BackboneâBase Interactions Critical to Quantum Stabilization of

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Backbone-Base Interactions Critical to Quantum Stabilization of Transfer RNA Anticodon Structure Rachel N. Witts, Emily C. Hopson, Drew E. Koballa, Thomas A. Van Boening, Nicholas H. Hopkins, Eric V. Patterson, and Maria Colleen Nagan J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp400084p • Publication Date (Web): 06 Jun 2013 Downloaded from http://pubs.acs.org on June 14, 2013

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The Journal of Physical Chemistry

Backbone-Base Interactions Critical to Quantum Stabilization of Transfer RNA Anticodon Structure Rachel N. Witts, Emily C. Hopson, Drew E. Koballa, Thomas A. Van Boening, Nicholas H. Hopkins, Eric V. Patterson and Maria C. Nagan* Department of Chemistry, Truman State University, 100 East Normal, Kirksville, MO 63501 *To whom correspondence should be addressed. Tel.: 660-785-4084; Fax: 660-785-4045; Email: [email protected]

ACS Paragon Plus Environment


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2 Abstract Transfer RNA (tRNA) anticodons adopt a highly ordered 3’-stack without significant base overlap. Density functional theory at the M06-2X/6-31+G(d,p) level in combination with natural bond orbital analysis was utilized to calculate the intramolecular interactions within the tRNA anticodon that are responsible for stabilizing the stair-stepped conformation. Ten tRNA x-ray crystal structures were obtained from the PDB databank and were trimmed to include only the anticodon bases. Hydrogenic positions were added and optimized for the structures in the stair-stepped conformation. The sugar-phosphate backbone has been retained for these calculations, revealing the role it plays in RNA structural stability. It was found that electrostatic interactions between the sugar-phosphate backbone and the base provide the most stability, rather than the traditionally-studied interbase stacking. Base-stacking interactions, though present, were weak and inconsistent. Aqueous solvation was found to have little effect on the intramolecular interactions.

Keywords: base-stacking, tRNA, anticodon, DFT, Natural Bond Orbital analysis, modified bases


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3 Introduction Stacking interactions occur in a variety of contexts in RNA and are key to forming RNA tertiary structure.1-3 Transfer RNA, the first RNA crystallized, possesses a number stacking motifs in its loop regions, at the top near the aminoacylation site and at the bottom where the anticodon bases all stack in a stair-stepped conformation.4,5 In RNA tetraloops,6-8 thermodynamic stability is gained from two loop bases stacking on the closing G:C base pair of the proximal helix. Sometimes stacking interactions are required to facilitate interaction of RNA helices in unusual structures such as pseudoknots9 or the adenosine platform first found in the P4-P6 domain of Tetrahymena thermophila self-splicing intron.10,11 Stacking interactions can also occur across the helix from one base to a base in the opposite strand, in a base-stacking motif called a purine-purine cross-strand stack.12 In general, interaction energies between stacked pairs are controlled mainly by the twist angle as opposed to the vertical separation.13,14 Base stacking, a noncovalent interaction essential in stabilizing nucleic acid structure1517

including RNA, has been examined experimentally in many contexts. Thermal denaturation

experiments indicate purine bases stack more strongly than pyrimidine bases, presumably because of larger surface area overlap.18 Thermodynamic parameters for single-strand stacking can be determined by calorimetry or by the temperature dependendence of spectroscopic properties. However, these methods measure the reversible transition from stacked to unstacked in single-stranded dinucleotides and thus give variable results because this transition occurs over a wide temperature range. For example, single-strand stacking for a poly(A) dinucleotide calculated by calorimetry gives a ΔH° that ranges from -3.0 kcal/mol19 to -8.5 kcal/mol.20 Conversely, experimental ΔG of vertical stacking in aqueous solution show small differences in the stability of different dimers. The values range from -1.5 kcal/mol for a GA stack, as



