Relative Populations of Some Tautomeric Forms of 2

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B: Biophysical Chemistry and Biomolecules

Relative Populations of Some Tautomeric Forms of 2#-Deoxyguanosine-5-Fluorouridine Mismatch Saeed Amini Komijani J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b00818 • Publication Date (Web): 02 Apr 2018 Downloaded from http://pubs.acs.org on April 3, 2018

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The Journal of Physical Chemistry

Relative Populations of Some Tautomeric Forms of 2′-Deoxyguanosine-5-Fluorouridine Mismatch Saeed K. Amini* Chemistry and Chemical Engineering Research Center of Iran, Tehran, Iran [email protected]

Abstract Importance of 2′-deoxyguanosine-uridine mispair as the most occurring mismatch in transcriptional studies of RNAs from DNAs is multiplied when 5-halo-substituted uridine species cause to serious increase in probability of its occurrence. Many studies relate this higher probability to existence of possible tautomeric and ionic forms of its constituent bases. According to these statements, relative populations of mismatches between 5-fluorouridine and both keto and enol forms of 2′-deoxyguanosine are computed by using conformational search. In order to have a complete scan of all of high probable conformers in a moderate computational time, an extensive conformational search methodology is employed here which benefits from advantages of both molecular dynamics (MD) simulations and quantum mechanics (QM) calculations. Population of enolic tautomer of normal wobble orientation is about 0.057% of that of its keto tautomer, whereas population of enolic tautomer of reverse wobble orientation is about 0.0054% of that of its keto tautomer. Totally, reverse wobble orientation is about six times more populated than normal wobble orientation. Calculated populations are in good agreement with experimental populations of closely related compounds. Reliability of applied methodology is certified in part by good agreement obtained between some experimental data such as NMR parameters and corresponding Boltzmann weighted average (BWA) data of most probable conformers. Validation of this methodology is certified with high accuracy by applying it on the substituted diuridine pairs, where experimental populations are available. Not only calculated populations and NMR parameters of this test are in very good agreement with experimental data, but also they free of ambiguities mentioned by experimentalists.

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1 Introduction The RNA polymerase’s recognition of complementary nucleobases through formation of specific hydrogen bonds (HBs) in RNAs’ transcription from DNAs is one of the most fundamental processes in biology which allows reliable readout of genetic information. However, association between nucleobases in nucleic acid helices is not restricted to Watson-Crick (WC) hydrogen bonding scheme, known as canonical pairing pattern. Just a little time after Watson and Crick’s base pairing proposal, they stated that WC-like mispairs could be the base of spontaneous mutations.1, 2 Donohue was who predicted structures of some non-canonical base pairs for first time, especially those of thymine in pair with either thymine or guanine.3 It is well known that other geometries such as Hoogsteen, wobble (Wb), sheared and reverse of all of mentioned patterns are frequently observed depending on the nucleic acid system and its matrix.4 Whereas, some of non-canonical pairs can cause to observation of both genome mutations and nucleobase mismatches in different steps of gene expression, some other only cause to observation of nucleobase mismatches in different steps of gene expression. Guanine-uracil (G•U) mismatch is the most occurring non-canonical pair that probability of its occurrence is increased by substituting uracil’s hydrogen at C5 position by halogens (XU). In order to reduce this probability and diminish its resulting deficiency in body’s operation, different aspects of G•U formation such as geometry and affecting factors are interesting for biologists. On the other hand, resultant pairs are of so particular interest that their structures and HB models attain special role in cancer therapy. Although FU is not considered as a mutagen, but it is demanding to get more information about its pairs’ topologies because of its undeniable importance in mismatch studies and its growing participation by increase of its industrial sources. Pairing of 2′-deoxyguanosine with 5-fluorouridine (dG•rFU) in transcription process, as initial step of gene expression, is very interesting because each base bears a different sugar moiety and one can observe a mismatch between an RNA type nucleoside and a DNA one. Although, resultant defects of this mispair can be reduced by getting more information about its mechanism of action during gene expression, existing studies do not provide enough information about its geometry. To this end it seems very interesting to survey topology of this substituted hetero-mismatch by using sophisticated structural studies. Since the wobble hypothesis and first observation of the guanine-uracil (G•U) wobble pair, it is subject to many studies.5, 6 This is due to its specific structural and functional aspects and important roles in RNAs such as mRNA codon recognition by tRNA anticodon. This conformational family of base pairing is suggested also for relatively high occurrence of mismatch between 2′-deoxyguanosene and uridine during genome transcription. Due to critical importance of adequate HB formation in mismatching, prediction of HB geometry of G•U base pair is the main focus of many experimental and theoretical investigations. However, assessment of structure and energy of probable HBs of a particular base pair inside a special nucleic acid structure encounters specific difficulties for each system and ACS Paragon Plus Environment

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employed methodology. Although, some reliable studies propose that either tautomeric forms or an equilibrium between tautomeric and ionic forms have incontrovertible role in the occurrence of G•U mispair, but some other studies propose different HB models for it such as single HB pattern.7-15 Although, reported geometries of most of these studies are in accordance with WC-like mispairing proposal and mentioned reliable reports about some special RNA and DNA structures, lack of a general rule about HB patterns that cover all of the G•U mispairs’ geometries sounds serious difficulties in nucleic acid studies.2 Specially, in the case of RNA transcription and HB formation between uridine and 2′-deoxyguanosine, inability to direct observation of this mismatching process causes its active and stable geometries to be unresolved completely up to now. Too many studies report structures and stabilities of mismatches between guanine and substituted uracil in different conditions by using a variety of experimental and theoretical methods. Same as G•U mismatch, there is not an accepted and popular rule about HB model in this case, too. Although, almost all studies, mainly from two decades ago, state that G•BrU and G•FU mispairs are in ionic forms, a new study state that both enolic tautomers and ionic forms of G•BrU can be responsible for its mutagenic property.16-22, 11 This situation becomes worsen about G•FU mispair because existence of only two olden experimental studies about its HB model cannot explain its special function in many cases, specially in transcription time which dG•rFU mispairing takes place and new surveys remain necessary for this special case.19, 21 Despite vast use of computational chemistry to predict pair species of G•U and G•XU mismatches in different situations, only four studies are about G•FU beside three studies reported about interaction between explicit water molecules and FU.23-29 Comparison of computational studies of G•U and G•XU with too many experimental and computational studies on the relevant single bases shows that activation energies of enolization or ionization of single bases are one order of magnitude larger than those of pairs, yet when they are considered in pair with water. For example, although calculations on the microsolvated state of BrU show that its enolic form in microsolvated and in conclusion in real water solvation state is more stable than its keto form, experimental results verify that population of its enolic form is only about 0.004% of keto form in a typical RNA duplex.30, 11 Thus, structural and energetic parameters obtained from single bases are not applicable for base pairing and three studies that report water-FU species cannot produce reliable data for G•FU.27-29 On the other hand, base pair formation within oligonucleotide duplexes under more physiological conditions is governed by the mutual orientation of bases as enforced by the sugar-phosphate backbone and its conformation. Four mentioned computational studies about G•FU by using MP2 method, which use a little different basis sets, report approximately same interaction energies for each unique orientation of this pair.23-26 One of these studies also reports ∆G values between wobble G•FU and its tautomeric orientations.23 These ∆G values may be considered as reliable amounts for this pair as truncated part of nucleic acid duplexes because of ACS Paragon Plus Environment


