Literature DB >> 31312699

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

Alexander A Lomzov1,2, Maxim S Kupryushkin1, Andrey V Shernyukov3,2, Mikhail D Nekrasov1, Ilya S Dovydenko1, Dmitry A Stetsenko1,2, Dmitrii V Pyshnyi1,2.   

Abstract

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].

Entities:  

Keywords:  Circular dichroism; Diastereomer assignment phosphodiesterase digestion assay; Molecular dynamics; NMR; Phosphoryl guanidine oligonucleotide; RP-HPLC; Spatial structure

Year:  2019        PMID： 31312699      PMCID： PMC6609727          DOI： 10.1016/j.dib.2019.104148

Source DB:  PubMed          Journal:  Data Brief        ISSN： 2352-3409


Specifications Table 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

Data

Data reported here describe the features of diastereomers of a trideoxynucleotide 5′-TpCpA-3′ modified at the phosphate group near the 3′-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.
Fig. 1

RP-HPLC analysis of reaction mixtures of native TpCpA (blue) and modified TpC*pA (red, * - position of modyfied phosphate) deoxyribotrinucleotides reaction mixture after detritilation. Upper chromatographic profiles are analytical and lower is preparative. Details of experiments see in section Material and methods.

RP-HPLC analysis of reaction mixtures of native TpCpA (blue) and modified TpC*pA (red, * - position of modyfied phosphate) deoxyribotrinucleotides reaction mixture after detritilation. Upper chromatographic profiles are analytical and lower is preparative. Details of experiments see in section Material and methods. MALDI-TOF MS spectra of oligonucleotides. Matrix-assisted laser desorption ionization – 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).
Fig. 2

MALDI-TOF MS spectra of native (A) and PG-modified ‘fast’ (B) and ‘slow’ (C) trinucleotides.

MALDI-TOF MS spectra of native (A) and PG-modified ‘fast’ (B) and ‘slow’ (C) trinucleotides.

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).
Fig. 3

RP-HPLC profiles of TpCpA, TpCp*A(‘fast’) and TpCp*A (‘slow’) before SVPD digestion. Data on oligonucleote analysis after digestion shown in Fig. 2 in Ref. [1].

RP-HPLC profiles of TpCpA, TpCp*A(‘fast’) and TpCp*A (‘slow’) before SVPD digestion. Data on oligonucleote analysis after digestion shown in Fig. 2 in Ref. [1].

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).
Fig. 4

Circular dichroism spectra of native (dashed line), ‘fast’ (thick line) and ‘slow’ (thin line) trinucleotides at 95 °C (A) and comparison with 25 °C (B).

Circular dichroism spectra of native (dashed line), ‘fast’ (thick line) and ‘slow’ (thin line) trinucleotides at 95 °C (A) and comparison with 25 °C (B).

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 (Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9, Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, Table 10).
Fig. 5

Fragments 1H NMR spectra (600 MHz, D2O). For each fragment ‘slow’ (top), ‘fast’ (middle) and their 1:1 mixture (down).

Fig. 6

1H–1H COSY NMR spectra of the ‘fast’ isomer.

Fig. 7

1H–1H COSY NMR spectra of the ‘slow’ isomer.

Fig. 8

1H–1H NOESY NMR (mixing time 0.8s, T = 8.0 °C) spectra of the ‘fast’ isomer.

Fig. 9

1H–1H NOESY NMR (mixing time 0.8s, T = 8.0 °C) spectra of the ‘slow’ isomer.

Table 1

Chemical shits of ‘fast’ diastereomer, ppm.

dTdCd(p*A)
H1'6.256.106.44
H2––8.19
H2'2.271.932.91
H2''2.422.362.62
H3'4.744.884.77
H4'4.114.224.27
H5–5.96–
H5' & H5''3.79 и 3.734.04 и 4.004.18 и 4.20
H67.427.61–
H71.83––
H8––8.37
C1'88.1888.3486.21
C2161.56159.84155.53
C2'40.2440.4841.20
C3'78.1278.5573.09
C4178.63168.61151.56
C4'87.9686.5087.79
C5114.4999.16121.36
C5'63.9667.4368.20
C6139.19143.75158.17
C715.50––
C8––142.49
(CH3)2–N–CN-Cp*A2.86
(CH3)2–N–CN-Cp*A42.38
-C <svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="20.666667pt" height="16.000000pt" viewBox="0 0 20.666667 16.000000" preserveAspectRatio="xMidYMid meet"><metadata> Created by potrace 1.16, written by Peter Selinger 2001-2019 </metadata><g transform="translate(1.000000,15.000000) scale(0.019444,-0.019444)" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 480 0 480 0 0 40 0 40 -480 0 -480 0 0 -40z M0 280 l0 -40 480 0 480 0 0 40 0 40 -480 0 -480 0 0 -40z"/></g></svg> N-Cp*A165.88
TpC−0.82
Cp*A0.37
Table 2

Coupling constants31P–13C of ‘fast’ diastereomer, Hz.

dT
dC
d(p*A)
TpCTpCCp*ACp*A
C2'2.902.90
C3'5.305.50
C4'6.88.87.68.8
C5'05.506.8
C=N-Cp*A = 7.5
Table 3

Chemical shits of ‘slow’ diastereomer, ppm.

dTdCd(p*A)
H1'6.236.096.44
H2––8.19
H2'2.272.022.88
H2''2.422.422.63
H3'4.734.924.75
H4'4.114.204.26
H5–5.96–
H5' & H5''3.74 и 3.793.98 и 3.994.17 и 4.22
H67.417.64–
H71.82––
H8––8.33
C1'88.1488.4286.25
C2160.69159.89155.52
C2'40.2240.741.27
C3'78.1178.6473.20
C4177.5168.65151.56
C4'88.0286.4087.82
C5114.4599.1121.40
C5'63.9468.1067.29
C6139.29143.84158.2
C715.37––
C8––142.40
(CH3)2–N–CN-Cp*A2.87
(CH3)2–N–CN–C*pA42.42
-CN-Cp*A165.91
TpC−0.89
Cp*A0.21
Table 4

Coupling constants31P–13C of ‘slow’ diastereomer, Hz.

dT
dC
d(p*A)
TpCTpCCp*ACp*A
C2'2.802.60
C3'5.406.50
C4'6.68.56.98.9
C5'05.105.8
C=N-Cp*A = 7.7
Table 5

Coupling constants 1H–1H, 1H–31P of adenosine monophosphate of ‘fast’ diastereomer, Hz.

H1'H2'H2''H3'H4'H5' & H5''Cp*A
H1'*6.36.70000
H2'6.3*14.06.4000
H2''6.714.0*4.9000
H3'06.44.9*4.400
H4'0004.4*3.92.1
H5' & H5''00003.9n.d..n.d..
Cp*A00002.1n.d..*
Table 6

Coupling constants 1H–1H, 1H–31P of adenosine monophosphate of ‘slow’ diastereomer, Hz.

H1'H2'H2''H3'H4'H5' & H5''Cp*A
H1'*6.56.50000
H2'6.5*14.06.4000
H2''6.514.0*4.8000
H3'06.44.8*4.300
H4'0004.3*3.92.1
H5' & H5''00003.911.54.9, 4.0
Cp*A00002.14.9, 4.0*
Table 7

Coupling constants 1H–1H, 1H–31P of timidine of ‘fast’ diastereomer, Hz.

