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- Human DNA Polymerase ι Incorporates dCTP Opposite Template G...Human DNA Polymerase ι Incorporates dCTP Opposite Template G via a G.C+ Hoogsteen Base Pair
Structure, Volume 13, Issue 10, 1 October 2005, Pages 1569-1577
Deepak T. Nair, Robert E. Johnson, Louise Prakash, Satya Prakash and Aneel K. Aggarwal
SummaryHuman DNA polymerase ι (hPolι), a member of the Y family of DNA polymerases, differs in remarkable ways from other DNA polymerases, incorporating correct nucleotides opposite template purines with a much higher efficiency and fidelity than opposite template pyrimidines. We present here the crystal structure of hPolι bound to template G and incoming dCTP, which reveals a G.C+ Hoogsteen base pair in a DNA polymerase active site. We show that the hPolι active site has evolved to favor Hoogsteen base pairing, wherein the template sugar is fixed in a cavity that reduces the C1′-C1′ distance across the nascent base pair from ∼10.5 Å in other DNA polymerases to 8.6 Å in hPolι. The rotation of G from to is then largely in response to this curtailed C1′-C1′ distance. A G.C+ Hoogsteen base pair suggests a specific mechanism for hPolι’s ability to bypass N-adducted guanines that obstruct replication.
Summary | Full Text | PDF (517 kb) - The Paperclip Triplex: Understanding the Role of Apex Residu...The Paperclip Triplex: Understanding the Role of Apex Residues in Tight Turns
Biophysical Journal, Volume 91, Issue 7, 1 October 2006, Pages 2552-2563
Lou-sing Kan, Laura Pasternack, Ming-Tsair Wey, Yu-Yu Tseng and Dee-Hua Huang
AbstractIn this study, we investigate the role of the apex nucleotides of the two turns found in the intramolecular “paperclip” type triplex DNA formed by 5′-TCTCTCCTCTCTAGAGAG-3′. Our previously published structure calculations show that residues C-A form a hairpin turn via Watson-Crick basepairing and residues T-C bind into the major groove of the hairpin via Hoogsteen basepairing resulting in a broad turn of the T-T 5′-pyrimidine section of the DNA. We find that only the CC/G apex triad (and not the TA/T apex triad) is required for intramolecular triplex formation, is base independent, and occurs whether the purine section is located at the 5′ or 3′ end of the sequence. NMR spectroscopy and molecular dynamics simulations are used to investigate a bimolecular complex (which retains only the CC/G apex) in which a pyrimidine strand 5′- TCTCTCCTCTCT-3′ makes a broad fold stabilized by the purine strand 5′-AGAGAG-3′ via Watson Crick pairing to the T-T and Hoogsteen basepairing to T-T of the pyrimidine strand. Interestingly, this investigation shows that this 5′-AGAGAG-3′ oligo acts as a new kind of triplex forming oligonucleotide, and adds to the growing number of triplex forming oligonucleotides that may prove useful as therapeutic agents.
Abstract | Full Text | PDF (289 kb) - An Incoming Nucleotide Imposes an anti to syn Conformational...An Incoming Nucleotide Imposes an anti to syn Conformational Change on the Templating Purine in the Human DNA Polymerase-ι Active Site
Structure, Volume 14, Issue 4, 1 April 2006, Pages 749-755
Deepak T. Nair, Robert E. Johnson, Louise Prakash, Satya Prakash and Aneel K. Aggarwal
SummarySubstrate-induced conformational change of the protein is the linchpin of enzymatic reactions. Replicative DNA polymerases, for example, convert from an open to a closed conformation in response to dNTP binding. Human DNA polymerase-ι (hPolι), a member of the Y family of DNA polymerases, differs strikingly from other polymerases in its much higher proficiency and fidelity for nucleotide incorporation opposite template purines than opposite template pyrimidines. We present here a crystallographic analysis of hPolι binary complexes, which together with the ternary complexes show that, contrary to replicative DNA polymerases, the DNA, and not the polymerase, undergoes the primary substrate-induced conformational change. The incoming dNTP “pushes” templates A and G from the to the conformation dictated by a rigid hPolι active site. Together, the structures posit a mechanism for template selection wherein dNTP binding induces a conformational switch in template purines for productive Hoogsteen base pairing.
Summary | Full Text | PDF (625 kb) - …more
Copyright © 2007 The Biophysical Society. All rights reserved.
