Proc. Natl. Acad. Sci. USAVol. 93, pp. 649-653, January 1996Biochemistry

Interresidue hydrogen bonding in a peptide nucleicacid-RNA heteroduplex

(antisense/hybrid duplex/DNA backbone analog)

RHONDA A. TORRES AND THOMAS C. BRUICEDepartment of Chemistry, University of California, Santa Barbara, CA 93106

Contributed by Thomas C. Bruice, September 25, 1995

ABSTRACT Previous molecular mechanics calculationssuggest that strands of peptide nucleic acids (PNAs) andcomplementary oligonucleotides form antiparallel duplexesstabilized by interresidue hydrogen bonds. In the computedstructures, the amide carbonyl oxygen nearest the nucleobase(07') forms an interresidue hydrogen bond with the backboneamide proton of the following residue, (n + 1)Hl'. Of the 10published two dimensional 1H NMR structures of a hexamericPNA-RNA heteroduplex, PNA(GAACTC)-r(GAGUUC), 9 ex-hibit two to five potential interresidue hydrogen bonds. In ourminimized average structure, created from the coordinates ofthese 10 NMR structures, three of the five possible interresi-due hydrogen bond sites within the PNA backbone display thecarbonyl oxygen (07') and the amide proton (n + 1)H1'distances and N1'-H1'-(n - 1)07' angles optimal forhydrogen bond formation. The finding of these interresiduehydrogen bonds supports the results of our previous molec-ular mechanics calculations.

In viable peptide nucleic acids (PNAs), the ribose-phos-phodiester backbone of oligonucleotides is replaced with apeptidic backbone consisting ofN-(2-aminoethyl)glycine units,with the glycine nitrogen connected to a purine or pyrimidinebase by an acetyl linker (Scheme 1) (1-5).

P02I°2

oCH X

n

X=H DNAX=OH RNA

PNA

Scheme I

These neutral, homomorphous DNA analogs recognize theirDNA and RNA complements by Watson-Crick and Hoogs-teen base pairing, thereby maintaining the specificity associ-ated with natural oligonucleotides. This type of PNA has beenshown to bind target DNA or RNA strand(s) with a greateraffinity than observed between innate oligonucleotide strands.These polymeric structures also display greater stabilityagainst nuclease activity and greater sensitivity to base-pairmismatches when compared with their DNA and RNA oligo-nucleotide counterparts. These features make these PNAsattractive as putative genetic regulatory agents. Much interest

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has, therefore, been generated regarding the structure of theseanalogs when bound to their DNA or RNA target sequences.Although detailed binding information regarding the PNA

strand in heteroduplex structures has been presented, the greatstability of PNA-nucleic acid hybrids has not been fullyexplained (2, 5). It has been attributed, in part, to a reductionin electrostatic repulsion that is associated with the replace-ment of the negatively charged phosphodiester backbone witha neutral PNA strand. It does not appear, however, that thestability of hybrids can be explained by the elimination ofelectrostatic charge alone (6-8). In our previous molecularmodeling studies (9, 10), we suggested that the conformationof the PNA strand is predefined by nucleobase pairing whichwas proposed to give rise to interresidue hydrogen bondingbetween the acyl oxygen (07') of the first residue and thebackbone amide proton (Hi') of the following residue, asshown in Scheme 2.

B1 B2x3 to,0>

H X1 |yHON a NLN N N

0 0

Scheme IIn

Formation of this hydrogen bond was suggested to contributeto the stability of the PNA strand when PNA binds to itsoligonucleotide complement (9, 10) and is believed to exist dueto the high effective concentrations of the amide donor andacceptor groups created in the "cooperative process" thatoccurs when oligonucleotide strands anneal (11). 'H NMRstructural information of PNA-DNA and PNA-RNA hetero-duplexes has recently been presented (12, 13), providingdetailed experimental data of the conformation adopted by thePNA strand within a hybrid duplex. However, the PNAbackbones in these NMR studies are not yet well restrained bythe experimental data because it was not possible to assign thebackbone prostereogenic methylene protons. It was concludedin both NMR studies that the amide protons in the PNAbackbone do not participate in internal hydrogen bonds. Aspredicted by model building and molecular mechanics (9, 10),it was found in both NMR studies that the PNA strand bindsantiparallel, and the primary amide within the PNA backboneadopts a single conformation when bound to its complemen-tary oligonucleotide strand.The subject of the most complete structural study to date

that describes in detail the conformation of the PNA backbone

Abbreviation: PNA, peptide nucleic acid.