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observed agreements among all of these studies in the case of interaction energies.23 However, populations of different tautomers obtained from these ∆G values are very far from experimental populations of different tautomeric orientations of both G•T and G•BrU mispairs in typical nucleic acid structures.11 Rigid structure of nucleobases due to their aromatic character and lack of large conformational flexibility in considered pairs of these four calculations may cause all of them to produce similar geometries for single bases which can result in their similar pair geometries and similar ∆G values. Thus, all of these calculations seem unreliable maybe due to ignorance of sugar backbone and its conformational flexibility. In accordance to many studies that report enolic form of guanine is less unstable than enolic forms of 5-sustituteduracil with respect to their relevant keto forms, experimental results of Kimsey et al. in typical nucleic acid structures show that populations of all of rGenol•rU, dGenol•dT and dGenol•dBrU mispairs are larger than those of G•Uenol, G•Tenol and G•BrUenol, respectively.11 Despite these findings, none of these computations report calculations considering Genol in G•FU and it is interesting to consider this sort of pairing and its reverse one in the survey of dG•rFU pair. Considering mentioned statements about lack of and drawbacks of available data about G•FU mispair, it is interesting to predict populations of different orientation and tautomeric species of dG•rFU by extensive molecular dynamics (MD) simulations supported by quantum mechanics (QM) calculations. Use of deoxyribose and ribose sugar parts, respectively in conjunction with guanine and uracil, can completely resemble structures that occur in physiological transcription time. Applied methodology will allow for inclusion of both the conformational search and sugar moiety’s inclusion. Reliability of obtained results can be certified by comparison of their some resultant data with experimental values. To validate applied methodology more rigorously, it is first used for an approximately solved problem i.e. to determine relative populations of different orientations of substituted diuridine pairs.31 According to these reliability certification and validation processes, obtained populations of dG•rFU can be used with high credibility in biological studies and proposed methodology can be used anywhere experimental studies are either impossible or expensive and time consuming.

2 Computational Details: In accordance with experimental conditions, deoxyribiose parts of methyl substituted diuridine (dithymidine) are acetylated in their O3′ and O5′ positions and ribiose parts of flourine and bromine substituted diuridines are acetylated in their O2′, O3′ and O5′ positions.31 Three different orientations of each pair are subject to QM geometry optimization which include reverse wobble rXU•rXU (rWbrXU•rXU), wobble rXU•rXU (WbrXU•rXU) and reverse wobble star rXU•rXU (rWb*rXU•rXU) conformational families according to experimentalists’ proposal (Figure 1).31 Different rXU•rXU conformational families are converted to dXU•dXU ones in the case of dithymidine mispair. ACS Paragon Plus Environment

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Nomenclature of conformational families is in accordance with those reported by Lee and Gutell.32 In order to obtain most probable conformers of each orientation of each substituent, QM dihedral angle scan for most important out of ring bonds is applied on these nine optimized orientations. Totally 729 rotamers are scanned for each orientation of each substituent by rotating each of glycosidic, C4′-C5′ and C5′-acetyl bonds by 120˚. Ten representative conformers among these 729 conformers of each orientation of each substituent which have minimum energy within a specific window are selected for following treatments (Figure 2). Resultant ninety conformers are subject to QM geometry optimization to obtain optimized conformers and to distinguish between unique conformers and those conformers that are repeated for several times. These unique conformers are saved for following quenched MD simulations by eliminating the similar optimized conformers.

Figure 1. Geometries of three different orientations of diuridine pairs with right, middle and left conformational families denoted as rWb, Wb and rWb*, respectively. Black, blue, red, white and yellow colors represent carbon, nitrogen, oxygen, hydrogen and substituted atoms, respectively. Existing strong hydrogen bonds are presented by dashed lines. The 2′-O-acetyl group is substituted by H in the case of dithymidine.

Figure 2. Typical positions of ten selected representative conformers are denoted by asterisks in dihedral angle scan profile of wobble orientation of dithymidine. ACS Paragon Plus Environment


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Obtained conformers from quenched MD simulations are subject to energy minimization by molecular mechanical (MM) method and employed force field. Among these molecular mechanically optimized conformers of each orientation of each substituent those within a 10 kJ/mole window of relevant minimum energy are selected for following QM calculations. In this step, extensive QM calculations started by QM geometry optimization of these selected conformers are applied. Unique conformers are selected among these QM optimized conformers and in order to confirm them as local minima and provide their computed free energy values, they are subject to frequency calculations at 113K. Their chemical shielding values are also calculated on the GIAO condition for comparison with experimental data.33 All QM calculations and MD simulation are run in the solution state with Conductor-Like Polarizable Continuum Model (CPCM) and explicit solvent model, respectively.34-36 The CPCM’s parameters of Freon solvent at 113K are obtained from relevant dielectric, density and solvation thermodynamics data.37-42 All QM calculations are run at B3LYP/6-311++G(p,d) level of theory except to dihedral scan that is run at HF/3-21G level of theory. In the MD simulations, charmm36 general force field (CGenFF36) is applied for both the substituted nucleosides and Freon solvent.43 The CGenFF36 parameters for O-protected ribose and deoxyribose moieties are obtained from ParamChem.44,

45

The

CGenFF36 values for Freon components and bromine and fluorine substituted nucleosides are parameterized employing Force Field Toolkit (FFTK) implemented in the Visualized Molecular Dynamics (VMD) software.46,