Biophysical Journal, Volume 92, Issue 3, 769-786, 1 February 2007
doi:10.1529/biophysj.105.079723
Biophysical Theory and Modeling
Orientation Preferences of Backbone Secondary Amide Functional Groups in Peptide Nucleic Acid Complexes: Quantum Chemical Calculations Reveal an Intrinsic Preference of Cationic D-Amino Acid-Based Chiral PNA Analogues for the P-form
Christopher M. Topham*, 1, 
, 
and Jeremy C. Smith†, ‡
* Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique UMR 5089, Toulouse, France
† Computational Molecular Biophysics, IWR, Universität Heidelberg, Heidelberg, Germany
‡ Oak Ridge National Laboratory/University of Tennessee Center for Molecular Biophysics, Oak Ridge National Laboratory, Oak Ridge, Tennessee
Address reprint requests to Christopher M. Topham.1 Christopher M. Topham’s present address is Novaleads, Centre de Bioinformatique de la Haute Garonne, CEEI Théogone, 10 avenue de l’Europe, 31520 Ramonville-Saint-Agne, France.
Abstract
Geometric descriptions of nonideal interresidue hydrogen bonding and backbone-base water bridging in the minor groove are established in terms of polyamide backbone carbonyl group orientation from analyses of residue junction conformers in experimentally determined peptide nucleic acid (PNA) complexes. Two types of interresidue hydrogen bonding are identified in PNA conformers in heteroduplexes with nucleic acids that adopt A-like basepair stacking. Quantum chemical calculations on the binding of a water molecule to an O2 base atom in glycine-based PNA thymine dimers indicate that junctions modeled with P-form backbone conformations are lower in energy than a dimer comprising the predominant conformation observed in A-like helices. It is further shown in model systems that PNA analogs based on D-lysine are better able to preorganize in a conformation exclusive to P-form helices than is glycine-based PNA. An intrinsic preference for this conformation is also exhibited by positively charged chiral PNA dimers carrying 3-amino-D-alanine or 4-aza-D-leucine residue units that provide for additional rigidity by side-chain hydrogen bonding to the backbone carbonyl oxygen. Structural modifications stabilizing P-form helices may obviate the need for large heterocycles to target DNA pyrimidine bases via PNA·DNA-PNA triplex formation. Quantum chemical modeling methods are used to propose candidate PNA Hoogsteen strand designs.Introduction
More than a decade ago, Nielsen and co-workers described an electrostatically neutral chimera between nucleic acids (the nucleobases) and (pseudo-) peptides (the backbone), termed “polyamide nucleic acids”, or PNAs 1,2. These molecules, which are resistant to both nuclease and proteinase attack 3, comprise a backbone that is structurally homomorphous to the deoxyribose phosphate backbone, containing achiral N-(2-aminoethyl) glycine (aeg) units to which the nucleobase is attached via a methylene carbonyl linker (Fig. 1). PNAs form specific and highly thermally stable complexes with complementary single-stranded DNA or RNA, mediated by Watson-Crick hydrogen bonding 4. Unique among oligonucleotide analogs, PNAs are additionally able to strand invade double-stranded DNA 5,6. Their remarkable strand invasion properties, alongside demonstrations that modular PNA-conjugate constructs can cross cellular 7,8,9 and nuclear 10,11 membrane barriers, have made PNAs promising lead candidate molecules for the therapeutic control of gene expression 12,13,14,15,16.
Figure 1
Schematic representation of a PNA residue junction with atom and dihedral angle nomenclature. Chiral PNA analogs based on D-aminoethylamino acid units carry side-chain (R) replacements of the pro-R hydrogen in the glycine moiety of the prototype (aeg) design.In practice, the efficient targeting of double-stranded DNA via (PNA·DNA-PNA) triplex formation remains essentially limited to homopurine (pu) DNA tracts 17. The use of pseudocomplementary (pc) PNA oligomers, comprising 2,4-diaminopurine and 4-thiouracil base replacements for adenine and thymine to disfavor unwanted self association of the pcPNA strands, allows mixed (pu/py) DNA sequences with a minimum 50% AT content to be targeted by a double duplex invasion mechanism 18. The future incorporation of pseudocomplementary guanine and cytosine nucleobase analogs is expected not only to increase the number of DNA sequences that can be targeted, but should also permit the covalent tethering of pcPNA oligomers as bis-pcPNAs so as to reduce the molecularity of the strand displacement process 14.
