Pyrimidine-modified Oligonucleotides ... Modification. Calc. Found ..... opposite strand (strand A), the backbone of the modified nucleotide exhibits a stretched.
S1
Structural Basis of Duplex Thermodynamic Stability and Enhanced Nuclease Resistance of 5'-C-Methyl Pyrimidine-modified Oligonucleotides Alexander V. Kel’in,† Ivan Zlatev,† Joel Harp,‡ Muthusamy Jayaraman,† Anna Bisbe,† Jonathan O’Shea, † Nate Taneja, † Rajar M. Manoharan, † Saeed Khan,§ Klaus Charisse, †
Martin A. Maier,† Martin Egli,‡ Kallanthottathil G. Rajeev,*† Muthiah Manoharan*†
†
Alnylam Pharmaceuticals, 300 Third Street, Cambridge, Massachusetts 02142, United States
§
Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095,
United States ‡
Department of Biochemistry and Center for Structural Biology, Vanderbilt University, School of Medicine,
Nashville, TN, 37232, United States
Table of contents Sequence and mass spectral data of oligonucleotides
S2
Melting temperature measurements
S3
SVPDE stability measurements and graphs
S4-S6
X-ray crystallography, Materials and Methods, Diffraction Data and Crystal S7-S14 Structure of Nucleosides References
S14-S15
NMR Spectra of Compounds
S16-127
LC-MS traces of oligonucleotides reported in Table S1.
S128-148
S2 Table S1. Mass spectral data of the oligonucleotides used in this study Oligonucleotide 5′-d(TACAGTCTATGT)-3′
Mass Calc. 3635.40
Found 3634.74
Modification Unmodified strand for Tm measurement
5′-d(TACAGQCTATGT)-3′ 5′-d(TACAGQCTATGT)-3′ 5′-d(TACAGQCTATGT)-3′ 5′-d(TACAGQCTATGT)-3′ 5′-d(TACAGQCTATGT)-3′ 5′-d(TACAGQCTATGT)-3′ 5′-d(TACAGQCTATGT)-3′ 5′-d(TACAGQCTATGT)-3′ 5′-d(ACATAGACTGTA)-3′ 5′-r(UACAGUCUAUGU)-3′ 5′-r(UACAGQCUAUGU)-3′ 5′-r(UACAGQCUAUGU)-3′ 5′-r(UACAGQCUAUGU)-3′ 5′-r(UACAGQCUAUGU)-3′ 5′-r(UACAGQCUAUGU)-3′ 5′-r(UACAGQCUAUGU)-3′ 5′-r(UACAGQCUAUGU)-3′ 5′-r(UACAGQCUAUGU)-3′ 5′-r(UACAGQCUAUGU)-3′ 5′-r(ACAUAGACUGUA)-3′ dT19Q dT19Q dT19Q dT19Q dT19Q dT19Q dT19 ●Q dT19 ●Q dT19 ●Q dT19 ●Q dT19 ●Q dT19 ●Q dT19 QQ dT18QQ dT18QQ dT18QQ dT18QQ dT18QQ dT18QQ dT18QQ dT18Q●Q dT18Q●Q dT18Q●Q dT18Q●Q dT18Q●Q dT18Q●Q dT18Q●Q dT18Q●Q 5′-r(CGAAQUCG) 5′-r(CGAAQUCG) 5′-r(CGAAQUCG)
3639.36 3653.39 3653.39 3651.40 3665.43 3665.43 3649.43 3649.43 3653.43 3757.27 3759.26 3773.29 3773.29 3771.30 3785.32 3785.32 3755.30 3769.33 3769.33 3803.35 6039.89 6039.89 6051.92 6051.92 6035.93 6035.93 6055.96 6055.96 6067.99 6067.99 6051.99 6051.99 6029.83 6057.88 6057.88 6053.90 6081.95 6081.95 6049.96 6049.96 6045.89 6073.95 6069.96 6098.02 6098.02 6037.96 6066.02 6066.02 2525.58 2525.58 2507.59
3638.93 3652.93 3653.02 3650.95 3664.77 3664.82 3648.87 3648.82 3652.75 3756.59 3758.72 3772.77 3772.82 3770.77 3784.75 3784.72 3754.74 3768.69 3768.75 3802.64 6038.40 6038.30 6051.27 6051.28 6035.23 6035.16 6054.50 6054.30 6067.26 6067.18 6051.32 6051.32 6029.16 6055.90 6056.20 6053.11 6081.14 6081.27 6049.12 6049.32 6043.23 6072.40 6069.26 6097.38 6097.36 6036.36 6065.28 6065.27 2524.62 2524.62 2505.40