47

The rectangular simulation box is of dimensions of 41×41×49Å

including nucleoside pair and about 510 molecules of each of CHF3 and CHClF2. Number of Freon components are tolerated equally by ±4 in different simulations due to box dimension’s limitation. The non-bonded cutoff, switch and pair list distances are set to 10Å, 9Å and 13Å, respectively. The SETTLE rigid bonds algorithm is applied on any bond including hydrogen atom.48 Temperature is fixed by Langevin dynamics with 1 ps-1 damping coefficient.49 Pressure is fixed at 1atm by Langevin piston.50 Each time step is set to 2 fs. The particle mesh Ewald (PME) summation with 1Å dimensions is applied for long range electrostatic interaction calculations in periodic boundary conditions (PBC).51 Each simulation is started with a 1000 steps of energy minimization by using conjugate gradient method. Each conformer is equilibrated for 500 ps in constant temperature and pressure (NPT) ensemble after slowly heating up to 450K. To reach correct system density, another 5 ns simulation is run in NPT ensemble at 450K. To keep planar structure of each nucleoside pair during these steps, the positions of three carbon atoms of each nucleoside are fixed (either C2 or C4 of base depending on the orientation of the relevant nucleoside to avoid fixing carbon atoms involved in HB plus C6 of base and C1′ of sugar). After equilibration at 450K, each conformer is simulated by quenching method for 600 ps releasing constraints on fixed atoms and structures are recorded after each 1 ps to optimize by using applied FF.52 ACS Paragon Plus Environment

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Figure 3. Geometries of four different orientations of 2′-deoxyguanosine-5-fluorouridine nucleoside mispair with conformational families denoted as WC-like, Wb, rWC-like and rWb from right to left, respectively. Black, blue, red, white and yellow colors represent carbon, nitrogen, oxygen, hydrogen and fluorine atoms, respectively. Existing strong hydrogen bonds are presented by dashed lines.

Four different orientations of nucleosides are used in the case of dG•rFU mispair which include wobble (Wb), enolic WC-like (WC-like), reverse wobble (rWb) and reverse enolic WC-like (rWC-like) conformational families (Figure 3). Nomenclature of conformational families is in accordance with those reported by Lee and Gutell.32 All condition mentioned for substituted diuridine pairs are met here except those stated in following sentences. In order to resemble physiological conditions for dG•rFU mispair, following changes are applied. Totally 256 rotamers are scaned for each orientation by rotating each of glycosidic and C4′-C5′ bonds by 90˚. Water is used as solvent with TIP3P model for MD simulations and QM calculations are run at 310K which is temperature of human’s body. Neither ribose nor deoxyribose moieties are O-protected. Because of breakage of nucleoside pair in the absence of mentioned fixed atoms constraints by their strong interactions with water molecules, fixed atoms constraints are conserved during production step, as well. Optimized conformers of each orientation of each tautomer obtained from quenched MD conformational search within 30 kJ/mole window of relevant minimum energy are selected for following treatments.53 All QM calculations are run using ORCA and molecular mechanical calculations and MD simulations are run using NAMD.54, 55 Simulation results are analyzed by VMD.47

3

Results and Discussions:

3.1 The O-protected 5-substituted diuridine pairs Janke and Weisz studied hydrogen-bonded species of O-protected 5-substituted diuridine nucleoside pairs by lowering the temperature of Freon solvent up to 113K to slow the HB exchange and observe individual species for more detailed characterization.31 Their study includes some ambiguities such as inability to find actual populations of each orientation or uncertainty to reject existence of Wb and rWb* orientations. These ambiguities can be surmounted by using a rigorous computational methodology that confirms their certain results, as well. To this end, computationally extensive methodology described in the computational detail is applied here. In their experimental conditions and according to their statement, results do not show noticeable contribution of enol tautomers to the NMR spectra of used diuridine pairs which is consistent with other recently published studies.31, 11, 56 So, in order to obtain probability of each orientation in experimental conditions, use of only keto forms of each substituted diuridine pairs is sufficient and it is not necessary to consider either ionized or enolic species. Because keto form of uridine cannot incorporate in three strong HBs simultaneously, orientations in which either ACS Paragon Plus Environment


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one or two nucleosides are interacted from their sugar moieties can be excluded here and use of those three orientations of Figure 1 is adequate.57, 58 At first, a QM optimized conformer of each orientation of each pair is subject to non-relaxed QM dihedral angle scan (Figure 1).31 In order to overcome discrepancies accompanying non-relaxed scanning and to have conformers with a variety of dihedral angles, ten representative conformers of each orientation which have minimum energy within a specific window of scan are selected (Figure 2). After QM geometry optimization followed by elimination of similar optimized conformers of these selected ninety conformers, representative conformers of each orientation of each substituent are reduced to only 3, 3, 4, 3, 3, 4, 4, 3 and 4 unique conformers for each of rWbrBrU•rBrU, WbrBrU•rBrU, rWb*rBrU•rBrU,

rWbrFU•rFU,

WbrFU•rFU,

rWb*rFU•rFU,

rWbdMeU•dMeU,

WbdMeU•dMeU,

rWb*dMeU•dMeU, respectively. These conformers are saved for following MD simulations. Choose of too long procedure and too many conformers of each orientation before MD simulation guarantees obtaining a maximum scan of minimum energy conformers in a simulation time of the order of ps.59 Its importance becomes more apparent in the end of MD conformational search where different representative conformers of each orientation do not produce same number of most stable conformers. Specially, some conformers do not show any contribution in the most stable conformers of corresponding orientation. So, inaccurate use of these noncontributing conformers as only starting points of MD conformational search can be resulted in imperfect search of most stable conformers of related orientation. Use of quenched conformational search at high temperature, 450K, causes to overcome to the barriers of conformational changes and to pass from barriers either in between local minima or between each of them and global minimum.60 Releasing the fixed coordinates’ constraints in production step of quenched MD simulations is resulted in observation of a variety of changes upon the relatively planar conformers of equilibration steps. Although, specific orientation of relevant pair is maintained during production steps of some simulations despite some deviations from planarity, in some other simulations they are interchanged to other specific orientations of that pair and in the some other simulations after tens or hundreds of ps they are either broken completely or reconnected from atoms other than those of specific orientations. Of course, observation of this wide spread of changes is not strange because simulation temperature is so high in comparison to experimental temperature that thermal motions can overcome HB barriers to break them.31 This is another reason that why several starting conformers of each orientation should be used in this pair study in which two molecules should be in close interaction with each other during production step without any constraint. Two different criteria can be considered in selecting conformers with low potential energy values. First is a summation over dinucleoside’s total energy (conformational and nonbonded energies) and its nonbonded energies with Freon solvent. Second is to consider just the dinucleoside’s total energy. For ACS Paragon Plus Environment