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650 Biochemistry: Torres and Bruice

is the hexameric PNARNA heteroduplex PNA(GAAC-TC).r(GAGUUC) (13). We employ the published structuraldata (13) concerning the PNA(GAACTC).r(GAGUUC) het-eroduplex to specifically explore the hydrogen bonding dis-tances and angles between the 07' and (n + 1)H1' function-alities. As predicted in our previous molecular modelingstudies (9, 10), the calculated 07'-(n + 1)H1' distances andNi'-Hi'-(n - 1)07' angles establish that the carbonyloxygen, 07', and the amide proton, (n + 1)H1', are orientedto favor interresidue hydrogen bonding.

MATERIALS AND METHODSThe 10 structures of the hexameric PNARNA heteroduplex,PNA(GAACTC)-r(GAGUUC), generated by simulated an-nealing with NMR constraints (generously provided by S. C.Brown and coworkers of Glaxo, Inc.), are now available fromthe Brookhaven Protein Data Bank (accession code 179d).QUANTA (14) version 4.0 (1994) was used to visualize the 10NMR-refined heteroduplexes. All computational analyseswere performed on a Silicon Graphics (Mountain View, CA)4D/34OGTX workstation. The nomenclature for the PNAbackbone devised by Brown and coworkers (13) was main-tained in this study for ease of comparison. The Cartesiancoordinates of all 10 NMR-refined structures were averaged byusing QUANTA to obtain a preliminary average structure. Thepartial atomic charges of the nucleobase and backbone atomsin the PNA strand were obtained from the DNAH.RTF andAMINOH.RTF files, respectively, provided with QUANTA. Aresidue topology file (RTF) was written for the PNA strand,while a modified RNAH.RTF was used for the RNA strand.Modifications were made in the default equilibrium values forthe phosphoryl (>po2-) and sugar-phosphate backbone an-gles provided by CHARMM (14-16). These values were takenfrom the AMBER (17) force field (Version 3a) and used in theenergy minimization calculations. Notable bond angle param-eter modifications are from 109.470 (CHARMM) to 119.90(AMBER) and from 109.470 (CHARMM) to 102.60 (AMBER) forthe O1P-P-02P and 05'-P--03' angles, respectively (18).The RNA strand was minimized with sodium counterionsinitially placed -3 A from the phosphorous atom in eachphosphate moiety. A distance-dependent dielectric was usedthroughout the minimization procedure. Hydrogen bondingand nonbonded interactions were cut off utilizing a switchingfunction at 5.0A and 15.0 A, respectively, with the angle cutofffor hydrogen bonding interactions set at 900. The criterion forhydrogen bonding was defined as having a separation distancec3.0 A between the hydrogen atom and the acceptor (oxygen)atom and the angle between the donor-H acceptor-i.e.,

N-H O0 angle, known as 0-90° < 0 s 1800 (19). Watson-Crick distance constraints (20) between the nucleobases wereapplied. The PNA-RNA heteroduplex was subjected to 100steps of steepest descents (SD) minimization to remove themost severe steric conflicts associated with the preliminaryaverage structure to yield the average structure. The averagestructure was further minimized by using the adopted basisNewton-Raphson (ABNR) algorithm until a final step toler-ance of 1 x 10-9 kcal per 10 steps was achieved to obtain aminimized average structure. The Watson-Crick distance con-straints were gradually removed, and the final ABNR mini-mizations were performed without constraints. rms deviationsbetween the structures were calculated by using only the heavyatoms. The H6/H8 to H8'/H8" distances from each of the 10NMR-refined structures were compared with those in ouraverage structure and our minimized average structure andwere found to be within the limits of the NMR experiment(21).The NEWHEL93 (22) program, generously provided by R. E.