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4 measured by the solubility of solid-state adenine and deoxyguanosine,21 to 0.3 kcal/mol for a UU stack as determined by vapor pressure osmometry.22 Despite the large number of thermodynamic studies on interstrand stacking, the interaction energies of the π -interactions involved are seldom discussed. Originating primarily from London dispersion forces, accurate computational descriptions of base stacking interaction energies rely on the inclusion of electron correlation.23 More recent studies have focused on finding the most computationally efficient and accurate methods for describing noncovalent interactions.24-30 Most notably CCSD(T)31 with extrapolation to the complete basis set (CBS)32 is considered the gold standard, with highly accurate electron correlation descriptions, but because of computational cost is limited to nucleobase dimers. Calculating CCSD(T) interaction energies while extrapolating to the complete basis set at the MP2 level, termed the CBS(T) method,24 is the most accurate method available that can be applied to a number of nucleic acid base dimers.25,29,30 As an alternative, resolution of identity MP2 (RI-MP2)33 has been shown to be a more computationally efficient method for studying larger biomolecules.34 Fundamental to translation of the genetic code into proteins is correct recognition of the mRNA codon sequence by the three tRNA anticodon nucleotides (34-36). Crystal structures of tRNA,35-45 indicate that the anticodon bases adopt a highly ordered 3’-stack.46 Small anticodon stem-loops bound to the ribosome aminoacyl-site have also exhibited the same structure, which facilitates recognition by the ribosomal bases A1492, A1493 and A503.47,48 In molecular dynamics studies of the third human tRNA coding for lysine (tRNALys,3),49 it was noted that the 3’-stack of the tRNA anticodon has a unique base stacking interaction that does not involve a classic displaced base stacking structure but instead exhibits more of a stair-stepped


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5 conformation in which the three bases are planar and parallel to one another but there is no significant base-base overlap (Figure 1). To ascertain which interactions are responsible for stabilizing this stair-stepped conformation, quantum mechanical calculations have been carried out on ten tRNA anticodon structures, with and without the sugar-phosphate backbone. Natural bond orbital (NBO) analysis of the resulting wavefunctions is undertaken to discern the underlying stabilizing forces of the stair-stepped conformation.

Methods Anticodon Structures. Ten crystal structures of tRNAs alone or in complex with a protein that does not affect the anticodon structure were downloaded from the PDB databank.38,41,43,44,47,50-54 All tRNA structures (Table 1) were truncated to the anticodon trinucleotides, with 5’-OH groups and 3’ phosphates. Trinucleotides without backbone atoms, where the backbone is replaced with a methyl group, were also constructed. A’-Structures. Single-stranded structures of anticodon sequences adopting the standard RNA A’-form,55 which exhibits classic base-stacking interactions, were constructed for comparison to the stair-stepped conformation employing the program NUCGEN found in AMBER 8.56 Trinucleotide structures that include the backbone and those where the sugarphosphate backbone is replaced with a methyl group were also examined. Calculations. All electronic structure calculations were carried out with Gaussian09, Revision A.257 using the M06-2X density functional58 and the 6-31+G(d,p) basis set.59 This level of theory agrees within 0.5 kcal/mol against the CBS extrapolated CCSD(T) energies for the S22 database of Jurecka and coworkers.25,58 The S22 set consists of common biomolecules



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6 where the intermolecular interactions are dominated by hydrogen bonding interactions, dispersion interactions, or a mixture of the two. Importantly, M06-2X/6-31+G(d,p) returns good results exclusive of zero-point energy and basis set superposition error (BSSE) corrections, making it an ideal level of theory for studying noncovalent interactions in systems where it would be prohibitively expensive to determine the zero point vibrational energy and BSSE. Systems described in the studies contained herein contained 1326 to 2194 basis functions; therefore computational efficency was extremly important. Additionally, evaluation of intramolecular interactions presents a challenge not encountered during the evaluation of intermolecular interactions. For example, it is impossible to separate into monomeric, noninteracting species the phosphate from the sugar from the nucleobase without changing the fundamental nature of the system. Highly accurate analyses such as BSSE-corrected CCSD(T)/CBS interaction energies and SAPT analysis60 are intractable for intramolecular interactions. Others have found similar excellent correlations between NBO E(2) interaction energies and AIM analysis, total interaction energies and other markers commonly used to define non-bonded interactions such as hydrogen bonding and π stacking.61-68 Thus, second-order perturbation energies available through NBO analysis serve as a reasonable tool to understand the nonbonded interactions of the anticodon stem loop. Hydrogen atoms were added to the structures obtained from the PDB using GaussView.69 The hydrogenic positions were then optimized while the positions of the heavy atoms were held constant. Following optimization, the second order perturbation theory70 feature of the NBO population analysis71 was used to identify molecular orbital interactions. The stabilization energy [E(2)] between any two NBOs (equation 1) is taken to be a property of the occupancy of the donor NBO (qi), the energy difference between the donor and acceptor