The amenability of the aeg PNA structural framework to chemical modification affords considerable advantages in the development of a pharmacologically efficacious antigene agent 19,20. The incorporation of charge and chirality, and manipulation of PNA backbone flexibility provide opportunities to increase solubility and bioavailability, and to improve selective binding to DNA targets. PNA binding to double-stranded DNA is effectively kinetically controlled 21, and the decrease in the magnitudes of association rate constants with increase in salt concentration 22,23,24 makes binding inhibition at physiological ionic strength a potential obstacle to in vivo applications. Marked improvements in binding rates at elevated ionic strengths obtained with bis-PNAs carrying positive charge either within the interchain linker or in the N- and C-terminal peptide tail sections 25,26 suggest that the judicious placement of charged groups on the aeg PNA backbone proper could similarly increase binding rates under physiological conditions without loss of sequence selectivity. The inherent flexibility of the prototype aeg PNA design 27,28,29,30, and the attendant entropy losses incurred upon binding to DNA, have at the same time prompted the search for conformationally constrained backbone skeletons that are preorganized so as to favor complex formation. Many strategies have focused on the covalent integration of (chiral) cyclic ring systems in designs that conform to the guiding principle of maintaining the base separation from and along the backbone by the same numbers of bonds as in DNA (see 20,31,32,33 for review and recent advances). In principle, rigidity may also be conferred without the creation of covalent cyclic structures by the attachment of one or more chemical substituents at pro-chiral carbon centers in the aeg PNA backbone (see Fig. 1). Much interest has been shown in the improved solubility and modified hybridization properties toward single-stranded DNA of PNA variants carrying positively charged chiral N-(2-aminoethyl) D-lysine units 34,35,36,37. A recent crystallographic study of an anti-parallel mixed-sequence PNA-DNA decamer heteroduplex, comprising a three-residue unit D-lysine “chiral box”, provides evidence that increased polyamide backbone conformational rigidity can be obtained through the introduction of chiral centers 38.
The availability of experimentally determined structures of PNA complexes provides a rich knowledge base for the rational design of conformationally restricted chiral PNA analogs bearing functional groups. Experimental studies of PNA complexes have thus far revealed the existence of two distinct morphological helix forms. Crystal structures of the D-lysine-based PNA-DNA heteroduplex 38, a homopyrimidine PNA·DNA-PNA (py·pu-py) triplex 39, and four mixed (pu/py) sequence self-complementary PNA-PNA duplexes 40,41,42,43 all possess an unusual low-twist angle helical morphology, known as the P-form. These under-wound structures are characterized by 16-fold (or 18-fold) helical repeats, according to whether (or not) a partner Watson-Crick DNA strand is present, and a pronounced displacement of the bases from the helix axis. In contrast to the P-form helices, which have average (local) twist angle values in the range 19–23°, antiparallel mixed-sequence heteroduplex aeg PNA-DNA octamer 44 and aeg PNA-RNA hexamer 27 NMR solution structures have significantly higher average (local) helix twist angles of 28° and 30°, respectively. Although the C2′-endo sugar puckering observed in the aeg PNA-DNA duplex is more typical of a B-form helix, both heteroduplexes adopt similar A-like helical topologies, with displacement of inclined Watson-Crick basepairs toward the minor groove.
We have previously reported that helix morphology exerts a significant influence on PNA backbone conformation and flexibility 45. The largest conformational variation is observed experimentally in rotations around the two bonds flanking the backbone secondary amide at junctions connecting residues at positions (i) and (i+1) in the PNA chain, described by the ɛ(i) and α(i+1) dihedral angles (see Fig. 1). Values of ɛ(i) and α(i+1) were shown to be highly correlated over certain ranges, providing the basis for a PNA backbone conformation classification scheme. Most notably from a design perspective, it was found that preferred Watson-Crick PNA backbone conformations in P-form helical structures differ from those in A-like helices, possibly due in part to differential solvation effects in the minor groove. These findings suggest that, in addition to restricting conformational flexibility, the incorporation of bulky and/or charged groups to the PNA backbone could be used to control the basepair stacking pattern through the selective stabilization of backbone conformers associated with a particular helical form. This thesis is supported by the existence of the D-lysine based PNA-DNA duplex as a P-form helix, rather than an A-like helical structure, although the possibility of the difference in helix morphology compared to the aeg PNA-DNA duplex studied by NMR 44 being the result of crystal packing, strand length difference and/or base sequence effects cannot as yet be completely ruled out 38. Conversely, a L-arginine based PNA T10 decamer was observed to bind a complementary DNA A10 sequence with 1:1 stoichiometry, rather than as a 2:1 (PNA·DNA-PNA) triplex as does its aeg PNA T10 counterpart 46. Notwithstanding the role of electrostatic effects or steric hindrance of Hoogsteen strand binding by the L-arginine side chain, a plausible explanation for this change in binding stoichiometry may be an inability of the 2-aminoethyl-L-arginine backbone to adopt conformations compatible with a P-form helix.
