restraints were performed for diastereomers of d(TpCp*A). The NOESY NMR restraints for two mixing times (0.4 and 0.8 s) and restraint penalties calculates as an average of last frames of every annealing cycle are shown were collected (Tables 11e15,

HPLC analysis and separation
Native and modified oligonucleotides were isolated by reverse-phased HPLC on an Agilent 1200 HPLC system (USA) using a Zorbax SB-C18 5 mm column 4.6 Â 150 mm. For native oligonucleotede linear gradient of buffer B (acetonitrile 0e50% in 20 mM triethylammonium acetate, pH 7.0), a flow rate of 2 ml min-1 was used. For separation of diastereomers complex gradient of buffer B (20% acetonitrile in 20 mM triethylammonium acetate, pH 7.0) according to Fig. 1 was used.
Fractions containing the appropriate peak were evaporated in vacuo, the bulk of triethylammonium acetate was removed by repeated coevaporations with ethanol. After evaporated until dryness oligonucleotides were dissolved in deionized water and stored at À20 C. Absorption spectra were recorded at wavelengths from 220 to 600 nm.

MALDI-TOF MS analysis
Matrix-assisted laser desorption ionization e time of flight mass spectroscopy (MALDI-TOF MS) was conducted on Reflex III, Autoflex Speed (Bruker, Germany) with 3-hydroxypicolinic acid as a matrix with positive ion detection scan mode.

Circular dichroism (CD) experiments
CD spectra were measured on a J-600 spectropolarimeter (Jasco, Japan) using temperaturecontrolled 1 mm pathlength quartz cell. The mesurments were performed in the range 190e330 nm at 25 C and 95 C. CD curves were recorded every 1 nm, bandwidth 2 nm and averaged over 5 scans. Oligonucleotides in concentration of 0.1 mM in milliQ water were used.

Ultraviolet (UV) spectra
UV spectra were recorded using 1 mL quartz cell with pathlength 1 cm on a UV-2100 spectrophotometer (Shimadzu, Japan). The mesurments were performed in the range 190e330 nm at 25 C. UV spectra were registered every 0.1 nm, bandwidth 1 nm. Oligonucluotides in concentration 12 mM in milliQ water were used.
Concentration of trinuclotides were determined from their UV absorbance using calculated molar extinction coefficients at 260 nm [5].

NMR analysis
All the spectra were acquired on a Bruker Avance 600 MHz spectrometer. The chemical shifts in NMR spectra were calibrated relative to DSS by substitution referencing [6]. The 1D and 2D experiments were performed at 25 C for assignments and included 1 H, 1 H{ 31 P}, 13 [7]. The assignments of signals in the spectra were carried out by their combined analysis. The spin systems of deoxyribose residues were identified from 1 He 1 H COSY (Figs. 6e7) and 1 He 1 H TOCSY spectra. The diastereotope assignment of the signals of proton H2' and H2'' were carried out on the basis of 1 He 1 H NOESY spectra (see Figs. 8 and 9) and by comparison with published chemicals shift ranges [8]. Distance restraints were derived from NOESY cross-peak intensities from spectra recorded at 8 C with mixing times 0.4 s and 0.8 s correspondingly. The fixed cytosine H5eH6 distance (2.5 Å) was used as internal reference to determine the quality of the calibration.
After the assignment of the signals in the 2D -spectra (the results are shown in Tables S1e10), the spin-spin interaction constants 1He1H were extracted from the 1H {31P} spectra and then the obtained data were used to measure the 1He31P constants from the 1H spectra.

Molecular dynamics simulations
The molecular dynamics (MD) simulations were performed using Amber 14 software [9]. Structures of TpCp*A were generated using xleap program (AmberTools 14) based on B-form DNA geometry. Particular atoms charges of modified nucleotides were calculated using RESP method based on structures optimized by Hartree-Fock method and 6-31G* basis in Gaussian'09 software [10]. Then the library files for Rp-and Sp-isomers of tetramethyl phoporylguanidine of 3 0 -adenosine were generated. NMR distance restraints were used for subsequent refinement of the structure. The upper bounds of the restraints were set to þ0.5 Å of the calculated NOE distance. The lower bounds of the restraints were set 1.8 Å. Structural calculations were performed using the sander module of Amber 14 as described in Ref. [11]. The generalized Born implicit solvent model with the equivalent of 0.1 M 1À1  ions, the weak-coupling algorithm of temperature regulation and integration time step of 0.001 ps were used. Simulation annealing protocol was applied and includes 100 cycles of heating to 800 K for 0.25 ns and following cooling to 300 K for 0.25 ns. Force constant 1 kcal/[mol$Å 2 ] for distance restraints was applied.
Trajectory analysis was performed using the cpptraj tool of Amber 16 [12]. Hierarchical cluster analysis was conducted for final structures of simulation annealing. Molecular graphics were prepared with the UCSF Chimera package [13]. Hierarchical cluster analysis was used for productive MD trajectory analysis of the DNA duplexes without terminal base pairs. The random sieve of 100 was applied.  Table 2 Coupling constants 31 Pe 13 C of 'fast' diastereomer, Hz. Table 4 Coupling constants 31 Pe 13 C of 'slow' diastereomer, Hz.   Table 9 Coupling constants 1 He 1 H, 1 He 31 P of cytidine monophosphate of 'fast' diastereomer, Hz.          Fig. 4 in Ref. [1].    Abbreviations [4]. #Cluster -Cluster number starting from 0 (0 is most populated). Frames -number of frames in cluster.
Frac -Size of cluster as fraction of total trajectory. AvgDist -Average distance between points in the cluster. Stdev -Standard deviation of points in the cluster. AvgCDist -Average distance of this cluster to every other cluster.