H1'H2'H2''H3'H4'H5' & H5''H6H7TpC
H1'*8.16.1000000
H2'8.1*14.16.300000
H2''6.114.1*3.000000
H3'06.33.0*3.20007.0
H4'0003.2*3.5000
H5' & H5''00003.512.5000
H6000000*1.10
H70000001.1*0
TpC0007.00000*
Table 8

Coupling constants 1H–1H, 1H–31P of timidine of ‘slow’ diastereomer, Hz.

H1'H2'H2''H3'H4'H5' & H5''H6H7TpC
H1'*8.06.1000000
H2'8.0*146.200000
H2''6.114*3.000000
H3'06.23.0*3.20007.0
H4'0003.2*3.5000
H5' & H5''00003.512.5000
H6000000*1.10
H70000001.1*0
TpC0007.00000*
Table 9

Coupling constants 1H–1H, 1H–31P of cytidine monophosphate of ‘fast’ diastereomer, Hz.

H1'H2'H2''H3'H4'H5' &H5''H5H6TpCCp*A
H1'*8.06.00000000
H2'8.0*14.26.3000000
H2''6.014.2*2.8000000
H3'06.32.8*2.700007.0
H4'0002.7*2.800n.d..n.d..
H5' & H5''00002.811.6004.80
H5000000*7.600
H60000007.6*00
TpC0000n.d..4.800*0
Cp*A0007.0n.d..0000*
Table 10

Coupling constants 1H–1H, 1H–31P of cytidine monophosphate of ‘slow’ diastereomer, Hz.

H1'H2'H2''H3'H4'H5' & H5''H5H6TpCCp*A
H1'*7.96.00000000
H2'7.9*14.26.3000000
H2''6.014.2*2.8000000
H3'06.32.8*2.800007.0
H4'0002.8*3.400n.d..n.d..
H5' & H5''00003.411.7004.2 4.60
H5000000*7.500
H60000007.5*00
TpC0000n.d..4.2, 4.600*0
Cp*A0007.0n.d..0000*
Fragments 1H NMR spectra (600 MHz, D2O). For each fragment ‘slow’ (top), ‘fast’ (middle) and their 1:1 mixture (down). 1H–1H COSY NMR spectra of the ‘fast’ isomer. 1H–1H COSY NMR spectra of the ‘slow’ isomer. 1H–1H NOESY NMR (mixing time 0.8s, T = 8.0 °C) spectra of the ‘fast’ isomer. 1H–1H NOESY NMR (mixing time 0.8s, T = 8.0 °C) spectra of the ‘slow’ isomer. Chemical shits of ‘fast’ diastereomer, ppm. Coupling constants31P–13C of ‘fast’ diastereomer, Hz. Chemical shits of ‘slow’ diastereomer, ppm. Coupling constants31P–13C of ‘slow’ diastereomer, Hz. Coupling constants 1H–1H, 1H–31P of adenosine monophosphate of ‘fast’ diastereomer, Hz. Coupling constants 1H–1H, 1H–31P of adenosine monophosphate of ‘slow’ diastereomer, Hz. Coupling constants 1H–1H, 1H–31P of timidine of ‘fast’ diastereomer, Hz. Coupling constants 1H–1H, 1H–31P of timidine of ‘slow’ diastereomer, Hz. Coupling constants 1H–1H, 1H–31P of cytidine monophosphate of ‘fast’ diastereomer, Hz. Coupling constants 1H–1H, 1H–31P of cytidine monophosphate of ‘slow’ diastereomer, Hz. 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 (Table 11, Table 12, Table 13, Table 14, Table 15, Fig. 12, Fig. 13, Fig. 14). The data on cluster analysis of the MD trajectories are shown in Table 16, Table 17, Table 18, Table 19. Molecular structures of the trinucleotides most represented in the MD simulation can be found in the Supplementary Data of this article. The structures flexibility was analyzed using RMSD map for the oligonucleotides structures after simulation annealing (Fig. 10 and Fig. 11).
Table 11

NOESY NMR restraints with mixing time 0.4s of ‘fast’ trinucleotide and MD restraint penalty values for Rp- and Sp-diastereomers.