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7 NBOs (εi and εj, respectively ), and the off-diagonal NBO Fock matrix element for the two orbitals (F(i,j)). F(i, j) 2 E(2) = qi ε j − εi

(1)

Thus, examination of the energy analysis results reveals both the magnitude of a particular € interaction as well as its origin. To assess the effect of solvent on the strength and contribution of each NBO interaction, the structures were optimized at the aforementioned level of theory employing the aqueous SMD72,73 solvation model and second order perturbation energies were calculated on the optimized structures as described above.

Results and Discussion To gauge the accuracy of the NBO E(2) approach, we compared our method to CCSD(T)/CBS results for the S22 database.25,58 The S22 database consists of 22 noncovalently bound dimers. Using the NBO E(2) approach, the interaction energy between the monomers is taken as the sum of the E(2) energies. NBO analysis on the S22 database gave good correlation with the CCSD(T)/CBS results, with slopes near 1.0 for the hydrogen bonded molecules, dispersion molecules, and mixed molecules that contain both hydrogen bonding and dispersion interactions (Figure 2, Table S1). These favorable correlations show that data obtained from the NBO analysis provides a qualitatively accurate picture of the noncovalent interactions. Futher base stacking analysis at the CCSD(T)/CBS level of theory has shown that for an AT stack and a GC stack in optimized, lowest energy geometries,74 the stacking interactions are similar to experimental values but are around 10% too low. The experimental value of stacking



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8 determined from the temperature dependence of equilibrium constants at relatively high temperatures for an unmethlyated AT stack is 14.0 kcal/mol75 compared to 9.75 kcal/mol calculated by NBO analysis for an unpaired AT stack from the S22 database with an optimized geometry. Thus, NBO analysis captures base stacking interactions with relative qualitative accuracy. Base Stacking Interactions in A’-form Standards. To determine if the methods employed give reasonable values for intramolecular interactions, standards containing unpaired, unmodified trinucleotides in A’-form geometries with sequences analogous to the tRNA anticodon sequences, with and without methyl groups replacing the sugar phosphate backbone were examined. Because of the localized nature of NBO analysis, a typical π-stacking interaction might be observed between, for instance, a uracil C6-C5 π bond and a cytosine C2-O2 π* bond rather than the entirety of both rings. π-stacking interaction energies summed over all three methylated A’-form standard bases, which is the sum of the stacking interactions between two pairs of stacked bases, ranged from 7.7 to 13.7 kcal/mol (Table 2). On average, π-stacking interactions between any two bases amounted to approximately 4.8 kcal/mol, which is consistent with previous base-stacking interaction energy estimates of unparied base stacks with optimized geometries at the MP2/6-31G*(0.25) level of theory, which range from 6.52 to 11.31 kca/mol.13 When the backbone is added to replace the methyl groups of the standards in A’-form geometries, base stacking is still present but to a lesser extent. π -stacking interaction energies over all three bases range from 1.1-6.1 kcal/mol while π -stacking between any pair of bases is approximately 2.0 kcal/mol. This is probably due to electron delocalization into the backbone. A similar phenonmenon was observed in quantum studies of B-DNA.76