Q = 2′-FU Q = (R)-C5′-Me-2′-FU Q = (S)-C5′-Me-2′-FU Q = 2′-OMeU Q = (R)-C5′-Me-2′-OMeU Q = (S)-C5′-Me-2′-OMeU Q = (R)-C5′-Me-dT Q = (S)-C5′-Me-dT Tm, unmodified complementary strand Unmodified strand for Tm measurement Q = 2′-FU Q = (R)-C5′-Me-2′-FU Q = (S)-C5′-Me-2′-FU Q = 2′-OMeU Q = (R)-C5′-Me-2′-OMeU Q = (S)-C5′-Me-2′-OMeU Q = dT Q = (R)-C5′-Me-dT Q = (S)-C5′-Me-dT Tm, unmodified complementary strand Q = (R)-C5′-Me-2′-FU Q = (S)-C5′-Me-2′-FU Q = (R)-C5′-Me-2′-OMeU Q = (S)-C5′-Me-2′-OMeU Q = (R)-C5′-Me-dT Q = (S)-C5′-Me-dT Q = (R)-C5′-Me-2′-FU Q = (S)-C5′-Me-2′-FU Q = (R)-C5′-Me-2′-OMeU Q = (S)-C5′-Me-2′-OMeU Q = (R)-C5′-Me-dT Q = (S)-C5′-Me-dT Q = 2′-FU Q = (R)-C5′-Me-2′-FU Q = (S)-C5′-Me-2′-FU Q = 2′-OMeU Q = (R)-C5′-Me-2′-OMeU Q = (S)-C5′-Me-2′-OMeU Q = (R)-C5′-Me-dT Q = (S)-C5′-Me-dT Q = 2′-FU Q = (S)-C5′-Me-2′-FU Q = 2′-OMeU Q = (R)-C5′-Me-2′-OMeU Q = (S)-C5′-Me-2′-OMeU Q = dT Q = (R)-C5′-Me-dT Q = (S)-C5′-Me-dT Q = (R)-C5′-Me-2′-FU Q = (S)-C5′-Me-2′-FU Q = dT
S3 Melting temperature measurements. All single strands (sense and antisense) were dissolved in nuclease-free water to a concentration of 1 mM. For the melting duplexes, 20 µL of each strand were mixed together, 100 µL of 10×PBS, pH 7.4 were added, followed by 860 µL of nuclease-free water, resulting in 1 mL of stock duplex in 1×PBS. The stock duplex was diluted ~8 × with 1×PBS buffer, and the concentration was adjusted to AU at 260 nm of 0.5 ODU/mL (± 5%) for each melting duplex. Melting point temperature (Tm) was experimentally determined on a DU 800 Series UV/Vis spectrophotometer equipped with the highperformance peltier temperature controller, the Micro Auto 6 Tm cell holder (six 325 µL Tm microcells with stopper) and the Tm analysis software. Duplexes were analyzed in the six 325 µL-samples format with duplex denaturation and renaturation profiles measured within a temperature range from 20.0 to 80.0 ⁰C with temperature ramping of 1.0 ⁰C/min. All Tm values were calculated using the first derivative method provided with the Tm analysis software and average values from two separate experiments (independent duplex preparations), each one consisting of two independent Tm measurements were calculated for each melting duplex. Average values were calculated using Microsoft Excel. SVPD stability measurements and graphs. DNA single strands solution were prepared at 0.1 mg/mL concentration in 50 mM TRIS-HCl (pH 8) buffer, containing 10 mM MgCl2. 1 mU/mL snake venom phosphodiesterase I (SVPD) was added to the DNA mixture prior to first injection and kinetics were monitored for 24 h at room temperature. Time points (0–24 h) were injected directly on the analytical HPLC ion-exchange column (one time point injection
S4 every 1 h) from the same sample preparation and analyzed by ion-exchange using a Dionex DNAPac PA200 analytical column at 30 ⁰C column temperature. A gradient of 37% to 52% 1 M NaBr, 10% CH3CN, 20 mM Na2PO4 buffer at pH 11, over 10 min with a flow rate of 1 mL/min was used to analyze each sample. Peak area integration at λ = 260 nm was used to calculate percentage of enzymatic degradation for each time point, as normalized per the area of the sample at time 0 h. Decay curves were plotted using XLFit in Microsoft Excel. Half-lives were determined at the time corresponding of 50% of total normalized integration area (50% of degradation). The enzymatic degradation method was calibrated at each run for a standard enzyme control oligonucleotide: dTdTdTdTdTdTdTdTdTdTdTdTdTdTdTdTdTdTdT●dT,
where
●
indicates
phosphorothioate (PS) diester linkage and dT is 2′-deoxythymidine nucleotide; with halflife of the enzyme control oligonucleotide being 2.5 h ± 0.3h. Each degradation experiment was run in duplicates. Representative decay curves and HPLC plots are shown below:
S5
(a)
(b)