#Residue number, Residue name, Atom NameResidue number, Residue name, Atom NameDistance, ÅRestraint penalty, kcal/mol Rp-isomerSp-isomer
11 dT H1'1 dT H3'6.140 ± 00 ± 0
21 dT H1'1 dT H63.120.06 ± 0.050.08 ± 0.05
31 dT H2'21 dT H1'4.030 ± 00 ± 0
41 dT H2'21 dT H2'12.740 ± 00 ± 0
51 dT H2'21 dT H3'3.570 ± 00 ± 0
61 dT H2'21 dT H4'4.610 ± 00 ± 0
71 dT H2'21 dT H64.090 ± 0.010 ± 0.01
81 dT H2'11 dT H1'3.520 ± 00 ± 0
91 dT H2'11 dT H3'3.440 ± 00 ± 0
101 dT H2'11 dT H62.870.08 ± 0.130.06 ± 0.12
111 dT H4'1 dT H1'4.100 ± 00 ± 0
121 dT H4'1 dT H3'3.460 ± 00 ± 0
131 dT H4'1 dT H64.920.03 ± 0.040.02 ± 0.04
141 dT H5'11 dT H3'3.680 ± 00 ± 0
151 dT H5'11 dT H4'2.670.01 ± 0.020.01 ± 0.02
161 dT H5'11 dT H64.600.18 ± 0.210.1 ± 0.17
171 dT H5'21 dT H3'3.820 ± 00 ± 0
181 dT H5'21 dT H4'3.320 ± 00 ± 0
191 dT H5'21 dT H64.840.19 ± 0.210.11 ± 0.17
201 dT H61 dT H3'3.900.16 ± 0.170.13 ± 0.16
211 dT M71 dT H62.850 ± 00 ± 0
222 dC H1'2 dC H2'13.150 ± 00 ± 0
232 dC H1'2 dC H5'14.490.01 ± 0.020.01 ± 0.02
242 dC H1'2 dC H5'24.610.03 ± 0.030.02 ± 0.03
252 dC H1'2 dC H63.240.07 ± 0.020.07 ± 0.02
262 dC H2'22 dC H1'3.240 ± 00 ± 0
272 dC H2'22 dC H2'12.710 ± 00 ± 0
282 dC H2'22 dC H3'3.390 ± 00 ± 0
292 dC H2'22 dC H56.290 ± 00 ± 0
302 dC H2'22 dC H63.680.03 ± 0.040.03 ± 0.04
312 dC H2'22 dC H5'15.280 ± 00 ± 0
322 dC H2'22 dC H5'25.700 ± 00 ± 0
332 dC H2'12 dC H5'14.450.01 ± 0.040.02 ± 0.05
342 dC H2'12 dC H5'24.300.01 ± 0.040.01 ± 0.04
352 dC H3'2 dC H1'5.760 ± 00 ± 0
362 dC H3'2 dC H2'13.210 ± 00 ± 0
372 dC H3'2 dC H5'13.210.04 ± 0.040.03 ± 0.04
382 dC H3'2 dC H5'23.180.02 ± 0.030.03 ± 0.04
392 dC H3'2 dC H64.170.01 ± 0.020.01 ± 0.03
402 dC H4'2 dC H5'12.310.04 ± 0.060.07 ± 0.07
412 dC H4'2 dC H5'22.430.01 ± 0.030.01 ± 0.03
422 dC H52 dC H2'14.690 ± 0.010 ± 0.01
432 dC H52 dC H5'15.840.05 ± 0.090.06 ± 0.1
442 dC H52 dC H5'26.560.04 ± 0.070.04 ± 0.07
452 dC H52 dC H62.500 ± 00 ± 0
462 dC H62 dC H2'12.820 ± 0.020.01 ± 0.03
472 dC H62 dC H5'13.800.04 ± 0.070.05 ± 0.08
482 dC H62 dC H5'23.830.11 ± 0.110.09 ± 0.11
493 d(p*A) H1'3 d(p*A) H83.840 ± 00 ± 0
503 d(p*A) H2'23 d(p*A) H1'3.170 ± 00 ± 0
513 d(p*A) H2'23 d(p*A) H2'12.850 ± 00 ± 0
523 d(p*A) H2'23 d(p*A) H3'3.300 ± 00 ± 0
533 d(p*A) H2'23 d(p*A) H84.240 ± 0.010 ± 0
543 d(p*A) H2'13 d(p*A) H1'3.210 ± 00 ± 0
553 d(p*A) H2'13 d(p*A) H3'2.940 ± 00 ± 0
563 d(p*A) H2'13 d(p*A) H83.090.01 ± 0.040 ± 0.02
573 d(p*A) H23 d(p*A) H1'5.200 ± 0.020 ± 0.02
583 d(p*A) H23 d(p*A) H2'16.460.04 ± 0.040.05 ± 0.04
593 d(p*A) H3'3 d(p*A) H1'4.230 ± 00 ± 0
603 d(p*A) H3'3 d(p*A) H83.850.13 ± 0.090.12 ± 0.08
613 d(p*A) H4'3 d(p*A) H1'3.590 ± 00 ± 0
623 d(p*A) H4'3 d(p*A) H2'14.360 ± 00 ± 0
633 d(p*A) H4'3 d(p*A) H2'23.880.01 ± 0.010.01 ± 0.01
643 d(p*A) H4'3 d(p*A) H86.020 ± 00 ± 0
651 dT H1'2 dC H5'14.140.13 ± 0.220.13 ± 0.19
661 dT H1'2 dC H5'24.290.17 ± 0.220.25 ± 0.23
671 dT H1'2 dC H65.250.07 ± 0.170.07 ± 0.15
681 dT H2'22 dC H64.240.07 ± 0.190.1 ± 0.23
691 dT H2'22 dC H5'14.270.03 ± 0.10.06 ± 0.14
701 dT H2'22 dC H5'24.450.01 ± 0.040.02 ± 0.04
711 dT H2'12 dC H55.450.18 ± 0.290.26 ± 0.29
721 dT H2'12 dC H64.780.08 ± 0.180.1 ± 0.18
731 dT H3'2 dC H5'13.610.16 ± 0.160.15 ± 0.18
741 dT H3'2 dC H5'23.630.29 ± 0.180.24 ± 0.17
751 dT H3'2 dC H64.660.1 ± 0.150.13 ± 0.15
761 dT H62 dC H5'15.310.17 ± 0.240.23 ± 0.23
771 dT H62 dC H5'26.060.14 ± 0.180.23 ± 0.23
781 dT M72 dC H55.860.35 ± 0.510.46 ± 0.47
791 dT H63 d(p*A) H2'15.830.35 ± 0.490.29 ± 0.48
802 dC H2'23 d(p*A) H84.850.13 ± 0.210.09 ± 0.24
812 dC H2'13 d(p*A) H84.950.13 ± 0.220.09 ± 0.24
822 dC H63 d(p*A) H2'16.730.1 ± 0.180.09 ± 0.15
833 d(p*A) H21 dT M75.300.54 ± 0.520.45 ± 0.44
843 d(p*A) H82 dC H5'15.560.19 ± 0.290.16 ± 0.24
853 d(p*A) H82 dC H5'26.100.11 ± 0.190.11 ± 0.16
861 dT H1'3 d(p*A) Me5.530.1 ± 0.20.05 ± 0.14
871 dT H4'3 d(p*A) Me4.540.58 ± 0.430.29 ± 0.42
881 dT H63 d(p*A) Me4.980.34 ± 0.380.17 ± 0.31
892 dC H1'3 d(p*A) Me4.280.08 ± 0.110.08 ± 0.1
902 dC H3'3 d(p*A) Me5.040 ± 00 ± 0
913 d(p*A) H23 d(p*A) Me4.520.79 ± 0.641.17 ± 0.44
923 d(p*A) H4'3 d(p*A) Me3.740.09 ± 0.120.26 ± 0.17
933 d(p*A) Me2 dC H5'14.080.06 ± 0.10.07 ± 0.17
943 d(p*A) Me2 dC H5'24.220.03 ± 0.080.02 ± 0.09
953 d(p*A) Me3 d(p*A) H83.720.45 ± 0.430.17 ± 0.34
963 d(p*A) Me2 dC H65.140.09 ± 0.10.07 ± 0.09
971 dT H62 dC H4'6.170.17 ± 0.220.29 ± 0.23
982 dC H1'2 dC H4'3.210.01 ± 0.020.01 ± 0.02
992 dC H2'22 dC H4'3.430.08 ± 0.050.08 ± 0.05
1002 dC H2'23 d(p*A) H5'13.430.33 ± 0.30.13 ± 0.21
1012 dC H2'23 d(p*A) H5'23.430.2 ± 0.20.16 ± 0.16
1022 dC H2'12 dC H4'4.000 ± 00 ± 0
1032 dC H3'2 dC H4'3.280 ± 00 ± 0
1042 dC H52 dC H4'5.650.27 ± 0.060.28 ± 0.06
1052 dC H4'3 d(p*A) Me2.920.07 ± 0.140.1 ± 0.17
1062 dC H62 dC H4'4.410.01 ± 0.030.02 ± 0.03
1073 d(p*A) H1'3 d(p*A) H5'14.370.04 ± 0.040.02 ± 0.03
1083 d(p*A) H1'3 d(p*A) H5'24.370.04 ± 0.050.06 ± 0.04
1093 d(p*A) H2'23 d(p*A) H5'14.600.06 ± 0.050.09 ± 0.05
1103 d(p*A) H2'23 d(p*A) H5'24.600.02 ± 0.030.03 ± 0.03
1113 d(p*A) H2'13 d(p*A) H5'13.730.06 ± 0.090.06 ± 0.07
1123 d(p*A) H2'13 d(p*A) H5'23.730.03 ± 0.060.03 ± 0.06
1133 d(p*A) H23 d(p*A) H5'15.480.67 ± 0.30.62 ± 0.25
1143 d(p*A) H23 d(p*A) H5'25.480.69 ± 0.230.82 ± 0.19
1153 d(p*A) H3'3 d(p*A) H5'12.980.07 ± 0.070.11 ± 0.06
1163 d(p*A) H3'3 d(p*A) H5'22.980 ± 0.010 ± 0.01
1173 d(p*A) H83 d(p*A) H5'14.380.16 ± 0.170.11 ± 0.15
1183 d(p*A) H83 d(p*A) H5'24.380.11 ± 0.170.18 ± 0.15
Table 12

NOESY NMR restraints with mixing time 0.4s of ‘slow’ trinucleotide and MD restraint penalty values for Rp- and Sp-diastereomers.