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9 Backbone Interactions in A’-Form Standards. The primary intermolecular stabilizing interaction in the A’-form standards occured between atoms of the sugar-phosphate backbone. The O2’-lone pair of a residue often interacted favorably with the C5’-H5’1/C5’-H5’2 σ* orbital (Figure 3a). This interaction was consistently 37-40 kcal/mol in magnitude with an average of 39 kcal/mol (Table 3). The small range of interaction energies is reasonable since the structures were all derived from the same crystal structure. The effect of O2’ in stabilizing RNA structure is well established77 and it is therefore not surprising that it is involved in the predominant stabilizing interaction. Also present consistently but to a lesser extent was an interaction between a backbone O1P, O5’, or O4’ lone pair and a C8-H8 or C6-H6 σ* of the base. This value ranged from 4.72 kcal/mol to 17.91 kcal/mol with an average around 11 kcal/mol. There is no sequence dependence seen with this interaction. Thus, in the A’-form structures, the backbone provides considerably more stabilization to the structure than do πstacking interactions. Base Stacking in Anticodon Structures. Base stacking interactions were present in the anticodon structures. Total interaction energies due to π-stacking ranged from 0.0 kcal/mol to 11.0 kcal/mol with the average close to that of the A’-form standards with the backbone (Table 2). However, the variation in interaction energies was large compared to that found in the A’form standards with the backbone and there was no correlation to sequence. As an example, 1XMO and 1FIR have essentially the same UUU bases, with the only difference arising in the identity of the modification of the 34th nucleotide, yet their π-interaction energies differ by 7.2 kcal/mol. In the case of 1WZ2, with the sequence CAA, stacking should be favorable due to the greater surface area overlap of two purine bases,18 yet the CAU sequence of 1YFG, with only one purine base, has 11.0 kcal/mol more stabilization. There is a lack of congruency between



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10 the interaction energies of A’-form standards structures and anticodon structures with the same sequence. For example, 2DR2 and 2TRA have the same π-interaction energy for the methylated A’-form standards, 8.8 kcal/mol, and similar π-interaction energies for the A’-form standards with the backbone, 4.0 and 4.9 kcal/mol respectively. In the anticodon confirmation 2DR2 has a π-interaction energy of 6.1 kcal/mol and 2TRA has a π-interaction energy of 3.0 kcal/mol. These observations regarding base stacking when detected in the anticodon are not suprising. The 3’-stack of the anticodon is necessary for correct recognition of the codon as well as for ribosomal recognition.48,78,79 Stronger stacking interactions will favor the preorganization of the RNA into a more canonical helical conformation18 because the majority of the base stacking interaction is between bases within a strand. A consistent, strong base stacking interaction could in theory disrupt the stair-stepped structure of the anticodon and further interrupt recognition of the anticodon by the ribosome. Backbone Interactions in Anticodon Structures. In contrast to the canonical RNA structure, the strongest and most consistent stabilizing interaction in the anticodon stair-stepped conformation occured between either a backbone O1P or O5’ lone pair and a C8-H8 or C6-H6 σ* of the base, depending upon if the base was a purine or pyrimidine (Figure 3b). The second order perturbation energy was on average 17.4 kcal/mol and as high as 47.2 kcal/mol in 2TRA. Some variability does exist among the structures with a standard deviation as large as the average. This is likely due to differences in structure but no obvious trend is noted. C-H...O hydrogen bonds have been observed in crystal structures of RNA molecules80,81 and computational studies have revealed the importance of these interactions in biological systems in general.

82-85

Another interaction, more moderate in nature, was observed between the

backbone O4’ lone pair and the C2’-H2’1 σ* (Figure 3c) for an average of 4.7 kcal/mol. Not all