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each orientation of each substituent, different conformers show very different interaction energies with solvent. Thus, considering the first criterion to find conformers within a 10 kJ window of minimum energy of each orientation of each substituent is resulted in less than 10 conformers for each orientation. On the other hand, second criterion’s energies of different conformers of each orientation of each substituent do not differ so much. Thus, one can select tens of conformers using this criterion which include most of those of first criterion. In order to include any low lying conformer, conformers with minimum energy are found by both criteria and used in following computations. Thus, 24, 16, 28, 47, 23, 29, 18, 45 and 14 conformers are selected for each of rWbrBrU•rBrU, WbrBrU•rBrU, rWb*rBrU•rBrU, rWbrFU•rFU,

WbrFU•rFU,

rWb*rFU•rFU,

rWbdMeU•dMeU,

WbdMeU•dMeU,

rWb*dMeU•dMeU,

respectively. In order to consider all conformers within a 10 kJ window of minimum energy of each orientation of each substituent, non-planar structures and those with hydrogen bonds from sugar moiety are maintained, as well. After QM optimization of all of these 244 conformers and elimination of the similar optimized conformers, only 8, 10, 12, 11, 9, 6, 10, 18 and 9 unique conformations are obtained for each of rWbrBrU•rBrU, WbrBrU•rBrU, rWb*rBrU•rBrU, rWbrFU•rFU, WbrFU•rFU, rWb*rFU•rFU, rWbdMeU•dMeU, WbdMeU•dMeU, rWb*dMeU•dMeU, respectively. Contribution of each conformer in the experimental NMR spectrum at 113K is determined by Boltzmann distribution function in which frequency calculations are employed to obtain free energies differences (∆G) between minimum energy value and corresponding conformer. Sum of these contributions is normalized to unit in two manners. At first, weight of chemical shift value of each conformer inside each orientation of each substituent is obtained by normalizing to unit the sum of contributions of conformers inside corresponding orientation of that substituent. At second, relative population of each orientation of each substituent is obtained by normalizing to unit the sum of contributions of its all conformers. Because the main focus of this test is resembling the experimentally determined relative position of each peak and its relative population, differences between calculated chemical shift (CS) values of each substituent are much more important than their absolute ones. In order to calculate chemical shift values, different chemical shielding values of standard (σstandard) are used for different substituents to have best matches with experimental chemical shift values and make comparisons easy to eye. Two calculated chemical shift values of each conformer are weighted by its calculated normalized contribution in the experimental NMR spectrum at 113K. Boltzmann weighted average (BWA) chemical shift values of two resonating protons of each orientation of each substituent are obtained by a summation over obtained weights of its conformers multiplied by corresponding chemical shift values. Total BWA chemical shift values of two resonating protons of each substituent are obtained by a summation over obtained relative populations of its orientations multiplied by corresponding BWA chemical shift values. In this step population of corresponding symmetric orientation of each resonating proton is multiplied by two because it includes two similar resonating protons.



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Table 1. Experimental and Boltzmann weighted average (BWA) 1H chemical shift values of substituted diuridines in ppm moreover to relative population of each orientation with a sum to unit for each substituent.a Orientation

BWA Chemical Shifts b

c

Diff.

Populations

A

B

Orien.

A:B

rWb

11.85

------

0.15

Wb

11.98

12.34

0.85

rWb*

------

12.52

Total

11.95

12.35

0.40

1.35:1

d

11.98

12.32

0.34

1.3:1

rWb

11.46

------

0.04

Wb

11.59

11.93

0.64

rWb*

------

12.00

Total

11.58

11.97

0.39

1:1.78

d

12.05

11.50

0.55

1.6:1

rWb

11.86

------

Wb

11.91

12.06

0.47

rWb*

------

12.22

0.02

dMeU•dMeU

Exp.

0.004

rBrU•rBrU

Exp.

0.32

rFU•rFU 0.51

Total

11.87

12.07

0.2

2.92:1

Exp.d

11.97

11.97

------

------

a: To convert calculated chemical shielding values to chemical shifts, σstandard values of 31.83ppm, 32.15ppm and 32.25ppm are used for rBrU•rBrU, rFU•rFU and dMeU•dMeU, respectively. b: Proton hydrogen bonded to O2. c: proton hydrogen bonded to O4. d: From reference 31.

These calculated BWA chemical shift values and Experimental data of different diuridine species are summarized in Table 1.31 Relative populations of three orientations of each substituent are presented in this table with a sum to unit beside relative populations of each resonating proton of that substituent. Comparison of relative positions and populations of experimental NMR spectra with calculated ones can at first confirm validation of applied methodology and at second help to remove some ambiguities and discrepancies mentioned by Janke and Weisz.31 As it is obvious, conformers with HB geometries non-coincident to relevant three orientations of each pair have no contribution in experimental NMR spectrum because of their very different shift values which are not seen in experiment and their number is equal to 3, 0, 1, 3, 3, 0, 0, 2 and 0 conformers for rWbrBrU•rBrU, WbrBrU•rBrU, rWb*rBrU•rBrU, rWbrFU•rFU,

WbrFU•rFU,

rWb*rFU•rFU,

rWbdMeU•dMeU,

WbdMeU•dMeU,

rWb*dMeU•dMeU,

respectively.31, 61 All of these conformers have probabilities less than %10-8. This certifies exclusion of these orientations as starting point as stated before keto form of uridine cannot incorporate in three strong HBs simultaneously and orientations in which either one or two nucleosides are interacted from their sugar moieties can be excluded from starting point.57, 58 Conformers with contribution more than %0.01 for each orientation are equal to 2, 8, 8, 4, 3, 1, 3, 7 and 0 conformers for rWbrBrU•rBrU, ACS Paragon Plus Environment