#Residue number, Residue name, Atom NameResidue number, Residue name, Atom NameDistance, ÅRestraint penalty, kcal/mol
RpSp
11 dT H1'1 dT H3'5.840 ± 00 ± 0
21 dT H1'1 dT H62.680.23 ± 0.040.22 ± 0.06
31 dT H2'21 dT H1'3.000 ± 00 ± 0
41 dT H2'21 dT H2'12.410 ± 00 ± 0
51 dT H2'21 dT H3'3.540 ± 00 ± 0
61 dT H2'21 dT H4'3.820.01 ± 0.010.01 ± 0.01
71 dT H2'21 dT H63.590.06 ± 0.060.05 ± 0.06
81 dT H2'11 dT H3'2.630 ± 00 ± 0
91 dT H2'11 dT H62.360.06 ± 0.090.07 ± 0.12
101 dT H4'1 dT H1'3.550 ± 00 ± 0
111 dT H4'1 dT H3'2.810 ± 00 ± 0
121 dT H4'1 dT H63.980.09 ± 0.060.1 ± 0.08
131 dT H5'11 dT H3'2.900.11 ± 0.070.11 ± 0.07
141 dT H5'11 dT H4'4.450 ± 00 ± 0
151 dT H5'11 dT H63.640.08 ± 0.130.12 ± 0.18
161 dT H5'21 dT H3'2.850.03 ± 0.060.03 ± 0.07
171 dT H5'21 dT H4'2.270.03 ± 0.050.03 ± 0.05
181 dT H5'21 dT H63.540.25 ± 0.180.26 ± 0.21
191 dT H61 dT H3'3.240.28 ± 0.110.28 ± 0.11
201 dT M71 dT H62.410 ± 00 ± 0
212 dC H1'2 dC H2'12.880.01 ± 0.010.01 ± 0.01
222 dC H1'2 dC H4'2.830.04 ± 0.040.04 ± 0.04
232 dC H1'2 dC H62.790.2 ± 0.030.2 ± 0.02
242 dC H2'22 dC H1'2.420 ± 00 ± 0
252 dC H2'22 dC H2'12.140 ± 00 ± 0
262 dC H2'22 dC H3'3.280 ± 00 ± 0
272 dC H2'22 dC H4'2.970.22 ± 0.060.21 ± 0.06
282 dC H2'22 dC H63.090.17 ± 0.080.17 ± 0.07
292 dC H2'12 dC H4'3.470.05 ± 0.030.04 ± 0.02
302 dC H3'2 dC H2'12.890 ± 00 ± 0
312 dC H3'2 dC H63.890.06 ± 0.070.05 ± 0.05
322 dC H52 dC H62.500 ± 00 ± 0
332 dC H62 dC H2'12.440.03 ± 0.060.02 ± 0.05
342 dC H62 dC H4'3.770.16 ± 0.070.13 ± 0.06
353 d(p*A) H1'3 d(p*A) H5'23.120.37 ± 0.10.38 ± 0.1
363 d(p*A) H1'3 d(p*A) H83.260.09 ± 0.040.09 ± 0.04
373 d(p*A) H2'23 d(p*A) H1'2.480 ± 00 ± 0
383 d(p*A) H2'23 d(p*A) H2'12.350 ± 00 ± 0
393 d(p*A) H2'23 d(p*A) H3'3.450 ± 00 ± 0
403 d(p*A) H2'23 d(p*A) H5'13.710.3 ± 0.080.3 ± 0.1
413 d(p*A) H2'23 d(p*A) H83.760.02 ± 0.050.02 ± 0.04
423 d(p*A) H2'13 d(p*A) H1'3.200 ± 00 ± 0
433 d(p*A) H2'13 d(p*A) H3'2.670 ± 00 ± 0
443 d(p*A) H2'13 d(p*A) H5'13.190.18 ± 0.120.19 ± 0.13
453 d(p*A) H2'13 d(p*A) H5'23.170.09 ± 0.120.12 ± 0.13
463 d(p*A) H2'13 d(p*A) H82.530.03 ± 0.10.02 ± 0.05
473 d(p*A) H3'3 d(p*A) H1'4.000 ± 00 ± 0
483 d(p*A) H3'3 d(p*A) H5'12.570.12 ± 0.130.14 ± 0.13
493 d(p*A) H3'3 d(p*A) H5'22.450.03 ± 0.040.04 ± 0.06
503 d(p*A) H3'3 d(p*A) H83.320.27 ± 0.130.25 ± 0.1
513 d(p*A) H4'3 d(p*A) H1'3.020.02 ± 0.030.02 ± 0.04
523 d(p*A) H4'3 d(p*A) H2'13.160.12 ± 0.030.13 ± 0.03
533 d(p*A) H4'3 d(p*A) H2'23.180.16 ± 0.060.16 ± 0.06
543 d(p*A) H83 d(p*A) H5'13.790.3 ± 0.250.25 ± 0.2
553 d(p*A) H83 d(p*A) H5'23.840.18 ± 0.190.19 ± 0.2
561 dT H3'2 dC H63.810.47 ± 0.280.53 ± 0.27
572 dC H2'23 d(p*A) H5'23.760.17 ± 0.160.22 ± 0.21
582 dC H2'13 d(p*A) H84.130.65 ± 0.460.54 ± 0.44
593 d(p*A) H82 dC H4'3.690.5 ± 0.490.54 ± 0.47
601 dT H1'2 dC H5'13.390.31 ± 0.320.35 ± 0.22
611 dT H1'2 dC H5'23.390.18 ± 0.160.17 ± 0.26
622 dC H2'22 dC H5'13.120.52 ± 0.510.51 ± 0.11
632 dC H2'22 dC H5'23.120.42 ± 0.420.44 ± 0.08
642 dC H2'12 dC H5'13.260.22 ± 0.20.21 ± 0.12
652 dC H2'12 dC H5'23.260.14 ± 0.140.16 ± 0.1
662 dC H3'2 dC H5'12.830.14 ± 0.130.13 ± 0.07
672 dC H3'2 dC H5'22.830.01 ± 0.010.02 ± 0.06
682 dC H4'2 dC H5'11.890.1 ± 0.10.1 ± 0.06
692 dC H4'2 dC H5'21.890.11 ± 0.110.1 ± 0.06
702 dC H62 dC H5'13.170.2 ± 0.180.16 ± 0.14
712 dC H62 dC H5'23.170.3 ± 0.30.3 ± 0.16
721 dT H1'3 d(p*A) Me4.250.19 ± 0.150.2 ± 0.38
731 dT H4'3 d(p*A) Me3.680.4 ± 0.390.38 ± 0.53
742 dC H1'3 d(p*A) Me3.550.22 ± 0.230.2 ± 0.19
752 dC H4'3 d(p*A) Me2.800.18 ± 0.170.23 ± 0.25
762 dC H63 d(p*A) Me4.220.34 ± 0.380.44 ± 0.14
773 d(p*A) Me2 dC H5'13.090.4 ± 0.390.49 ± 0.4
783 d(p*A) Me2 dC H5'23.090.23 ± 0.230.32 ± 0.33
793 d(p*A) H23 d(p*A) Me3.900.85 ± 0.881.19 ± 0.62
803 d(p*A) H3'2 dC H5'13.100.95 ± 0.981.14 ± 0.52
813 d(p*A) H3'2 dC H5'23.100.84 ± 0.870.98 ± 0.48
Table 13

NOESY NMR restraints with mixing time 0.8s of ‘fast’ trinucleotide and MD restraint penalty values for Rp- and Sp-diastereomers.