Figure S1. Time course HPLC profile upon incubation with SVPD full length ON remaining with time between 0-24 h (a) dT18dT●dT and (b) dT18Y●Y, where dT is thymidine; Y is (S)-C5′-Methyl-2′-deoxy-2′fluorouridine and ● indicates phosphorothioate linkage
S6
(a)
(b)
(c)
Figure S2. Exonuclease activity on dT18QQ and dT18Q●Q upon incubation with SVPD. % of full length ON remaining with time between 0-24 h upon incubation with SVPD, where Q is for: (a) (R)- or (S)-5′methyl-2′-deoxythymidine; (b) (R)- or (S)-5′-methyl-2′-methoxyuridine and (c) (R) or S)-C5′-Methyl-2′deoxy-2′-fluorouridine, and ● indicates phosphorothioate linkage
S7
X-ray crystallography, Materials and Methods, Diffraction Data and Crystal Structure of Nucleosides Crystallization The (R)-C5′-Me-2′-FU-containing duplex [r(CGAAXUCG)]2 (X=(R)-C5′-Me-2′-FU) was crystallized by sitting-drop vapor diffusion with 1 mM oligonucleotide mixed 1:1 with a solution of 10% 2-methyl-2,4pentanediol (MPD), 40 mM sodium cacodylate pH 5.5, 20 mM hexamine cobalt(III) chloride, 12 mM sodium chloride, 80 mM potassium chloride and equilibrated against a reservoir of 40% MPD. The (S)-C5′-Me-2′-FU-containing duplex [r(BrCGAAXUCG)]2 (X=(S)-C5′-Me-2′-FU) with a 5′-terminal 5bromo-C nucleoside (BrC) was crystallized by sitting-drop vapor diffusion with 1 mM oligonucleotide mixed 1:1 with a solution of 10% MPD, 40 mM sodium cacodylate pH 5.5, 20 mM hexamine cobalt(III) chloride, 20 mM MgCl2 and equilibrated against a reservoir of 40% MPD. The reference dT-containing duplex [r(CGAAdTUCG)]2 was crystallized by sitting-drop vapor diffusion with 1 mM oligonucleotide mixed 1:1 with a solution of 10% MPD, 40 mM sodium cacodylate pH 5.5, 20 mM hexamine cobalt(III) chloride, 80 mM sodium chloride, 20 mM magnesium chloride and equilibrated against a reservoir of 40% MPD. X-ray diffraction data collection Nucleoside: The diffraction intensities were measured at 100(2) K on a X-ray diffractometer system equipped with multilayer mirror optics and Cu-IµS micro-focus radiation source (λ=1.54178 Å). The cell parameters were obtained from the least-squares refinement of carefully centered reflections in a wide 2θ range. The frames were integrated with a V8.34A software package, using a narrowframe algorithm. Data were corrected for absorption effects using a multi-scan method, SADABS, V2012. . All non-hydrogen atoms were refined anisotropically, and hydrogens atoms were introduced in calculated positions with isotropic thermal parameters set 20% to 30% greater than their bonding partners. The structures were refined with full-matrix least–squares on F2 until convergence. Oligonucleotide Duplex: Diffraction data for the (R)-C5′-Me-2′-FU duplex were collected in-house on a rotating-anode X-ray generator equipped with a CCD area detector and a cryostat. Diffraction data
S8 for the reference dT- and (S)-C5′-Me-2′-FU-containing duplex crystals were collected using synchrotron radiation at the Life Sciences-Collaborative Access Team, LS-CAT, 21-ID-D beam line at the Advanced Photon Source, Argonne National Laboratory. All crystals were flash-cooled directly from the crystallization mother liquor and maintained at 100 K during data collection. Data for a brominated (S)-C5′-Me-2′-FU crystal were collected near the Br K-edge (wavelength 0.91833 Å). Data reduction was performed using Proteum software for the in-house data and using HKL20001 for all synchrotron data. Selected