Dickerson (University of California, Los Angeles), was used todetermine the helical parameters of the PNA-RNA duplexstructure following the energy minimizations. The programwas run with coordinates in the Brookhaven Protein DataBank format. It was necessary to change the names of some ofthe atoms in the PNA strand to execute the program. Atomname changes in the PNA backbone include C (PNA) renamedto P, 0 (PNA) renamed to 01P, Ni' (PNA) renamed to 05',and C8' (PNA) renamed to Ci'. The atoms used to define thehelices were the Cl' atoms in the RNA strand, the C8' atomsin the PNA strand, the N9 atoms of the purines, and the Niatoms of the pyrimidines of both strands in the duplex. Themajor and minor groove widths were determined by calculat-ing the backbone carbonyl carbon (PNA) to phosphorous(RNA) distances across each groove. The turn angle (0/bp),axial rise (A/bp), and helical rise (bp/repeat) parameters weredirect outputs of the program, while the pitch height (A/repeat) was calculated as the product of the helical rise andaxial rise values. The sugar puckers of the ribose rings in theRNA strand were determined by inspection.

Separate semiempirical calculations (unpublished data)were performed to model the dependence of the hydrogenbond strength on the N-H 0--C hydrogen bond betweenan amide proton of one formamide molecule and the amideoxygen of a second formamide molecule, similar to a studyperformed by Novoa and Whangbo (23). The energies of thestructures were initially calculated by using the SAM1 geom-etry-optimization method (24-26) in the AMPAC 5.0 (27) pro-gram. This was followed by single-point gas-phase energycalculations with AM1 (28) and with the SM2.1/AM1 solva-tion model (29) to correct the single-point gas-phase energiesto a water dielectric. The energies of the structures weredetermined as a function of the N-H--0 angle (at fixed

Table 1. Helical parameters calculated from the NMR* and computationalt PNARNA heteroduplex structuresTurn Axial Unit Average Average

Heteroduplex angle, rise, Helical rise, height, minor-groove major-groove Sugarstructure 0/bp A/bp residue/turn A/turn width, A width, A puckertModel 1* 26.8 2.15 13.44 28.92 14.71 20.43 C3'-endoModel 2* 24.4 2.20 14.76 32.45 15.54 21.04 C3'-endoModel 3* 22.4 2.45 16.09 39.39 15.31 21.23 C3'-endoModel 4* 22.8 2.60 15.78 41.05 15.56 21.09 C3'-endoModel 5* 31.6 1.70 11.40 19.25 15.99 20.77 C3-endoModel 6* 28.3 2.03 12.73 25.87 16.40 21.58 C3'-endoModel 7* 25.6 2.03 14.05 28.59 16.33 21.25 C3'-endoModel 8* 29.0 1.72 12.39 21.27 16.06 21.77 C3'-endoModel 9* 29.6 2.28 12.17 27.80 16.12 20.26 C3'-endoModel 10* 26.7 2.18 13.48 29.33 15.29 20.49 C3'-endoAveraget 26.6 2.15 13.53 29.06 15.62 21.09 C3'-endoMinimizedt 24.4 2.40 14.73 35.35 15.65 20.26 C3'-endo

*Calculated from ref. 13 data.tRefers to the results of this computational study.fRefers to the RNA strand in each structure only.

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Table 2. Comparison of conformational angles* important forinterresidue hydrogen bonding in the initial average structureand the lowest energy structure of the PNA-RNA heteroduplex

Residue Xl a cotGl -2 - (+3) 78 35 (+21) 81 ± 35 (+28) 176A2 6 +5 (+2) 72 25 (-12) 76 ± 25 (+13) 178A3 3 +5 (+17) 74 55 (-6) 74 ± 55 (-5) 180C4 9±15(-3) 76±50(+29) 76±50(-23) 172T5 4 +15 (+11) 85 30 (-6) 85 ± 30 (-13) 172C6 2 + (+1) - -

The first value in each column was obtained (13) from lowest energy,simulated annealing structure (of the 10 NMR-refined structures). The± range listed includes only a selected number of the NMR-refinedstructures, as in ref. 13. The values in parentheses represent thedeviation observed in our initial average structure relative to thelowest energy NMR-refined structure.*All torsional angles given in degrees.tRefers to the torsional angle between this residue and the followingresidue listed (not reported in ref. 13).