#Residue number, Residue name, Atom NameResidue number, Residue name, Atom NameDistance, ÅRestraint penalty, kcal/mol
RpSp
11 dT H1'1 dT H3'4.650 ± 00 ± 0
21 dT H1'1 dT H62.800.59 ± 0.618.13 ± 1.68
31 dT H2'21 dT H1'3.460 ± 00 ± 0.01
41 dT H2'21 dT H2'12.680.1 ± 0.140.06 ± 0.1
51 dT H2'21 dT H3'3.550 ± 00 ± 0
61 dT H2'21 dT H4'4.350 ± 00 ± 0
71 dT H2'21 dT H63.590 ± 00.02 ± 0.09
81 dT H2'11 dT H1'4.120 ± 00 ± 0
91 dT H2'11 dT H3'3.050 ± 00 ± 0
101 dT H2'11 dT H62.660 ± 00 ± 0
111 dT H4'1 dT H1'3.650.04 ± 0.120.17 ± 0.31
121 dT H4'1 dT H3'3.190 ± 00 ± 0
131 dT H4'1 dT H64.497.82 ± 1.580.1 ± 0.21
141 dT H5'11 dT H3'3.330 ± 00.19 ± 0.49
151 dT H5'11 dT H4'2.820.11 ± 0.210.32 ± 0.42
161 dT H5'11 dT H64.090.51 ± 0.690.02 ± 0.11
171 dT H5'21 dT H3'3.420.19 ± 0.310.38 ± 0.58
181 dT H5'21 dT H4'2.780.32 ± 0.340.22 ± 0.44
191 dT H5'21 dT H63.960.33 ± 0.610.1 ± 0.33
201 dT H61 dT H3'3.446.01 ± 1.810.31 ± 0.5
211 dT M71 dT H62.590 ± 00 ± 0
222 dC H1'2 dC H2'13.190 ± 00 ± 0
232 dC H1'2 dC H5'13.990.05 ± 0.131.03 ± 0.62
242 dC H1'2 dC H5'24.071.42 ± 0.890 ± 0
252 dC H1'2 dC H62.865.02 ± 1.231.86 ± 1.05
262 dC H2'22 dC H1'2.720 ± 00 ± 0
272 dC H2'22 dC H2'12.510.12 ± 0.150.19 ± 0.2
282 dC H2'22 dC H3'3.240 ± 00 ± 0
292 dC H2'22 dC H54.482.34 ± 1.382.75 ± 1.52
302 dC H2'22 dC H63.242.37 ± 1.10 ± 0
312 dC H2'22 dC H5'14.640 ± 00 ± 0
322 dC H2'22 dC H5'24.810.04 ± 0.130 ± 0
332 dC H2'12 dC H5'14.040 ± 00 ± 0
342 dC H2'12 dC H5'23.930 ± 00 ± 0
352 dC H3'2 dC H1'4.650 ± 00 ± 0
362 dC H3'2 dC H2'12.980 ± 00 ± 0
372 dC H3'2 dC H5'12.930 ± 0.010 ± 0
382 dC H3'2 dC H5'22.914.07 ± 1.281.54 ± 0.85
392 dC H3'2 dC H63.890.7 ± 0.630.44 ± 0.45
402 dC H4'2 dC H5'12.149.65 ± 1.325.31 ± 1.5
412 dC H4'2 dC H5'22.140.05 ± 0.137.65 ± 1.49
422 dC H52 dC H2'14.010 ± 00.04 ± 0.09
432 dC H52 dC H5'14.810 ± 00.54 ± 0.59
442 dC H52 dC H5'24.841.31 ± 1.10 ± 0
452 dC H52 dC H62.500 ± 0.010 ± 0
462 dC H62 dC H2'12.630 ± 0.010 ± 0.01
472 dC H62 dC H5'13.430 ± 00.17 ± 0.26
482 dC H62 dC H5'23.420 ± 00.01 ± 0.05
493 d(p*A) H1'3 d(p*A) H83.412.03 ± 0.771.35 ± 0.66
503 d(p*A) H2'23 d(p*A) H1'2.640 ± 00 ± 0
513 d(p*A) H2'23 d(p*A) H2'12.830.04 ± 0.080.06 ± 0.12
523 d(p*A) H2'23 d(p*A) H3'3.600 ± 00 ± 0
533 d(p*A) H2'23 d(p*A) H83.920.58 ± 0.491.14 ± 0.72
543 d(p*A) H2'13 d(p*A) H1'3.380 ± 00 ± 0
553 d(p*A) H2'13 d(p*A) H3'3.140 ± 00 ± 0
563 d(p*A) H2'13 d(p*A) H82.820.23 ± 0.380.09 ± 0.21
573 d(p*A) H23 d(p*A) H1'4.060.41 ± 0.491.9 ± 1.08
583 d(p*A) H23 d(p*A) H2'14.475.3 ± 2.650.73 ± 0.78
593 d(p*A) H3'3 d(p*A) H1'3.970.02 ± 0.070.02 ± 0.07
603 d(p*A) H3'3 d(p*A) H83.460 ± 01.34 ± 1.11
613 d(p*A) H4'3 d(p*A) H1'3.150.4 ± 0.460.13 ± 0.26
623 d(p*A) H4'3 d(p*A) H2'13.591.53 ± 0.751.24 ± 0.71
633 d(p*A) H4'3 d(p*A) H2'23.500 ± 00.34 ± 0.47
643 d(p*A) H4'3 d(p*A) H84.220 ± 0.030.07 ± 0.16
651 dT H1'2 dC H5'13.700.79 ± 0.813.59 ± 1.59
661 dT H1'2 dC H5'23.720 ± 00.84 ± 0.69
671 dT H1'2 dC H64.360.11 ± 0.220.16 ± 0.33
681 dT H2'22 dC H63.940 ± 00 ± 0
691 dT H2'22 dC H5'13.970 ± 00 ± 0
701 dT H2'22 dC H5'24.090 ± 00 ± 0
711 dT H2'12 dC H54.540 ± 0.030 ± 0
721 dT H2'12 dC H63.780.02 ± 0.090 ± 0
731 dT H3'2 dC H5'13.350.11 ± 0.210 ± 0
741 dT H3'2 dC H5'23.340.44 ± 0.490.44 ± 0.5
751 dT H3'2 dC H64.080.34 ± 0.461.2 ± 0.88
761 dT H62 dC H5'14.520 ± 0.020 ± 0.03
771 dT H62 dC H5'24.650 ± 00.73 ± 0.82
781 dT M72 dC H54.460.05 ± 0.190 ± 0.03
791 dT H63 d(p*A) H2'15.380.01 ± 0.130.35 ± 0.57
802 dC H2'23 d(p*A) H84.282.01 ± 1.310 ± 0
812 dC H2'13 d(p*A) H84.351.44 ± 0.780 ± 0
822 dC H63 d(p*A) H2'14.721.38 ± 1.110.14 ± 0.35
833 d(p*A) H21 dT M74.870.29 ± 0.490 ± 0
843 d(p*A) H82 dC H5'15.930 ± 00 ± 0
853 d(p*A) H82 dC H5'25.410 ± 00.97 ± 0.83
861 dT H1'3 d(p*A) Me4.920 ± 00 ± 0
871 dT H4'3 d(p*A) Me4.320.79 ± 0.820.17 ± 0.39
881 dT H63 d(p*A) Me4.840.21 ± 0.390 ± 0
892 dC H1'3 d(p*A) Me4.020.05 ± 0.160.04 ± 0.17
902 dC H3'3 d(p*A) Me3.860 ± 00 ± 0
913 d(p*A) H23 d(p*A) Me4.071.46 ± 1.170.87 ± 0.95
923 d(p*A) H4'3 d(p*A) Me3.420.18 ± 0.430.45 ± 0.56
933 d(p*A) Me2 dC H5'13.710 ± 0.030 ± 0
943 d(p*A) Me2 dC H5'23.820 ± 00.34 ± 0.51
953 d(p*A) Me3 d(p*A) H83.020 ± 0.030.24 ± 0.46
963 d(p*A) Me2 dC H64.640.03 ± 0.110 ± 0
Table 14

NOESY NMR restraints with mixing time 0.8s of ‘slow’ trinucleotide and MD restraint penalty values for Rp- and Sp-diastereomers.