crystal data and data collection parameters are summarized in Table S1. Structure determination and refinement The structures of the reference dT-containing and the (R)-C5′-Me-2′-FU-containing duplexes were determined by molecular replacement using the program PHASER2 with an A-form RNA octamer duplex as the search model. The structure of the (S)-C5′-Me-2′-FU duplex was phased by Br-SAD using the program HKL2MAP and SHELXC/D/E.3-6 Refinements were performed using PHENIX7 or REFMAC58, and manual fitting was performed using COOT.9 Final refinement parameters are summarized in Table S1. Crystals of the (R)-C5′-Me-2′-FU-containing and reference duplexes are both of space group P43212 with two independent duplexes per asymmetric unit. The presence of two duplexes per asymmetric unit in the structure of the (R)-C5′-Me-2′-FU duplex allows analysis of the conformation of four independent octamers that contain the trimer 5'-AXU (X=(R)-C5′-Me-2′-FU). Superimposition of the four trinucleotides revealed nearly identical conformations (Figure 5A; main paper). When one of the corresponding 5'-A(dT)U trimers of the four independent strands of the reference duplex was overlaid as well, it was clear that 5'-(R)-C-methylation causes only minor adjustments in the backbone geometry (Figure 5B; main paper). Indeed, the backbone torsion angles of (R)-C5′-Me-2′-FU nucleotides fall into the standard sc-/ap/sc+/sc+/ap/sc- ranges (α to ζ) with the sugar 2'-fluoro-2'-deoxyribose exhibiting a C3'-endo pucker (North range). The (S)-C5′-Me2′-FU-containing duplex also crystallized in space group P43212, but the a and b unit cell constants are shorter compared to the dT and (R)-C5′-Me-2′-FU duplex crystals and the asymmetric unit
S9 contains a single duplex. Moreover, the conformations of the two strands differ in the region of the modified nucleotide. The backbone torsion angles in one of them (strand B) fall into the standard sc/ap/sc+/sc+/ap/sc- range, although there are subtle adjustments in the angles to avoid a short contact between the (S)-C5′-methyl group and the 2′-hydroxyl group of the 5’ nucleotide. In the opposite strand (strand A), the backbone of the modified nucleotide exhibits a stretched conformation with all three angles α, β, and γ falling into the ap range. Four cobalt hexamine ions fill the major groove of the (S)-C5′-Me-2′-FU-containing duplex. Data deposition Coordinates and structure factors have been deposited in the Protein Data Bank (www.rcsb.org) with ID codes 5DER ((R)-C5′-Me-2′-FU), 5D8T ((S)-C5′-Me-2′-FU) and 5DEK (reference dT). Illustrations All figures were generated with the program UCSF Chimera.10
S10
Table S2. Selected crystal data, X-ray diffraction data collection and refinement data.
Structure
(R)-C5′-Me-2′-FU
dT Reference
(S)-C5′-Me-2′-FU
Wavelength [Å]
1.5418
0.9786
0.91833
Resolution range [Å] 25.34 - 1.80 (1.87 - 1.80)
39.16 - 1.99 (2.06 - 1.99)
30.00 - 1.20 (1.22 - 1.20)
Space group
P 43 21 2
P 43 21 2
P 43 21 2
Unit cell (a, b, c) [Å]
44.12, 44.12, 86.93
44.06, 44.06, 85.40
31.17, 31.17, 84.33
Total reflections
75,612
77,721
193,718
Unique reflections
8,451 (799)
6,053 (561)
13,758
Multiplicity
8.9 (5.1)
12.8 (7.5)
8.1(4.3)
Completeness (%)
99.75 (97.80)
97.80 (95.57)
99.7(100.0)
Mean I/sigma(I)
11.58 (2.26)
9.61 (1.94)
19.55 (4.85)
Wilson B-factor
16.77
39.25
5.3
R-merge
0.103 (0.419)
0.126 (0.682)
0.145 (0.584)
R-p.i.m.