C-O-H angles of 1200 and 1790) and as a function of theC-0--H angle (at the determined optimal N-H-0 angleof 1790 from the previous calculations). The O.H distancebetween the formamide molecules was held fixed for eachreaction trajectory, and these distances ranged from 2.11 A to3.50 A, to include the interresidue hydrogen-bonding distancesproduced from the energy minimization calculations. Thedihedral angle between the two formamide molecules wasfixed at 00, 900, and 1800 for the reaction trajectory calculations.The energy dependence on the dihedral angle was found to beinsignificant.

RESULTS AND DISCUSSIONExamination of the 10 two-dimensional 1H NMR-refinedstructures (13) of PNA(GAACTC)-r(GAGUUC) revealedthat the C7' carbonyl groups were directed toward the Cterminus of the PNA strand in an antiparallel fashion (Scheme2), in accord with our previous molecular modeling studies (9,10). The average rms deviations between the initial averagestructure (obtained by averaging the Cartesian coordinates ofthe 10 NMR-refined structures) and each of these 10 refinedstructures were calculated to be 1.0 A. Further minimizationof the initial average structure (see Materials and Methods)gave the minimized average structure. The average rms devi-ation between our minimized average structure and each of the10 NMR-refined structures was subsequently determined to be1.4 A.

The helical parameters of the following 12 structures weredetermined by using the NEWHEL93 (21) program: 10 NMR-refined PNA-RNA heteroduplex structures (13), our initialaverage structure, and our minimized average structure (Table1). All structures examined displayed a C3'-endo sugar pucker,as expected for an A-type RNA conformation. The remaininghelical parameters, however, were found to closely resembleB-type helical conformations rather than A-type helical con-formations. Such mixed characteristics can be expected tooccur in a heteroduplex of this length (20). The helical param-eters of both the initial average and minimized averagestructures were found to be within those values calculated forthe helical parameters that we report (Table 1) for each of the10 NMR-refined structures.

All the published conformational angle parameters of thePNA(GAACTC)-r(GAGUUC) heteroduplex (13) comparewell to those of our initial average structure (data not shown).Because the NMR spectroscopic data did not allow for as-signment of the prostereogenic hydrogens in the PNA back-bone, with the exception of H8' and H8", we restrict ourdiscussion to the torsional angles Xi, 8, a, and w (Scheme 2).These are the important torsional angles for interresiduehydrogen bonding. As can be seen in Table 2, all of thesetorsional angles in the initial average structure, with the excep-tion of one, XI of residue PNA(A3), are well within theconformational angle parameter ranges reported (13). In theinitial average structure, the 07'-(n + 1)H1' distances andthe Ni'-Hi'-(n - 1)07' angles were found to be orientedsuch that interresidue hydrogen bonding (Scheme 2) wasobserved for all potential sites in the PNA backbone. As shownin Fig. 1, the N-terminal to C-terminal 07'-(n + 1)Hl'distances are 2.31 A, 2.92 A, 2.45 A, 2.26 A, and 2.31 A,respectively. The distances and angles between potential hy-drogen bond donors and acceptors (Table 3) within the PNAbackbone of the initial average structure are all consistent withthe distance and angle requirements for hydrogen bondinggiven in Materials and Methods (19).