#Residue number, Residue name, Atom NameResidue number, Residue name, Atom NameDistance, ÅRestraint penalty, kcal/mol
RpSp
11 dT H1'1 dT H3'4.650 ± 00 ± 0
21 dT H1'1 dT H3'4.320 ± 00 ± 0
31 dT H1'1 dT H62.749.62 ± 1.939.52 ± 1.92
41 dT H2'21 dT H1'3.350 ± 00 ± 0
51 dT H2'21 dT H2'12.900.07 ± 0.110.07 ± 0.1
61 dT H2'21 dT H3'3.430 ± 00 ± 0
71 dT H2'21 dT H4'4.120 ± 0.030 ± 0.01
81 dT H2'21 dT H63.540.3 ± 0.480.34 ± 0.52
91 dT H2'11 dT H3'2.980 ± 00 ± 0
101 dT H2'11 dT H62.580 ± 0.010 ± 0.03
111 dT H4'1 dT H1'3.590.15 ± 0.410.13 ± 0.37
121 dT H4'1 dT H3'3.210 ± 00 ± 0
131 dT H4'1 dT H64.360.06 ± 0.210.07 ± 0.24
141 dT H5'11 dT H3'3.380.19 ± 0.420.16 ± 0.37
151 dT H5'11 dT H4'3.120 ± 0.010 ± 0.02
161 dT H5'11 dT H64.030.07 ± 0.30.08 ± 0.28
171 dT H5'21 dT H3'3.460.12 ± 0.260.12 ± 0.28
181 dT H5'21 dT H4'2.780.29 ± 0.560.25 ± 0.45
191 dT H5'21 dT H63.800.21 ± 0.440.23 ± 0.52
201 dT H61 dT H3'3.350.32 ± 0.630.3 ± 0.55
211 dT M71 dT H62.480.01 ± 0.040.01 ± 0.04
222 dC H1'2 dC H2'13.160 ± 00 ± 0
232 dC H1'2 dC H4'2.980.19 ± 0.370.28 ± 0.47
242 dC H1'2 dC H62.935.3 ± 1.285.22 ± 1.32
252 dC H2'22 dC H1'2.640 ± 00 ± 0
262 dC H2'22 dC H2'12.490.12 ± 0.150.13 ± 0.16
272 dC H2'22 dC H3'3.560 ± 00 ± 0
282 dC H2'22 dC H4'3.300.01 ± 0.050 ± 0.02
292 dC H2'22 dC H63.182.58 ± 1.252.57 ± 1.25
302 dC H2'12 dC H4'3.700.16 ± 0.240.17 ± 0.26
312 dC H3'2 dC H2'13.330 ± 00 ± 0
322 dC H3'2 dC H64.270 ± 00 ± 0
332 dC H52 dC H62.500.1 ± 0.170.1 ± 0.16
342 dC H62 dC H2'12.700 ± 0.020 ± 0.03
352 dC H62 dC H4'3.990.55 ± 0.680.55 ± 0.67
363 d(p*A) H1'3 d(p*A) H5'23.910.93 ± 0.70.98 ± 0.74
373 d(p*A) H1'3 d(p*A) H83.273.46 ± 1.123.53 ± 1.22
383 d(p*A) H2'23 d(p*A) H1'2.630 ± 00 ± 0
393 d(p*A) H2'23 d(p*A) H2'12.820.03 ± 0.060.03 ± 0.07
403 d(p*A) H2'23 d(p*A) H3'3.510 ± 00 ± 0
413 d(p*A) H2'23 d(p*A) H5'16.020 ± 00 ± 0
423 d(p*A) H2'23 d(p*A) H83.810.09 ± 0.230.07 ± 0.18
433 d(p*A) H2'13 d(p*A) H1'3.270 ± 00 ± 0
443 d(p*A) H2'13 d(p*A) H3'2.940 ± 00 ± 0
453 d(p*A) H2'13 d(p*A) H5'13.570.46 ± 0.610.42 ± 0.63
463 d(p*A) H2'13 d(p*A) H5'23.460 ± 0.010 ± 0
473 d(p*A) H2'13 d(p*A) H82.730 ± 00 ± 0.02
483 d(p*A) H3'3 d(p*A) H1'3.910.04 ± 0.110.03 ± 0.09
493 d(p*A) H3'3 d(p*A) H5'13.010 ± 00 ± 0
503 d(p*A) H3'3 d(p*A) H5'23.090 ± 0.010 ± 0.01
513 d(p*A) H3'3 d(p*A) H83.351.18 ± 1.121.37 ± 1.17
523 d(p*A) H4'3 d(p*A) H1'3.090.27 ± 0.460.17 ± 0.33
533 d(p*A) H4'3 d(p*A) H2'13.362.67 ± 0.982.68 ± 1.06
543 d(p*A) H4'3 d(p*A) H2'23.471.26 ± 0.881.3 ± 0.83
553 d(p*A) H83 d(p*A) H5'14.100.03 ± 0.130.03 ± 0.13
563 d(p*A) H83 d(p*A) H5'24.330 ± 00 ± 0
571 dT H3'2 dC H64.020.04 ± 0.210.04 ± 0.18
582 dC H2'23 d(p*A) H5'24.350 ± 00 ± 0
592 dC H2'13 d(p*A) H84.520.32 ± 0.620.5 ± 0.7
603 d(p*A) H82 dC H4'4.090.03 ± 0.160 ± 0.02
Table 15

Total restraint penalty energies (in kcal/mol) calculated for the last frame of MD simulation annealing.

Mixing time‘fast’ TpCp*A
‘slow’ TpCp*A
RpSpRpSp
0.4s10.52 ± 2.0210.45 ± 2.2813.35 ± 1.4814.30 ± 1.43
0.8s25.47 ± 3.5423.71 ± 2.8631.20 ± 3.8531.45 ± 4.10
Fig. 12

Distribution of total NMR distance energy penalties obtained in molecular dynamics simulation annealing analysis using distance restraints obtained from NOESY NMR data for ‘fast’ and ‘slow’ diastereomers applied for Rp- and Sp-substituted trinucleotides with mixing times 0.4 and 0.8 s. The distributions with the different penalties for the diastereomers are shown in Fig. 4 in Ref. [1].

Fig. 13

Comparison of average restraint penalty energy values (in kcal/mol) calculated for final structures of 500 annealing simulations for the ‘fast’ and ‘slow’ isomers (NOESY NMR mixing time 0.4 s). The number in horizontal axis corresponds to the serial number of restraint in Tables S11–S12 Bars are standard deviations.