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WbrBrU•rBrU,


rWb*rBrU•rBrU,

rWbrFU•rFU,

WbrFU•rFU,

rWb*rFU•rFU,

rWbdMeU•dMeU,

WbdMeU•dMeU, rWb*dMeU•dMeU, respectively. Most probable and most shielded peak with CS value of 11.95ppm is related to that proton which is hydrogen bonded to O2 in dMeU•dMeU nucleoside pair (dithymidine pair). Experimentally proposed hydrogen types responsible for observed two peaks and their relative positions are certified by these calculations because their calculated relative populations and chemical shift values are consistent with experimental data.31 According to calculated populations, rWb orientation of dithymidine is about forty times more populated than its rWb* orientation. This puts in challenge the Janke and Weisz statement that rWb and rWb* orientations participate unequally in experimental spectrum because population of rWb* can be ignored according to its very low population obtained from these calculations. Calculated and experimental differences between CS values of two resonating protons are 0.4ppm and 0.34ppm, respectively, which are in very good agreement with each other.31 Also, calculated and experimental relative populations of two resonating protons in the NMR spectrum are 1:1.35 and 1:1.3, respectively, which are very close to each other.31 These observations completely certify the reliability of applied methodology. On the other hand, conformers with populations more than %0.01 show CS values inside a very narrow span (differences less than 0.1ppm) for each resonating proton in its either asymmetric (Wb) or symmetric orientations (rWb or rWb*). These very small differences certify importance of applied methodology in distinguishing between symmetric and asymmetric orientations because they could not be distinguished by simple NMR experiments due to overlap of their peaks. Contribution of rWb* orientation is found to be less than %0.4 and its contribution seems to be very hard to distinguish by experiment.31 Although, experimental results show that relative populations of two resonating protons are changed just from 1:0.94 to 1:1.3 in going from acylated 2'-deoxyuridine to acetylated dithymidine, comparison of this calculation and experimental results of acylated 2'-deoxyuridine shows that relative populations of two symmetric species changes from 1:1 to 1:40 and total contribution of asymmetric species changes from %44 to %85.61, 62 On the other hand precise predictions of relative populations by applied methodology in comparison to very bad predictions by two calculations which use diuracile pairs as their representatives in nucleic acid helices, accent to the use of such extensive computations of this methodology in order to obtain reliable data.63, 64 Most probable and less shielded peak with CS value of 11.97ppm is related to that proton which hydrogen bonded to O4 in the rBrU•rBrU nucleoside pair. Considering these calculations, experimentally proposed hydrogen types responsible for observed two peaks and their relative positions should be corrected because their calculated relative populations and chemical shift values are in contrast to experimental data.31 Although, contribution of rWb orientation is found to be only %4 and its relative population is about one-eighth of that of rWb* orientation, but these data certify Janke and Weisz statement that all orientations participate in experimental spectrum.31 Calculated and experimental ACS Paragon Plus Environment


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differences between CS values of two resonating protons are 0.39ppm and 0.55ppm, respectively, which are in relatively good agreement with each other.31 By correcting the proton types of experimental spectrum, calculated and experimental relative populations of each resonating proton in the NMR spectrum are 1:1.78 and 1:1.6, respectively, which are relatively close to each other.31 Since force field of rBrU is parameterized by FFTK, these increases in differences in comparison to those of dithymidine can be related to differences in parameterization’s algorithms applied in original CGenFF36 and FFTK.43, 46 Conformers with populations more than %0.01 show CS values inside a very narrow span of the order of that of dithymidine (differences less than 0.08ppm) for each resonating proton in its either asymmetric (Wb) or symmetric orientations (rWb or rWb*). Although, relative intensity of peak of that proton which is hydrogen bonded to O2 is increased in going from diuridine to dithymidine, calculated results show that it is decreased in going from diuridine to rBrU•rBrU and it is in contrast to Janke and Weisz conclusion.61 This observation may be related to electron withdrawing character of Br and its more vicinity to O4 atom which makes it less negative than O2 atom and in conclusion more susceptible for HB formation. This change in susceptibility in HB formation is more obvious from relative populations of symmetric species.62 While, rWb*:rWb is changed from 1:1 to 1:40 in going from acylated 2'-deoxyuridine to acetylated dithymidine, it is changed to 8:1 in going to rBrU•rBrU.62 In the case of rFU•rFU nucleoside pair, that proton which is hydrogen bonded to O2 is about three times more populated than that proton which is hydrogen bonded to O4. Obtaining small difference between chemical shift values of two resonating protons (0.2ppm) and observing a single experimental peak with more than 0.2ppm bandwidth, imply hiding of peak of protons which is hydrogen bonded to O4 under single experimental peak of very larger populated proton in 113K. This conclusion is extensible to higher temperatures because all conformers resulted from MD conformational search produce same chemical shielding values irrespective of their relative populations which can be changed by temperature changes. Although, conformers with hydrogen bonds from sugar moiety show very different CS values, but their relative populations are less than %10-10 in this temperature and it seems hard for them to pass populations of other conformers up to room temperature to put this conclusion in doubt. Thus, Janke and Weisz statement that it seems unlikely the hiding of peak of protons which is hydrogen to O4 under single experimental peak because of observation of single peak in the whole temperature range of slow exchange, sounds incorrect.31 Of course, in accordance with their statement, proton which is hydrogen bonded to O2 resonates in higher field than other one.31 Interestingly, changes in calculated relative populations of two resonating protons and changes in calculated relative populations of different orientations in going to diuridine are consistent with those of dithymidine.61, 62 Totally good agreement is observed between experimental and calculated data.31 Interchange of experimental relative positions and populations of two resonating protons of bromine substituted pair implies the importance of use of computations beside experiment to explain its results more precisely. ACS Paragon Plus Environment

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This interchange in between chemical shift positions is in agreement with the finding that more shifted proton is that one which is hydrogen bonded to O4 and results of dithymidine, rFU•rFU and nonsubstituted diuridine.31,

61, 62

Two experimental studies propose asymmetric orientation as most

populated species in non-substituted diuridines but with approximately same and high populations for symmetric orientations.61,

62

Its relative population is reduced by increasing electronegativity of

substituent and while it is more populated than symmetric orientations in rBrU•rBrU and dithymidine species, it is less populated than one of them in rFU•rFU. Very different chemical shift values of structures that do not coincide to any of specific orientations beside their very higher Gibbs free energy values certify lack of their observation in experiment at 113K. Totally, applied methodology is validated by these comparisons and can be used in following part.