Comparisons of the torsion angles Xi, 8, s, and co (Table 4)in the minimized average structure (Fig. 2) to those anglesreported (13) establishes that only the torsion angle s ofresidue PNA(A3) is outside the given range. Of the fiveinterresidue hydrogen bonds that were present in the initialaverage structure, three remain in the minimized average struc-ture. The two interresidue hydrogen bonds in the A3-C4-T5region are no longer present (Fig. 2). The portion of the averagestructure presented in the literature (13) is this A3-C4-T5region. Examination of Tables 4 and 5 reveals that theNl'-Hl'--(n - 1)07' angles are near the suggested (31)

N,'

s c % * f

FIG. 1. Stereoview of our initial average structure of the PNA(GAACTC).r(GAGUUC) heteroduplex. The PNA strand (shown here in black)displays all of the five potential interresidue hydrogen bonds. The distances are given from N terminus to C terminus. The RNA strand is shownin gray. Molecular modeling was conducted as described in Materials and Methods. The picture was made using SYBYL 6.1 (30).

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Table 3. Interresidue hydrogen bonds observed in the initialaverage structure

Residues Distance, A* Angle, ot

Gl-A2 2.31 131A2-A3 2.92 134A3-C4 2.45 147C4 T5 2.26 138T5-C6 2.31 142

*Distance between the carbonyl oxygen of the first residue listed andthe amide proton of the second residue listed.

tAngle between the carbonyl oxygen of the first residue listed and theamide proton and nitrogen of the second residue listed.

optimal angle of 1600 for the three remaining interresiduehydrogen bonds observed in the minimized average structure.Comparison of the initial average structure with the mini-

mized average structure suggests that lack of interresiduehydrogen bonding in the A3-C4-T5 region of the latterappears to result from removal of close contacts introduced inthe PNA backbone when the initial average structure wascreated from the 10 NMR-refined structures. The values forthe interresidue hydrogen bond conformational angles (XI, 8,s, and c) in combination with the rms deviation data and theappearance of the A3-C4-T5 region suggests that theminimized average structure that we have obtained is virtuallyidentical to the average structure presented in the literature(13). It is important to note that both our initial averagestructure and our minimized average structure represent pos-sible conformations of the PNA backbone. However, ourminimized average structure possesses structural features sim-ilar to the published portion (13) of the average structure.To determine the dependence of the N-H .0=C hydro-

gen bond strength upon the 07'-(n + 1)Hl' distances andNi'-Hi'-(n - 1)07' angles, separate experiments modelingthe hydrogen bond between two formamide molecules wereconducted. These calculations confirm that the three hydrogenbonds in the minimized average structure with Ni'-Hi'-(n- 1)07' angles of 1510 and the 07'-(n + 1)Hl' distances of2.12 A, 2.13 A, and 2.17 A (Fig. 2) are very near the optimaldistances and angles for hydrogen bonding predicted by thesemiempirical solvation calculations. Thus, there are threevery reasonable hydrogen bonds in our minimized averagestructure.

Table 4. Comparison of conformational angles* important forinterresidue hydrogen bonding in the minimized average structureand the lowest energy structure of the PNA'RNA heteroduplexResidue Xi S E ,t

G6 -2 +5 (-2) 78 + 35 (+13) 81 35 (-8) -180A2 6 5 (+O) 72 +25 (+10) 76 25 (-14) -173A3 3 5 (+3) 74 +55 (+13) 74 +55 (-91) -172C4 9 + 15 (+13) 76 +50 (-8) 76 +50 (+48) 161T5 4 + 15 (+0) 85 + 30 (+0) 85 + 30 (+22) -179C6 2+5(+3)

The first value in each column was obtained (13) from lowest energy,simulated annealing structure (of the 10 NMR-refined structures). The+ range includes only a selected number of the NMR-refined struc-tures, as in ref. 13. The values in parentheses represent the deviationobserved in our minimized average structure relative to the lowestenergy NMR-refined structure.*AII torsional angles given in degrees.tRefers to the torsional angle between this residue and the followingresidue listed (not reported in ref. 13).