Fig. 14

Comparison of average restraint penalty energy values (in kcal/mol) calculated for final structures of 500 annealing simulations for the ‘fast’ and ‘slow’ isomers (NOESY NMR mixing time 0.8 s). The number in horizontal axis corresponds to the serial number of restraint in Tables S11–S12 Bars are standard deviations.

Table 16

Summary of the cluster analysis of Rp-isomer using NOESY NMR restraints of ‘slow’ trinucleotide with mixing time 0.4s.

#ClusterFramesFracAvgDistStdevAvgCDist
0125080.4893.4870.9615.056
151930.2033.1690.8084.95
238290.153.6730.8235.041
311140.0443.1890.7775.083
410300.043.5950.785.082
59370.0373.550.7644.962
66510.0253.2530.7955.178
72040.0083.5060.8345.031
81130.0043.410.8174.972
9230.0012.3920.6175.171

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.

Table 17

Summary of the cluster analysis of Sp-isomer using NOESY NMR restraints of ‘fast’ trinucleotide with mixing time 0.4s.

#ClusterFramesFracAvgDistStdevAvgCDist
0155440.6072.4850.7065.075
143800.1712.7050.6194.839
223900.0933.1550.7834.924
315310.063.2090.7644.974
48540.0332.9640.6975.165
58010.0313.0180.7555.035
6670.0032.6950.6634.684
7230.0012.2370.6894.846
8902.3410.5834.728
9301.4220.1725.073
Table 18

Summary of the cluster analysis of Rp-isomer using NOESY NMR restraints of ‘slow’ trinucleotide with mixing time 0.8s.

#ClusterFramesFracAvgDistStdevAvgCDist
0121930.4762.4340.4823.536
1109520.4282.0030.4664.101
210050.0392.3050.5233.889
36970.0272.6060.5153.51
44690.0182.1670.5113.899
52310.0092.3810.4413.925
6310.0012.0910.4953.483
7180.0012.1130.4373.653
8401.410.2374.188
9201.79803.967
Table 19

Summary of the cluster analysis of Sp-isomer using NOESY NMR restraints of ‘fast’ trinucleotide with mixing time 0.8s.

#ClusterFramesFracAvgDistStdevAvgCDist
0252640.9872.3370.4743.895
11500.0062.40.5084.439
21020.0041.9930.4983.958
3500.0021.6030.3984.764
4140.0012.1650.6174.185
5602.2320.3573.935
6502.2360.524.481
7502.1750.6394.084
8302.0550.4284.231
9302.2030.4594.247
Fig. 10

RMSD map for 500 final structures after simulation annealing. Mixing time 0.4 s.

Fig. 11

RMSD map for 500 final structures after simulation annealing. Mixing time 0.8 s.

NOESY NMR restraints with mixing time 0.4s of ‘fast’ trinucleotide and MD restraint penalty values for Rp- and Sp-diastereomers. NOESY NMR restraints with mixing time 0.4s of ‘slow’ trinucleotide and MD restraint penalty values for Rp- and Sp-diastereomers. NOESY NMR restraints with mixing time 0.8s of ‘fast’ trinucleotide and MD restraint penalty values for Rp- and Sp-diastereomers. NOESY NMR restraints with mixing time 0.8s of ‘slow’ trinucleotide and MD restraint penalty values for Rp- and Sp-diastereomers. Total restraint penalty energies (in kcal/mol) calculated for the last frame of MD simulation annealing. Distribution of total NMR distance energy penalties obtained in molecular dynamics simulation annealing analysis using distance restraints obtained from NOESY NMR data for ‘fast’ and ‘slow’ diastereomers applied for Rp- and Sp-substituted trinucleotides with mixing times 0.4 and 0.8 s. The distributions with the different penalties for the diastereomers are shown in Fig. 4 in Ref. [1]. Comparison of average restraint penalty energy values (in kcal/mol) calculated for final structures of 500 annealing simulations for the ‘fast’ and ‘slow’ isomers (NOESY NMR mixing time 0.4 s). The number in horizontal axis corresponds to the serial number of restraint in Tables S11–S12 Bars are standard deviations. Comparison of average restraint penalty energy values (in kcal/mol) calculated for final structures of 500 annealing simulations for the ‘fast’ and ‘slow’ isomers (NOESY NMR mixing time 0.8 s). The number in horizontal axis corresponds to the serial number of restraint in Tables S11–S12 Bars are standard deviations. Summary of the cluster analysis of Rp-isomer using NOESY NMR restraints of ‘slow’ trinucleotide with mixing time 0.4s. 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. Summary of the cluster analysis of Sp-isomer using NOESY NMR restraints of ‘fast’ trinucleotide with mixing time 0.4s. Summary of the cluster analysis of Rp-isomer using NOESY NMR restraints of ‘slow’ trinucleotide with mixing time 0.8s. Summary of the cluster analysis of Sp-isomer using NOESY NMR restraints of ‘fast’ trinucleotide with mixing time 0.8s. RMSD map for 500 final structures after simulation annealing. Mixing time 0.4 s. RMSD map for 500 final structures after simulation annealing. Mixing time 0.8 s.

Experimental design, materials and methods

Synthesis

Standard phoshoramidite solid-phase synthesis of all modified and unmodified oligonucleotides containing phosphodiester linkages (PO) was carried out on an ASM-800 DNA/RNA synthesizer (‘‘Biosset’’, Russia). Oligonucleotides were synthesized at 1 μmol scale, using standard commercial 2-cyanoethyl deoxynucleoside phosphoramidites and CPG solid supports (Glen Research, USA). Oligonucleotides with internucleotide tetramethyl phosphoryl guanidine group was synthetized as described in Refs. [2], [3].

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 × 150 mm. For native oligonucleotede linear gradient of buffer B (acetonitrile 0–50% 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 −20 °C. Absorption spectra were recorded at wavelengths from 220 to 600 nm.

MALDI-TOF MS analysis

Matrix-assisted laser desorption ionization – 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.

SVDPE digestion assay

Oligonucleotides were incubated with SVDPE at 37 °C. Reaction micture contaned 3 μg of SVDPE, 20 mM Tris-HCl, pH 9.0, 5 mM MgCl2 and 0.1 mM oligonucleotide [4]. Aliquots of 20 μl withdrawn at 0.5, 1, 24, 48 and 150h, heated to 65 °C for 30 min, then analyzed by RP-HPLC.

Circular dichroism (CD) experiments

CD spectra were measured on a J-600 spectropolarimeter (Jasco, Japan) using temperature-controlled 1 mm pathlength quartz cell. The mesurments were performed in the range 190–330 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 190–330 nm at 25 °C. UV spectra were registered every 0.1 nm, bandwidth 1 nm. Oligonucluotides in concentration 12 μM 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 1H, 1H{31P}, 13C{1H}, 31P, 31P{1H}, 1H × 1H COSY, 1H × 13C HMBC, 1H × 31P HMBC, 1H × 1H TOCSY, 1H DOSY, 1H × 1H NOESY (with mixing time 0.25 s), 1H × 1H ROESY (with mixing time 0.25 s). Spectra were processed on Bruker Topspin and analyzed in CCPNMR2 [7]. The assignments of signals in the spectra were carried out by their combined analysis. The spin systems of deoxyribose residues were identified from 1H–1H COSY (Fig. 6, Fig. 7) and 1H–1H TOCSY spectra. The diastereotope assignment of the signals of proton H2' and H2'' were carried out on the basis of 1H–1H NOESY spectra (see Fig. 8, Fig. 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 H5–H6 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 S1–10), the spin-spin interaction constants 1H–1H were extracted from the 1H {31P} spectra and then the obtained data were used to measure the 1H–31P 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′-adenosine were generated. NMR distance restraints were used for subsequent refinement of the structure. The upper bounds of the restraints were set to +0.5 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 1−1 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·Å2] 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.