3.2 The 2′-deoxyguanosine-5-uridine pair The very good execution of applied methodology in previous section makes it applicable to propose probable geometries of dG•rFU nucleoside mismatch, although its low temperature NMR study is impossible and restricted by guanosine’s strong propensity for self-aggregation. Main focus of this part is to obtain most probable HB structures in transcription time between 2′-deoxyguanosine and 5fluorouridine because it is difficult to do experimentally. Kimsey et al. propose four appreciably populated tautomeric and ionic forms for rG•rU, dG•dBrU and dG•dT mismatches in RNA and DNA helices without ruling out the existence of some other species.11 Hu et al. investigated three normal and neutral mismatches between 5-bromouracil and guanine using DFT calculations and reported Genol•BrU as most stable species for this base pair.65 Many other studies report that G to Genol tautomerization is more probable than U to Uenol tautomerization, yet in their unpaired states. Based on their calculations, Brovarets and Hovorun state that Genol•U base pair is higher lived than G+•U- and G•Uenol base pairs and G•XUenol base pair to Genol•XU base pair transformation’s barrier is small and reachable in body temperature.66 They also state that G•Tenol base pair is dynamically unstable and is transformed to Genol•T base pair despite its thermodynamic stability.66, 67 On the other hand, Brovarets et al. state that Genol•Tenol base pair is an unstable and very low lived species.66, 68 These computational results beside Kimsey et al. experimental report of smaller populations for G•XUenol species in comparison to Genol•XU species in different RNA and DNA structures accent that observation of dGenol•rFU may be more probable than dG•rFUenol in transcription time (11). Reverse orientations of these proposed forms that include antiparallel glycosidic bonds by 180o rotation of one of nucleosides relative to other one seem important, too, because of their observation in some nucleic acid helices and complementation of dG•rFU study by their inclusion.69-73 Only three computational studies report reverse G•U base pair species and just one of them is about reverse G•FU base pair which reports its interaction energy without any comparison with normal wobble orientation.74, 75, 24 Thus, totally four orientations comprising two ACS Paragon Plus Environment


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normal and reverse orientations of one of Wb (dG•rFU) and WC-like (dGenol•rFU) pairing schemes are selected here for extensive investigation as most probable neutral tautomers of 2′-deoxyguanosine-5fluorouridine mispair. In the first two orientations, both bases are in their keto forms and make either wobble or reverse wobble orientations (Figure 3). In the second two orientations, guanosine in its enol form pairs with 5-fluorouridine via three HBs to make either a WC-like or its reverse orientation (Figure 3). Water is used as solvent to both include probable interactions in transcription time between RNA polymerase and nucleosides and produce data which are comparable with experimental data obtained from nucleic acid helices solvated in water because it is impossible to use hydrophobic pocket of RNA polymerase in this extensive study. Obtained populations can assess those of transcription time to high certainty by including water interactions which can resemble HBs between RNA-polymerase machinery with DNA and mRNA. This statement is based on some facts that other factors have small effects on the HB structures. For example it is stated that base stacking effects as another factor in double helix formation is ignorable because its effect is negligible in comparison to HB geometry and conformational variety.76, 77 Thus, data on these nucleoside pairs, modified by the sugar residue, can be transferred to transcription result. Total unique conformers which are saved for MD simulations by eliminating the similar optimized geometries of 10 candidate conformers of each orientation from dihedral angle scan are equal to 8, 8, 6 and 4 for WC-like, Wb, rWC-like and rWb, respectively. Totally, 60, 84, 59 and 32 unique conformers are obtained from MD simulations in the 30kJ window of their lowest energy conformers for each of WC-like, Wb, rWC-like and rWb, respectively. Similar to diuridine pairs, non-planar structures and those having hydrogen bonds from sugar moieties are maintained for following manipulations. Total number of unique conformers is reduced to 17, 13, 18 and 17 by eliminating the similar conformers resultant from QM geometry optimization of previous 235 conformers for each of WC-like, Wb, rWClike and rWb, respectively. Moreover to these 65 unique conformers, some other unique conformers including four distinct orientations and appreciable probabilities are resulted from QM geometry optimization of previous 235 conformers (Figure 4). These four orientations are characterized as cis Watson-crick/sugar edge (cisWC/SE), cis Watson-crick/sugar edge assisted by O5′ (cisWC/SE-O5′), trans Watson-crick/sugar edge (transWC/SE) and single bifurcated HB (singleHB). In their three orientations, dG incorporates in HB with its all three groups incorporated in C•G base pair and rFU incorporates with assist of its sugar moiety. The last orientation includes a single bifurcated HB in accordance with previous studies in which O4 of rFU is hydrogen bonded to both of amino and imino protons of dG at 2.06Å and 1.98Å, respectively.13-15


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Figure 4. Geometries of four rare orientations of dGenol•rFU nucleoside pair with appreciable probabilities and structures from right to left denoted as are transWC/SE, cisWC/SE, cisWC/SE-O5′ and singleHB, respectively. Black, blue, red, white and yellow colors represent carbon, nitrogen, oxygen, hydrogen and fluorine atoms, respectively. Existing strong hydrogen bonds are presented by dashed lines. Table 2. Experimental and Boltzmann weighted average (BWA) 1H, 15N and 19F chemical shift values of dGenol•rFU in ppm. The ∆G values of different orientations in kJ/mole with respect to rWb orientation as most stable one and resultant relative populations are also included.a Pop. ∆G 1

H of NH

F

rU dG

1

H of NH2

H1 H2

15

N of NH

F

rU dG

19

F

BWA Exp. BWA Exp. BWA Exp. BWA Exp. BWA Exp. BWA Exp. BWA Exp. BWA Exp. BWA Exp.

WC-like 0.0000785 0.00016b, 0.00036b, 0.00136b 23.91 19.5b, 16.2b, 15.9b 14.00 11.58 6.87 4.21 160.71 190.94 -101.57

Wb 0.138 >0.99b 3.99 0.0b 12.20 12.23c, 12.16d 11.55 10.52c, 11.02d 4.15 6.05c, 6.21d 4.15 6.05c, 6.21d 159.01 159e 144.0 144e -97.8 -88.23c,-88.55c, -97.8d

rWC-like 0.0000437

rWb 0.809

transWC/SE 0.0132

cisWC/SE 0.000162

cisWC/SE-O5′ 0.0389

singleHB 0.000525

24.62

0.0

5.67

17.01

4.61

13.98

13.90

12.22

7.09

10.45

12.31

7.24

11.12

11.95

9.36

10.25

10.25

9.67

7.20

4.21

5.98

5.64

6.89

6.18

4.26

4.18

4.29

3.91

4.17

4.02

161.28

158.34

156.25

158.41

162.03

154.59

191.09

143.77

141.33

144.22

140.36

143.44

-99.51

-103.24

-99.11

-97.92

-97.64

-102.14 1

a: To convert calculated chemical shielding values to chemical shifts, σstandard values of 31.37ppm, 231.63ppm and 248.49are used for H, respectively. b: From reference 11 from left to right for dG•dBrU, rG•rU and dG•dT, respectively. c: From reference 19 for dG•dFU. d: From reference 78 for rG•rFU. e: From reference 11 for rG•rU.