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11 of the trinucleotides possess this interaction. Additionally, the magnitude of the interaction is not correlated with that of the O1P/O5’ lone pair interaction with C8-H8/C6-H6 σ*. However it is one of the few interactions observed across most of the trinucleotides and therefore, in the cell, probably contributes modestly to the overall stability of the tRNA anticodon structure. It is helpful to note that for large interaction energies, the overlap of the two Fock matrix elements, F(i,j), is consistently near 0.1, which is one to two orders of magnitude larger than for most other interactions. This simply indicates significant overlap of the two interacting NBOs. Since the E(2) formula has F(i,j)2 in the numerator, the reported E(2) values are quite large when the overlap is significant. This significant overlap is due at least in part to the fact that the donor orbital in each case is on an anionic phosphate oxygen, and the tails of these orbitals extend some distance from the atom. Similar overestimation of the interaction energy can be seen when comparing NBO E(2) energies to CCSD(T)/CBS interaction energies for the formic acid dimer (Table S1). Given the good correlation between NBO E(2) interaction energies and those from other, more rigorous methods, our results qualitatively correlate with important interactions, even if they might overestimate them. No sequence-specific trends were observed in the trinucleotides. For instance, 1WZ2, 1YFG and 2TRA all exhibited the largest interaction energies between the O1P/O5’ lone pair and the C8-H8/C6-H6 σ* yet their sequences are CAA, CAU and GUC, respectively. It is reasonable though to not observe sequence-specific trends since all tRNAs adopt similar structures. The interactions between the backbone and the base observed in these studies, indicate a basis for uniform structure formation. In the cell, ribosomal nucleotides recognize the overall anticodon-codon base paired structure.48 The uniform stair-stepped anticodon structure, which has been found now with a variety of different codon-anticodon pairs in the



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12 ribosome,48,78,79 allows for correct anticodon-codon positioning in the context of the ribosome. Sugar-phosphate backbone hydrogen bonding with RNA bases has been observed in a number of RNA tertiary motifs.86,87 Interaction energies for these hydrogen bonds between base acceptor atoms and the phosphate backbone in these systems showed stabilization energies that exceed standard Watson-Crick hydrogen bonding stabilization.88 In addition to classic base stacking and base pair hydrogen bonding, interactions with the RNA backbone are also important for stabilization and must be considered for a complete picture of the stabilizing interactions of nucleic acids. Effect of Solvent. Comparison of the gas phase and solvated interaction energies show the influence solvent has on the interactions within the molecule. Overall, solvation has a minimal effect relative to the magnitude of the second order perturbation interaction energies. Solvation decreased the base-stacking interactions by an average of 1.3 kcal/mol in most of the anticodons examined (Table 4), consistent with previous findings that inclusion of an aqueous environment causes a modest decrease in the interaction energy due to solvent polarization.77 In the interactions between a lone pair on O1P/O5’ and the antibonding σ orbital of the C8-H8/C6H6 bonds, the interaction energy decreased on average 0.6 kcal/mol between the gas and aqueous phases. While the interaction energy between O4’ lone pair and the backbone C2’H2’1 σ* orbital was consistently found to increase across all anticodons, the effect was small (0.4 kcal/mol). Considering that some of the stabilization energies are on the order of 20 kcal/mol, solvation with water decreases interaction energies by about 10% overall. Effect of Modified Bases. Most tRNA molecules contain naturally occurring modified nucleic acid bases,89 with modifications observed often at the 34th and 37th positions.47,90 Modifications vary in chemical structure from very simple addition of a methyl group to


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13 addition of an amino acid.91 A universal role of modified bases in RNA structure has not been determined. However in the specific case of the 5-methylaminiomethyluridine (mnm5U) modification at position 34 of 1XMO, it seems that the modification quantum mechanically stabilizes the anticodon structure. The O1P/O5’ lone pair interaction with C8-H8/C6-H6 σ* in 1XMO was only a little over 1 kcal/mol, which is rather low when compared to the other systems. However, the modification in this molecule compensates by interaction of the O3’ lone pair of Residue 33 with the N-H σ* of mnm5U34 (Figure 4) with a stabilization energy of 60.4 kcal/mol in the gas phase and 48.7 kcal/mol in the presence of water. This strong modificationbackbone interaction substitutes for the backbone-base interaction observed in the other anticodon structures.