Specifications Table

Subject areaChemistry, Physical Chemistry, Biology
More specific subject areaBiochemistry and physical chemistry of nucleic acids
Type of dataTable, graph, figure
How data was acquiredHigh pressure liquid chromatography, mass spectroscopy, Circular Dichroism spectroscopy, 1D and 2D NMR, Molecular dynamics simulations
Data formatRaw and analyzed data
Experimental factorsNative trinucleotide d(TpCpA) and individual diastereomers of mono-substituted phosphoryl guanidine oligonucleotide d(TpCp*A) were purified and analyzed
Experimental featuresIndividual 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 locationInstitute of Chemical Biology and Fundamental Medicine of Siberian Branch of the Russian Academy of Sciences, 8 Lavrentiev Ave., Novosibirsk, 630090, Russian FederationNMR data were collected in N.N. Vorozhtsov Novosibirsk Institute of Organic Chemistry of Siberian Branch of the Russian Academy of Sciences, 9 Lavrentiev Ave., Novosibirsk, 630090, Russian Federation
Data accessibilityData is with this article
Related research articleA. A. Lomzov, M. S. Kupryushkin, A. V. Shernyukov, M. D. Nekrasov, I. S. Dovydenko, D. A. Stetsenko, D. V. Pyshnyi, Diastereomers of a mono-substituted phosphoryl guanidine trideoxyribonucleotide: Isolation and properties, Biochem. Biophys. Res. Commun., 514,2019, 807–811[1].
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

  8 in total

1.  UCSF Chimera--a visualization system for exploratory research and analysis.

Authors:  Eric F Pettersen; Thomas D Goddard; Conrad C Huang; Gregory S Couch; Daniel M Greenblatt; Elaine C Meng; Thomas E Ferrin
Journal:  J Comput Chem       Date:  2004-10       Impact factor: 3.376

2.  PTRAJ and CPPTRAJ: Software for Processing and Analysis of Molecular Dynamics Trajectory Data.

Authors:  Daniel R Roe; Thomas E Cheatham
Journal:  J Chem Theory Comput       Date:  2013-06-25       Impact factor: 6.006

3.  Recommendations for the presentation of NMR structures of proteins and nucleic acids--IUPAC-IUBMB-IUPAB Inter-Union Task Group on the standardization of data bases of protein and nucleic acid structures determined by NMR spectroscopy.

Authors:  J L Markley; A Bax; Y Arata; C W Hilbers; R Kaptein; B D Sykes; P E Wright; K Wüthrich
Journal:  Eur J Biochem       Date:  1998-08-15

4.  Enzymatic assignment of diastereomeric purity of stereodefined phosphorothioate oligonucleotides.

Authors:  M Koziołkiewicz; A Owczarek; E Gendaszewska
Journal:  Antisense Nucleic Acid Drug Dev       Date:  1999-04

5.  Diastereomers of a mono-substituted phosphoryl guanidine trideoxyribonucleotide: Isolation and properties.

Authors:  Alexander A Lomzov; Maxim S Kupryushkin; Andrey V Shernyukov; Mikhail D Nekrasov; Ilya S Dovydenko; Dmitry A Stetsenko; Dmitrii V Pyshnyi
Journal:  Biochem Biophys Res Commun       Date:  2019-04-15       Impact factor: 3.575

6.  Study of a DNA Duplex by Nuclear Magnetic Resonance and Molecular Dynamics Simulations. Validation of Pulsed Dipolar Electron Paramagnetic Resonance Distance Measurements Using Triarylmethyl-Based Spin Labels.

Authors:  Alexander A Lomzov; Eugeniy A Sviridov; Andrey V Shernuykov; Georgiy Yu Shevelev; Dmitrii V Pyshnyi; Elena G Bagryanskaya
Journal:  J Phys Chem B       Date:  2016-06-03       Impact factor: 2.991

7.  The CCPN data model for NMR spectroscopy: development of a software pipeline.

Authors:  Wim F Vranken; Wayne Boucher; Tim J Stevens; Rasmus H Fogh; Anne Pajon; Miguel Llinas; Eldon L Ulrich; John L Markley; John Ionides; Ernest D Laue
Journal:  Proteins       Date:  2005-06-01

8.  Phosphoryl guanidines: a new type of nucleic Acid analogues.

Authors:  M S Kupryushkin; D V Pyshnyi; D A Stetsenko
Journal:  Acta Naturae       Date:  2014-10       Impact factor: 1.845
  8 in total
  2 in total

1.  Control of backbone chemistry and chirality boost oligonucleotide splice switching activity.

Authors:  Pachamuthu Kandasamy; Graham McClorey; Mamoru Shimizu; Nayantara Kothari; Rowshon Alam; Naoki Iwamoto; Jayakanthan Kumarasamy; Gopal R Bommineni; Adam Bezigian; Onanong Chivatakarn; David C D Butler; Michael Byrne; Katarzyna Chwalenia; Kay E Davies; Jigar Desai; Juili Dilip Shelke; Ann F Durbin; Ruth Ellerington; Ben Edwards; Jack Godfrey; Andrew Hoss; Fangjun Liu; Kenneth Longo; Genliang Lu; Subramanian Marappan; Jacopo Oieni; Ik-Hyeon Paik; Erin Purcell Estabrook; Chikdu Shivalila; Maeve Tischbein; Tomomi Kawamoto; Carlo Rinaldi; Joana Rajão-Saraiva; Snehlata Tripathi; Hailin Yang; Yuan Yin; Xiansi Zhao; Cong Zhou; Jason Zhang; Luciano Apponi; Matthew J A Wood; Chandra Vargeese
Journal:  Nucleic Acids Res       Date:  2022-06-10       Impact factor: 19.160

2.  Impact of guanidine-containing backbone linkages on stereopure antisense oligonucleotides in the CNS.

Authors:  Pachamuthu Kandasamy; Yuanjing Liu; Vincent Aduda; Sandheep Akare; Rowshon Alam; Amy Andreucci; David Boulay; Keith Bowman; Michael Byrne; Megan Cannon; Onanong Chivatakarn; Juili Dilip Shelke; Naoki Iwamoto; Tomomi Kawamoto; Jayakanthan Kumarasamy; Sarah Lamore; Muriel Lemaitre; Xuena Lin; Kenneth Longo; Richard Looby; Subramanian Marappan; Jake Metterville; Susovan Mohapatra; Bridget Newman; Ik-Hyeon Paik; Saurabh Patil; Erin Purcell-Estabrook; Mamoru Shimizu; Pochi Shum; Stephany Standley; Kris Taborn; Snehlata Tripathi; Hailin Yang; Yuan Yin; Xiansi Zhao; Elena Dale; Chandra Vargeese
Journal:  Nucleic Acids Res       Date:  2022-06-10       Impact factor: 19.160
  2 in total

北京卡尤迪生物科技股份有限公司 © 2022-2023.