It is with this design philosophy in mind that we present here a structural analysis of the spatial orientation preferences of the backbone amide −NH and >CO groups at residue junctions in experimental structures of PNA complexes, classified according to backbone conformation. Ambiguities concerning the controversial participation of (i+1) backbone −NH groups in hydrogen bonding interactions with the carbonyl oxygen of the backbone-base linker at the preceding residue position (i), as proposed by Bruice and co-workers 47,48 but otherwise widely refuted (see, for example, 27,41,44,49), have been clarified using an operational definition of a hydrogen bond routinely employed in the classification of protein secondary structure 50,51. We have investigated the energetic and the structural changes involved in the binding of a water molecule to an O2 base atom in a model symmetry-restrained PNA thymine dimer (pTT) system using quantum chemical methods. Collectively the results are consistent with bound water molecules in the minor groove playing a structurally more important role in the stabilization of Watson-Crick PNA backbone conformation in aeg PNA P-form structures than in A-like hybrid double helices, where a tendency to form weak (i+1)/(i) interresidue hydrogen bonds of less than ideal geometry is evident in at least two PNA conformation classes. Our findings shed light on conflicting results obtained in molecular dynamics simulations of solvated aeg PNA-DNA duplexes 49,52.
In another series of quantum chemical calculations on a structurally modified pTT junctions, in which the pro-R hydrogen of the aeg PNA CA atom is replaced by aliphatic chains carrying positive charge (see Fig. 1), we exploit the symmetry-restrained dimer as a model with which to investigate local backbone preorganization. In contrast to the aeg PNA junction, the P-form backbone conformation common to the D-lysine “chiral box” and the PNA·DNA-PNA triplex can be identified at minima on the chiral dimer potential energy surfaces. Shortening of the D-lysine aliphatic chain allows direct electrostatic interaction with the PNA backbone carbonyl oxygen in this conformation, suggesting that the introduction of chiral PNA analogs based on either 3-amino-D-alanine or 4-aza-D-leucine should also favor P-form helix formation.
We conclude with a proposal that conformationally restricted structural modifications preferentially stabilizing P-form helices could be exploited to promote PNA·DNA-PNA triplex formation, and in particular to target mixed-sequence double-stranded DNA via triplex invasion mechanisms using small bases and other functional groups, rather than large heterocycles, attached to Hoogsteen strand carbonyl linkers to recognize DNA pyrimidine bases in the major groove. Quantum chemical modeling methods are used in support of candidate PNA Hoogsteen strand designs.
Computational methods
PNA residue junction conformer database
The database, comprising a total of 207 nonredundant PNA residue junction atom sets, was compiled from eight experimental structures of PNA complexes in the Protein Data Bank (PDB) 53. Of these, 50 and 56atom sets were, respectively, extracted from NMR solution structures of A-like mixed (py-pu) sequence PNA-RNA (PDB code 176D; 27) and PNA-DNA (PDB code 1PDT; 44) heterodimers. For comparative purposes, we also included data sets obtained by constrained energy minimization of the NMR models with PNA base atoms and all atoms of the nucleic acid strands held fixed (see Topham and Smith 45). The remaining atom sets were culled from PNA strands in P-form x-ray crystal structures of a PNA·DNA-PNA triplex (PDB code, 1PNN (32 sets); 39), a mixed-sequence PNA-DNA heteroduplex, comprising a three-residue unit D-lysine “chiral box” (PDB code, 1NR8 (9 sets); 38), and four self-complementary right-handed homoduplexes (PDB codes, 1PUP (16 sets), 40; 1QPY (28 sets), 41; 1HZS (10 sets), 42; 1RRU (6 sets), 43). Hydrogen atoms were added to the Protein Data Bank crystal structures using the HBUILD 54 facility in CHARMM 55, before energy minimization with all heavy atoms held fixed. PNA force field parameters were abstracted from the distributed all-atom CHARMM22 nucleic acid 56 and protein 57 sets, supplemented as previously reported 45.