0.034 (0.209)
0.038 (0.354)
0.036 (0.213)
Reflections for R-free 811
557
708
R-work
0.2525 (0.3235)
0.2358 (0.3464)
0.1214(0.138)
R-free
0.2945 (0.3781)
0.2809 (0.3985)
0.1536 (0.1690)
751
492
Number of non-H atoms 866 RNA
672
668
338
Ligands
8
10
4
Water
186
73
150
R.m.s.d. (bonds) [Å]
0.004
0.004
0.019
R.m.s.d. (angles) [°]
0.8
0.6
1.6
39.0
12.0
2
Average B-factor [Å ] 25.3 RNA
23.3
38.1
8.7
Ligands
39.6
57.2
16.2
Water
31.8
44.3
25.0
S11
Cambridge Crystallographic Data Centre Depository Information for Compounds: 1b, 1d, 4a, 4b, 4c, 1a, 3b, 3c, 3d, 7c and 8c Summary of Data CCDC 1435399 (1b) Formula: C15H25FN2O5Si Unit Cell Parameters: a 11.7332(2) b 7.11060(10) c 21.7964(3) P21 Summary of Data CCDC 1435400 (1d) Formula: C15H26N2O5Si Unit Cell Parameters: a 12.8467(17) b 6.8781(8) c 20.678(3) P21 Summary of Data CCDC 1435401 (4a) Formula: C17H30N2O6 Si,0.2(H2O) Unit Cell Parameters: a 18.4164(6) b 7.1764(2) c 16.1480(4) C2 Summary of Data CCDC 1435402 (4b) Formula: C16H27FN2O5Si Unit Cell Parameters: a 26.8444(13) b 6.9877(3) c 24.5888(13) C2 Summary of Data CCDC 1435403 (4c) Formula: 2(C17H30N2O5Si),H2O Unit Cell Parameters: a 6.4142(4) b 42.949(3) c 7.9259(5) P21 Summary of Data CCDC 1435404 (1a) Compound Name: Formula: C16H28N2O6Si Unit Cell Parameters: a 8.5192(4) b 24.7912(11) c 9.9306(5) P21 Summary of Data CCDC 1435405 (3b) Formula: C16H27FN2O5Si Unit Cell Parameters: a 5.9113(7) b 7.4445(10) c 21.509(3) P21 Summary of Data CCDC 1435406 (3c) Formula: C17H30N2O5Si, H2O Unit Cell Parameters: a 14.9336(14) b 6.4542(6) c 21.621(2) P21 Summary of Data CCDC 1435407 (3d) Formula: C16H28N2O5Si Unit Cell Parameters: a 22.5276(6) b 22.8393(6) c 7.7201(2) P21212 Summary of Data CCDC 1435408 (7c) Formula: C11 H16 N2 O5 Unit Cell Parameters: a 5.4882(3) b 14.0720(7) c 15.6434(8) P212121 Summary of Data CCDC 1435409 (8c) Formula: C11H16N2O5 Unit Cell Parameters: a 5.3929(2) b 14.5647(5) c 15.1113(5) P212121
S12
X-Ray structures of monomers.
S13
S14
Figure S3. The ellipsoid contour probability level is 50.at 100 ⁰K for compounds 1d, 3d, 4a, 4c, 7c and 8c, and that of compounds 1a is 50 at 298 ⁰K and 3c is 50 at 293 ⁰K.
References. (1) Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307. (2) McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M. D.; Storoni, L. C.; Read, R. J. J. Appl. Crystallogr. 2007, 40, 658. (3) Pape, T.; Schneider, T. R. J. Appl. Crystallogr. 2004, 37, 843. (4) Schneider, T. R.; Sheldrick, G. M. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2002, D58, 1772. (5) Sheldrick, G. M. Z. Kristallogr. 2002, 217, 644. (6) Sheldrick, G. M. 2003, Goettingen University. (7) Adams, P. D.; Afonine, P. V.; Bunkoczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.; Headd, J. J.; Hung, L. W.; Kapral, G. J.; Grosse-Kunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R. J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 213.