Conclusions Intramolecular interactions, which provide stabilization to the anticodon, are presented here. For the first time, the effect of the sugar-phosphate backbone was considered in the overall stability of the structure, and was examined in detail. Natural bond orbital analysis provides a reasonable estimate for the non-bonded interactions within RNA and agrees with both highlevel computational methods as well as available experimental data. Inclusion of the nucleic acid backbone has been rare in quantum studies because the computational expense increases significantly by including these atoms. However, a few studies have examined backbone effects in select systems using DFT methods. Quantum mechanicalmolecular mechanical (QM/MM) approaches corroborate experimental structural analysis, indicating that modifications to the backbone affect the local torsions in the backbone.92 Since these interactions have seldom been examined, few molecular dynamics programs take into



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14 account the electrostatics of the nucleic acid backbone. It is suggested then that parameters for the backbone electrostatics be determined and incorporated into future force fields. Analysis of the data presented here show that in addition to base stacking and hydrogen bonding, backbone-base interactions are critical for the structural stabilization of RNA. The strongest and most consistent interactions in both the anticodon structures and the A’-form trinucleotides are electrostatic interactions that involve the backbone. The calculated basestacking interaction energies suggest that the stair-stepped confirmation of the anticodon disrupts the π-interactions from the A’-form in some systems, but not in a sequence dependent manner. The overall lack of sequence dependence for the interactions observed provides further evidence for a uniform structure of the anticodon. Most importantly, when examining features of the tertiary structure of nucleic acids, electrostatic interactions that involve the sugarphosphate backbone must be taken into consideration to obtain an accurate picture of the stabilizing factors.

Acknowledgements Acknowledgement is made to Truman State University and the National Science Foundation for this work. This work was supported in part by NSF Grant CHE-074096, and by NSF Grants CHE-0821581 and CHE-0521063 as part of the MERCURY high-performance computer consortium (http://mercury.chem.hamilton.edu).

Supporting Information Presented in the supporting information are the geometries and SCF energies for all structures described, the correlation graph of CCSD(T)/CBS interaction energies completed by


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15 Jurecka25 versus NBO analysis interaction energies completed for the S22 database, and the second order pertubation theory interaction energies (kcal/mol) calculated by NBO analysis for the S22 database. This information is available free of charge via the Internet at http://pubs.acs.org.



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25 Bonded and Stacked Structures of Guanine---Cytosine, Adenine---Thymine, and Their 9- and 1-Methyl Derivatives: Complete Basis Set Calculations at the MP2 and CCSD(T) Levels and Comparison with Experiment. J. Am. Chem. Soc. 2003, 125, 15608-15613. (75)

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27 Groups on DNA Structure. J. Phys. Chem. B 2006, 111, 432-438.



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28 Table 1. Anticodon sequences and structures exhibiting the stair-stepped conformation. tRNA Species Anticodona Resolution (Å) PDBID Sourcef Asp S. cerevisiae GUC 3.0 2TRA 41 T 51 Cys E. coli GCA 2.6 1B23 P Glu T. thermophilus CUC 2.1 1N78 52 P 53 Leu P. horikoshii CAA 3.2 1WZ2 P 5 b 47 Lys E. coli mnm UUU 3.0 1XMO R 5 2 c 44 Lys B. taurus mcm s UUU 3.3 1FIR T Phe S. cerevisiae GmAAd 1.9 1EHZ 38 T 54 Trp B. taurus CCA 3.0 2DR2 P 5 e 50 Val E. coli cmo UAC 2.8 2UUB R fMet S. cerevisiae CAU 3.0 1YFG 43 T a b Anticodon sequence is written with nucleotides 34-36, left to right; 5methylaminiomethyluridine=mnm5U; c5-methoxycarbomoylmethyl-2-thiouridine=mcm5s2U; d 2'-O-methylguanine =Gm; e uridine-5-oxyacetic acid=cmo5U; fT=tRNA alone; P=complexed to protein; R=complexed to mRNA in the ribosome.


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29 Table 2. Total π-stacking interaction energy (kcal/mol). Values are summed over all trinucleotides. A’-form Methylated Anticodons Standards A’-form f with Backbone with Backbone PDBID Sequence Standards GCA 1B23 13.7 3.7 10.4 b GmAA 1EHZ 10.9 3.8 8.1 CUC 1N78 9.2 6.1 1.5 CAA 1WZ2 6.8 1.1 0.0 CAU 1YFG 9.7 5.3 11.0 mnm5UUUc 1XMO 7.7 4.6 7.8 5 2 d a mcm s UUU 1FIR — — 0.6 CCA 2DR2 8.8 4.0 6.1 GUC 2TRA 8.8 4.9 3.0 2UUB cmo5UACe 10.0 3.6 6.8 Average 9.5 ± 2.0 4.1 ± 1.4 5.5 ± 4.0 a b Sequence of unmodified A’-form standard is the same as 1XMO; 2'-O-methylguanine =Gm; c 5-methylaminiomethyluridine=mnm5U; d5-methoxycarbomoylmethyl-2-thiouridine=mcm5s2U; e uridine-5-oxyacetic acid=cmo5U; fA‘-form standards do not contain modified bases.