The observations (13) offered to refute our previous sug-gestion (9, 10) of N-H- 0=C bonds all focus on the T5amide proton of the PNA(GAACTC)-r(GAGUUC) structure:(i) lack of protection of this amide proton from exchange withH20 solvent; (ii) the appearance of "long amide hydrogen tocarbonyl oxygen distances (on average -2.8 A) and poorN-H--0 angles (-130°)" for hydrogen bonding in the 15N-labeled primary amide of residue T5; and (iii) the chemicalshifts appear inconsistent with those of amide protons partic-ipating in hydrogen bonding. These experiments are suggestiveof the lack of a persistent hydrogen bond at the T5 position.Actually, our minimized average structure, as well as thepublished average structure (13), shows hydrogen bond dis-tances and angles in the A3-C4-T5 region to be excessive.At the three remaining possible sites in the minimized averagestructure, interresidue hydrogen bonds were observed. Thus,from available data, interresidue hydrogen bonding may per-sist in some regions of the heteroduplex. In other regions,nonpersistence may relate to competitive hydrogen bondingwith water. The extent of interresidue hydrogen bonding thatoccurs may be dependent on the sequence and the length of thePNA strand. Concerning the lack of a chemical shift for theamide proton in residue T5, it has been suggested that a

FIG. 2. Stereoview of our minimized average structure of the PNA(GAACTC)-r(GAGUUC) heteroduplex. The PNA strand (in black) displaysthree (of the five possible) interresidue hydrogen bonds that persisted after the complete molecular modeling procedure (Materials and Methods).Interresidue hydrogen bond distances are given from N terminus to C terminus. The two interresidue hydrogen bonds that were found in theA3-C4-T5 region of the initial average structure are no longer present. The RNA strand is shown in gray. The picture made using SYBYL 6.1 (30).

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Table 5. Interresidue hydrogen-bond distances and anglesobserved in the minimized average structure

Residues Distance, A* Angle, at

Gl A2 2.13 151A2-A3 2.17 151A3-C4 3.44 103C4-T5 3.32 115T5-C6 2.12 151

*Distance between the carbonyl oxygen of the first residue listed andthe amide proton of the second residue listed.

tAngle between the carbonyl oxygen of the first residue listed and theamide proton and nitrogen of the second residue listed.

chemical shift may not be observed in the case of weakhydrogen bonding (19). In addition, it has also been shownexperimentally (31) that an amide proton equilibrating be-tween hydrogen bonding with another amide oxygen andhydrogen bonding with a water molecule can display a smallchemical shift dependence (A68/AT).

CONCLUSIONSEmploying the published 10 PNA(GAACTC)'r(GAGUUC)heteroduplex structures, obtained by two-dimensional 'HNMR (13), we have created a minimized average structure.Through the examination of the 10 NMR-refined structures, itwas observed that 9 of the 10 structures displayed 07'-(n +1)H1' distances and Ni'-HI'-(n - 1)07' angles such thatinterresidue hydrogen bonding was possible. Comparison (rmsdeviation calculations, helical parameters, and conformationalangle parameters) of our minimized average structure with theaverage structural data published (13) suggests that the twostructures are essentially identical. Our results show that of thepotential five interresidue hydrogen bonds, three have verynear ideal geometries for hydrogen bond formation. For theremaining possible two hydrogen bonds located in theA3-C4-T5 region both the 07'-(n + 1)H1' distances andNl'-Hi'-(n - 1)07' angles are not conducive to hydrogenbond formation. Applying experimental results from a singleresidue (T5) to all the residues in the PNA strand may be anoversimplification of the complexity of the binding of com-plementary oligonucleotide strands and the conformationalflexibility associated with the PNA backbone. On the basis ofour findings, we contend that the proposed interresidue hy-drogen-bonding motif is supported by the present NMR-refinement data (12, 13).

We would like to thank Dr. S. C. Brown from Glaxo, Inc., forproviding us with the coordinates of the 10 NMR-refined structuresprior to their release. We would also like to thank Helgi Adalsteinssonfor his work on the semiempirical calculations. R.A.T. is a recipient ofa National Institutes of Health Underrepresented Minority GraduateResearch Assistant Supplementary Grant. This study was supported bygrants from the National Institutes of Health and the Office of NavalResearch.

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