S15 (8) Murshudov, G.; Vagin, A.; Dodson, E. (1996) Application of Maximum Likelihood Refinement, in the Refinement of Protein structures, Proceedings of Daresbury Study Weekend. (9) Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Acta Crystallogr D Biol Crystallogr. 2010, 66, 486. (10) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. (2004) UCSF Chimera-a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605-1612.
S16 NMR Spectra of Compounds
S17
S18
S19
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S21
S22
S23
S24
S25
S26
S27
S28
S29
S30
S31
S32
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S35
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S45
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S47
S48
S49
S50
S51
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S53
S54
S55
S56
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S60
S61
S62
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S64
S65
S66
S67
S68
S69
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S71
S72
S73
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S75
S76
S77
S78
S79
S80
S81
S82
S83
S84
S85
S86
S87
S88
S89
S90
S91
S92
S93
S94
S95
S96
S97
S98
S99
S100
S101
S102
S103
S104
S105
S106
S107
S108
S109
S110
S111
S112
S113
S114
S115
S116
S117
S118
S119
S120
S121
S122
S123
NOESY
O NH N
HO O HO
F
O
S124
S125
S126
S127
S128
LC-MS traces of oligonucleotides reported in Table S1. 5′-d(TACAGTCTATGT)-3′ Calc: 3635.40
Found: 3634.74
S129
5′-d(TACAGQCTATGT)-3′ Calc: 3639.36 Q = 2′-FU
Found: 3638.93
S130
5′-d(TACAGQCTATGT)-3′ Calc: 3653.39 Q = (R)-C5′-Me-2′-FU
Found: 3652.93
S131
5′-d(TACAGQCTATGT)-3′ Calc: 3653.39 Q = (S)-C5′-Me-2′-FU
Found: 3653.02
S132
5′-d(TACAGQCTATGT)-3′ Calc: 3651.40 Q = 2′-OMeU
Found: 3650.95
S133
5′-d(TACAGQCTATGT)-3′ Calc : 3665.43 Q = (R)-C5′-Me-2′-OMeU
Found : 3664.77
S134
5′-d(TACAGQCTATGT)-3′ Calc : 3665.43 Q = (S)-C5′-Me-2′-OMeU
Found : 3664.82
S135
S136 5′-d(ACATAGACTGTA)-3′ Calc: 3653.43
Found: 3652.75
S137 5′-r(UACAGUCUAUGU)-3′ Calc: 3757.27
Found: 3756.59
S138 5′-r(UACAGQCUAUGU)-3′ Calc: 3759.26 Q = 2′-FU
Found: 3758.72
S139 5′-r(UACAGQCUAUGU)-3′ Calc : 3773.29 Q = (R)-C5′-Me-2′-FU
Found : 3772.77
S140 5′-r(UACAGQCUAUGU)-3′ Calc: 3773.29 Q = (S)-C5′-Me-2′-FU
Found: 3772.82
S141 5′-r(UACAGQCUAUGU)-3′ Calc: 3771.30 Q = 2′-OMeU
Found: 3770.77
S142 5′-r(UACAGQCUAUGU)-3′ Calc: 3773.29 Q = (R)-C5′-Me-2′-FU
Found: 3772.77
S143 5′-r(UACAGQCUAUGU)-3′ Calc: 3785.32 Q = (S)-C5′-Me-2′-OMeU
Found: 3784.72
S144 5′-r(UACAGQCUAUGU)-3′ Calc: 3755.30 Q = dT
Found: 3754.74
S145 5′-r(UACAGQCUAUGU)-3′ Calc : 3769.33 Q = (R)-C5′-Me-dT
Found : 3768.69
S146 5′-r(UACAGQCUAUGU)-3′ Calc: 3769.33 Q = (S)-C5′-Me-dT
Found: 3768.75
S147 5′-r(ACAUAGACUGUA)-3′ Calc: 3803.35
Found: 3802.64