15

N and

19

F,

Relative populations of different orientations of dG•rFU beside some experimental values of relevant structures including just normal orientations are summarized in Table 2.11 Numbers of conformers with contributions more than %0.01 are equal to 1, 12, 0 and 16 for WC-like, Wb, rWC-like and rWb orientations, respectively. Relative populations of Wb and WC-like orientations is 1:5.67*10-4 and this is changed to 1:4.1*10-4 by including the populations of three rare orientations that are geometrically in close vicinity to Wb orientation. These values are close to experimental relative populations of G:Genol in dG•dBrU, rG•rU and dG•dT pairs which are equal to 1:1.6*10-4, 1:3.6*10-4 and 1:13.6*10-4, respectively.11 Observed differences between these experimental data and calculated populations can be related to some different parameters. Although, calculations use ribose for 5-fluorouridine and deoxyribose for guanosine to resemble RNA’s transcription, all experimental pairs bear their own sugar moieties which are similar for both of their constituents.11 Observation of four times larger relative population for dGenol•dT in comparison to rGenol•rU can be mainly related to this factor and this can be accepted as reason of higher calculated population of dGenol•rFU in comparison to two other experimental data. Despite pH-independence of population of dGenol•dT at pH values of 6 up to 8, that of rG•rU is highly pH-dependent and is decreased by increasing pH values from 7 to 8.11 I.e. relative ACS Paragon Plus Environment


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population of rGenol•rU will increase by using computational pH and pass from that of dGenol•rFU. According to experimental data, increasing temperature by 20oC results in about two times increased populations of rGenol•rU and dGenol•dT species. If one consider same changes for dGenol•dBrU versus temperature, its population can become close to that of dGenol•rFU and will pass it by considering same sugar moieties for both substituents.11 Thus, it can be concluded that relative population of Genol•XU base pair is decreased by increasing the electronegativity of substituent if both bases bear same sugar moieties. Of course, all of these conclusions are based on the fact that experimental relative populations of Genol•XU and G•XUenol species are retained in different conditions. Finally, it seems interesting to certify reliability of obtained populations of these orientations in terms of energetic and NMR parameters with some clarifications. Table 2 also includes relative Gibbs free energy values for comparison with other available data.11 Calculated interaction energies in G•T and G•U base pairs show that reverse wobble orientation is about 0.4-1.7kJ/mole more stable than usual wobble orientation depending on applied computational levels of theory.74, 75 In accordance with these studies, Table 2 shows that rWb orientation is about 3.1kJ/mole more stable than Wb orientation. Also, Wb orientation is about 20.6kJ/mole more stable than WC-like orientation which is in good agreement with experimental and high level computational stability energies of 17.6-19.5kJ/mole, 15.9-23.4kJ/mole, and 16.2kJ/mole reported for G•BrU, G•T, and G•Upairs, respectively.11, 79 It is reported that interaction energies in cisWC/SE and transWC/SE base pairs of G•U are 39.3kJ/mole and 96.7kJ/mole, respectively.58,

80

Although, obtained HB pattern in

tranWC/SE orientation is same as that of Sponer et al., that of cisWC/SE is different and despite their study that guanosine’s amine nitrogen is hydrogen bonded to H2′, here one of guanosine’s amine protons is hydrogen bonded to O2′.58,

80

These observations beside BWA populations of these two

species imply that reported interaction energies of at least cisWC/SE was underestimated by Sponer et al. maybe because of lack of conformational search and reaching to global minimum of this orientation instead of its local minima in their study.58, 80 Of course, for cis orientation another structure assisted by O5′ instead of O2′ (cisWC/SE-O5′) with planar structure and higher population is found that cannot be consistent with helical structure of nucleic acid molecules and is not proposed by Sponer et al..58 A single HB orientation is also observed in accordance with experiments with appreciable population.13-15 As it is discussed, these observations about energetic parameters can be addressed as more evidences for reliability of calculated population values and makes them as valid populations for proposed orientations of dG•rFU. Boltzmann weighted average CS values of some nuclei of dG•rFU which have experimental data for some relevant structures are summarized in Table 2.11, 19, 78 Two experimental NMR studies in water solution show that 1H chemical shift of H3 of 5-fluorouracil is shifted to high field by1.14ppm and 1.71 ppm relative to H1 of guanine in typical dG•dFU and rG•rFU pairs, respectively.19, 78 Calculated BWA ACS Paragon Plus Environment

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chemical shift values of Wb orientation show best match with these data and existence of other orientations can be easily ruled out which is in accordance with conclusion of both experiments.19, 78 Chu and Horowitz state that CS value of 19F shifts by about 4.5-5ppm towards downfield in going from WC rA•rFU orientation to Wb rG•rFU orientation in typical tRNA structures.81 Observed 5.5ppm downfield shift in going from rWb orientation to Wb orientation in which O4 of rFU losses its HB with dG certifies that this shift is due to involvement of O4 of rFU in hydrogen bond with rG. According to HB donating groups and their structural parameters, O4 of rFU involves in relatively strong HBs in WClike and rWC-like orientations and same argument can be employed in explaining their highfield shifts relative to Wb orientation by 3.77ppm and 1.71ppm, respectively. Experimental data show that replacing rU by rFU in rG•rU pair of specific RNA duplex causes to just up field shift of CS value of H3 of U by 0.5ppm and has no effect neither on the CS value of H1 of G nor on any other reported values of G and U.78 Thus, comparison of calculated data can be done unambiguously with experimental chemical shift values of GU anywhere NMR data are unavailable for dG•rFU. Thus, conclusion about existence of Wb orientation considering relative positions of chemical shift values of H1 of guanine and H3 of 5-fluorouracil can be easily extended to some other experiments in which this difference is reported to vary from 0.6ppm up to 1.2ppm in typical rG•rU pairs.11, 13, 82-86 Two of these experiments from same authors state that observed rG•rU pair geometry is either consistent with singleHB or other single HB model based on observed and predicted chemical shift values of H5 and H1′ atoms of uracil.13, 85 Comparison of calculated CS values of H1 of guanine and H3 of 5-fluorouracil completely reject existence of singleHB orientation in their experiments because relative position of these two nuclei in experiment is reverse of that of computed singleHB model.13, 85 Existence of other proposed single HB model can also be ruled out because it may be produce same chemical shift values and Wb orientation is proposed as probable geometry for their pairs.13,