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30 Table 3. Primary interactions in trinucleotides that include the sugar phosphate backbone. All values are second order perturbation energies (kcal/mol) and summed over all three trinucleotides. A’-form Standards Anticodons LP O2' — LP O5’/O4’/OP1 — LP O5’/OP1 — LP O4’— C5’-H5’1/ C8-H8/C6-H6 σ* C8-H8/C6-H6 σ* C2’-H2’1 σ* C5’-H5’2 σ* gas PDBID gas gas aqueous gas aqueous 8.0 1B23 38.0 16.7 17.6 2.5 2.8 4.7 1EHZ 37.8 5.6 4.4 12.9 14.2 15.9 1N78 39.6 13.5 11.6 1.4 1.5 9.0 1WZ2 38.3 14.0 11.5 2.5 2.7 12.5 1YFG 40.3 41.5 38.1 7.5 7.9 16.4 1XMO 39.6 1.8 1.7 3.5 3.8 a — 1FIR — 35.6 21.7 9.0 9.8 9.5 2DR2 39.9 8.4 11.6 2.4 2.6 11.5 2TRA 37.9 47.2 43.4 0.0 0.0 12.6 2UUB 37.9 6.9 6.0 5.5 6.0 11.1 ± 3.7 17.4 ± 16.8 ± Average 38.8 ± 1.0 16.2 14.0 4.7 ± 4.0 5.1 ± 4.4 a Sequence of unmodified A’-form standard is the same as 1XMO.


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31 Table 4. Comparision of average interaction energies for gas phase and solution phase calculations of base stacking and backbone interactions (kcal/mol). Interaction

Gas average

Aqueous average

Base stacking

5.5 ± 4.0

4.2 ± 3.9

LP O5’/OP1 — C8-H8/C6-H6 σ*

17.4 ± 16.2

16.8 ± 14.0

LP O4’— C2’-H2’1 σ*

4.7 ± 4.0

5.1 ± 4.4



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32 Figure Captions Figure 1. Examples of base-base overlap in A’-form standards and anticodon trinucleotides as viewed from down the sugar phosphate backbone. a) A’-form standard of 2TRA, sequence GUC. b) 2TRA Anticodon. Figure 2. Correlation of CCSD(T)/CBS interaction energy on the S22 and second order perturbation energies from NBO analysis at the M06-2X/6-31+G(d,p) level of theory. Figure 3. Examples of interactions with the sugar-phosphate backbone in A’-form standards and anticodon trinucleotides. a) Back view of A’-form standard for 1FIR, O2’-LP C5’H5’1/C5’-H5’2 σ* (13.7 kcal/mol) b) Front view of 1EHZ anticodon, O4’of backbone to C2’H2’1 σ* (9.3 kcal/mol) and c) Front view of 2TRA anticodon, O1P/O5’ of backbone-C8H8/C6-H6 σ* of base (25.1 kcal/mol). Figure 4. Chemical structure of the modified base mnm5U and an example interaction of the O3’ LP p orbital interacting with the N-H σ* (41.8 kcal/mol).


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33

a

b

Witts et al., Figure 1



34

35.00 H-Bonding 30.00 Dispersion 25.00

NBO E(2) (kcal/mol)

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Mixed y = 1.3228x R² = 0.64803

20.00

15.00

10.00 y = 0.8426x R² = 0.84598 5.00 y = 0.7704x R² = 0.74833 0.00 0

5

10

15

20

25

CCSD(T)/CBS (kcal/mol)



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37 TOC Graphic


BackboneâBase Interactions Critical to Quantum Stabilization of

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