85

Difference between calculated CS

values of N1 of dG and N3 of rFU in the case of Wb orientation is in very good agreement with experimental data of this orientation in dG•dU and rG•rU misspaires.11, 85 Two experimental studies on rG•rU pair show about 1ppm differences between chemical shift values of two hydrogen atoms of amine group of rG that comparing them with calculated data reveals very interesting results.87, 88 Despite these experiments, calculated results for keto forms show no differences between CS values of these two nuclei and calculated differences in enol forms are very larger than these experimental data.87, 88 Other conformers of keto forms should produce such experimental spectra because these enol tautomers cannot be in major population in experimental conditions.87,

88

Thus their geometries may be one of

mentioned rare orientations of cisWC/SE or transWC/SE that show closest chemical shift values. Same as energetic parameters, these observations about CS values can be used as other evidences for reliability of calculated population values of dG•rFU species. ACS Paragon Plus Environment


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4 Conclusion Very good execution of applied methodology in the case of substituted diuridine nucleoside pairs makes it applicable anywhere there is not favor conditions for experiment or it is expensive to do. Thus, its application to find relative populations of different orientations of dG•rFU mismatch as a pair occurring in transcription time and without reliable data in literatures results calculated values that are in good agreement with experimental populations of relevant nucleoside pairs. According to obtained results, reverse wobble orientation is about six times more populated than normal wobble orientation and relative populations of keto to enol orientations are 1:5.7*10-4 and 1:5.4*10-4 for Wb and reverse rWb orientations, respectively. Comparisons of calculated energetic and NMR parameters of dG•rFU with experimental data of some relevant pairs provide more evidences about reliability of obtained populations.

Acknowledgements I acknowledge Iran National Science Foundation (INSF) for grant number 92027773.

5 References (1) Watson, J. D.; Crick, F. H. C. Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid. Nature 1953, 171, 737-738. (2) Watson, J. D.; Crick, F. H. C. The Structure of DNA. Cold Spring Harb. Symp. Quant. Biol. 1953, 18, 123-131. (3) Donohue, J. Hydrogen-Bonded Helical Configurations of Polynucleotides. Proc. Natl. Acad. Sci. 1956, 42, 60-65. (4) Wyatt, J. R.; Tinoco, I. J. RNA Structural Elements and RNA Function. The RNA World; Cold Spring Harbor Laboratory Press, 1993; Vol. 24, 465-496. (5) Crick, F. H. C. Codon-Anticodon Pairing: The Wobble Hypothesis. J. Mol. Biol. 1966, 19, 548-555. (6) Lander, J. E.; Jack, A.; Robertus, J. D.; Brown, R. S.; Rhodes, D.; Clark, B. F.; Klug, A. Structure of Yeast Phenylalanine Transfer RNA at 2.5 A Resolution. Proc. Natl. Acad. Sci. 1975, 72, 4414-4418. (7) Ogle, J. M.; Murphy, F. V.; Tarry, M. J.; Ramakrishnan, V. Selection of tRNA by the Ribosome Requires a Transition From an Open to a Closed Form. Cell 2002, 111, 721-732. (8) Demeshkina, N.; Jenner, L.; Westhof, E.; Yusupov, M.; Yusupova, G. A New Understanding of the Decoding Principle on the Ribosome. Nature 2012, 484, 256-260. (9) Rozov, A.; Demeshkina, N.; Westhof, E.; Yusupov, M.; Yusupova, G. Structural Insights Into the Translational Infidelity Mechanism. nat. Comm. 2015, 6, 7251. (10) Demeshkina, N.; Jenner, L.; Westhof, E.; Yusupov, M.; yusupova, G. New Structural Insights Into the Decoding Mechanism: Translation Infidelity Via a G.U Pair With Watson–Crick Geometry. FEBS Lett. 2013, 587, 1848-1857. (11) Kimsey, I. J.; Petzold, K.; Sathyamoorthy, B.; Stein, Z. W.; Al-Hashimi, H. M. Visualizing Transient Watson–Crick-Like Mispairs in DNA and RNA Duplexes. Nature 2015, 519, 315-320. (12) Rozov, A.; Westhof, E.; Ysupov, M.; Yusupova, G. The Ribosome Prohibits the G•U Wobble Geometry at the First Position of the Codon–Anticodon Helix. Nucleic Acids Res. 2016, 44, 6434-6441. (13) Chen, X.; McDowell, J. A.; Kierzek, R.; Krugh, T. R.; Turner, T. H. Nuclear Magnetic Resonance Spectroscopy and Molecular Modeling Reveal That Different Hydrogen Bonding Patterns Are Possible for GâU ACS Paragon Plus Environment

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Relative Populations of Some Tautomeric Forms of 2′-Deoxyguanosine-5-Fluorouridine Mismatch ACS Paragon Plus Environment

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Saeed K. Amini*



TOC 254x190mm (300 x 300 DPI)


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Figure 1. Geometries of three different orientations of diuridine pairs with right, middle and left conformational families denoted as rWb, Wb and rWb*, respectively. Black, blue, red, white and yellow colors represent carbon, nitrogen, oxygen, hydrogen and substituted atoms, respectively. Existing strong hydrogen bonds are presented by dashed lines. The 2′-O-acetyl group is substituted by H in the case of dithymidine. 254x190mm (300 x 300 DPI)



Figure 2. Typical positions of ten selected representative conformers are denoted by asterisks in dihedral angle scan profile of wobble orientation of dithymidine. 254x190mm (300 x 300 DPI)


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Figure 3. Geometries of four different orientations of 2′-deoxyguanosine-5-fluorouridine nucleoside mispair with conformational families denoted as WC-like, Wb, rWC-like and rWb from right to left, respectively. Black, blue, red, white and yellow colors represent carbon, nitrogen, oxygen, hydrogen and fluorine atoms, respectively. Existing strong hydrogen bonds are presented by dashed lines. 254x190mm (300 x 300 DPI)



Figure 4. Geometries of four rare orientations of dGenol•rFU nucleoside pair with appreciable probabilities and structures from right to left denoted as are transWC/SE, cisWC/SE, cisWC/SE-O5′ and singleHB, respectively. Black, blue, red, white and yellow colors represent carbon, nitrogen, oxygen, hydrogen and fluorine atoms, respectively. Existing strong hydrogen bonds are presented by dashed lines. 254x190mm (300 x 300 DPI)


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Relative Populations of Some Tautomeric Forms of 2

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