Structural analysis of PNA residue junctions
Analysis of (α, β, γ, δ, ɛ, and ω) backbone and (χ1, χ2, and χ3) backbone-base linker dihedral angles in the residue junction database was carried out according to the contiguous atom quartet definitions given previously 45. A {æ(i), β(i), γ(i), δ(i), β(i+1), γ(i+1), δ(i+1)} dihedral domain vector was defined at each junction between adjacent residue positions (i) and (i+1) in the PNA chains, where æ(i) is given by α(i+1)+ɛ(i). We refer to æ as the coupling constant. Negative-signed values of æ in the range (−150°>æ≥0°) are assigned to the {æ−} domain, positive-signed values in the range (0°>æ≥150°) are classed as {æ+}, and values in the range (150°>æ≥210°) are assigned to the {æ-trans} domain. β-domains are, respectively, categorized as {g+}, {trans} or {g−} for angles in the range (0°≥β<120°), (120°≥β<210°) or (210°≥β<360°). The γ and δ dihedrals are either assigned to the {+90°} domain for angle values falling in the range (0°≥(γ, δ)<180°), or the {−90°} domain for angles in the range (180°≥(γ, δ)<360°). The orientation of the carbonyl group of the PNA backbone secondary amide bond was measured using the pseudodihedral angle (ν) introduced by the Orozco and Laughton groups 49. Recast in terms of the atom nomenclature used previously 39,45, and retained here (see Fig. 1), ν is defined at the ith residue position (ν(i)) by the atom quartet, CF(i)–NB(i)–C(i)–O(i).
Our PNA residue junction classification scheme allows for the coarse classification of conformers according to the {æ} domain occupied, and assignment to a particular subset or class according to the flanking {β, γ, δ} torsion angle domain combination 45. Hierarchic flanking angle domain pattern searches are now more robustly conducted by the ordered examination of the four-component {γ(i), δ(i), β(i+1), γ(i+1)} and {δ(i), β(i+1), γ(i+1), δ(i+1)} vectors, before consideration of the {β(i), γ(i), δ(i)} and {δ(i+1), β(i+1), γ(i+1)} three-component vectors. Seven conformational classes are currently recognized (Table 1). It should be noted that all values of γ and δ were inadvertently systematically interchanged in our earlier analysis of PNA conformational preferences 45, and the {æ, β, γ, δ} vector defining the (æ+)-β-trans conformational class is accordingly redefined in Table 1 as {æ+, trans, +90°, −90°}. Three residue conformers in the 1PDT data set, previously assigned as (æ+)-β-g−, are now reclassified as (æ+)-β-trans, and one residue junction in the 1PUP data set, previously annotated (æ−)-P, is more appropriately considered as a member of a newly recognized class, (æ-trans)-P, uniquely observed in PNA-PNA duplexes. Structurally, these conformers resemble (æ−)-P conformers, with compensatory shifts in values β, δ, and ɛ(i) dihedral angles permitting minor groove water bridging between the base and the backbone −NH(i+1) group to be maintained. A statistical analysis of backbone dihedral angle values in all seven conformational classes is presented in Table 2.
| Table 1 Geometric criteria for PNA backbone conformation classification |
| Helix Morphology | Conformational Class | æ | β | γ | δ | Other Conditions | ||
|---|---|---|---|---|---|---|---|---|
| P-form | (æ−)-P | {æ−} | g+ | +90° | +90° | 180°≥α(i+1)<360° | ||
| P-form | (æ−)-Pminor | {æ−} | g+ | +90° | +90° | 0°≥α(i+1)<180° | ||
| P-form | (æ-trans)-P | {æ-trans} | g+ | +90° | +90° | |||
| A-like | (æ−)-β-g+ | {æ−} | g+ | +90° | +90° | |||
| A-like | (æ-trans)-β-g+ | {æ-trans} | g+ | +90° | +90° | |||
| A-like | (æ+)-β-trans | {æ+} | trans | +90° | −90° | |||
| A-like | (æ+)-β-g− | {æ+} | g− | −90° | −90° | |||
| Table 2 Statistical analysis of selected (pseudo-) dihedral angle values in seven conformational classes of PNA residue junction in experimental structures |
| Helix morphology | Conformational class | Median æ (°) | Average α(i+1) (°) | Average ɛ(i) (°) | Average ν(i) (°) | Median α(i+1)+ν(i) (°) | Median β (°) | Median γ (°) | Median δ (°) | n | (%) | ||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| P-form | (æ−)-P | −116 | −109±9.0 | −8±10.7 | +118±15.7 | +11 | +69 | +69 | +90 | 67 | 66.3 | ||
| (æ−)-Pminor | −127 | +75±10.1 | +156±11.6 | −56±11.7 | +19 | +67 | +69 | +89 | 25 | 24.8 | |||
| (æ-trans)-P | −162 | −109±11.2 | −54±14.1 | +81±8.3 | −29 | +95 | +74 | +120 | 7 | 6.9 | |||
| Others | 2 | 2.0 | |||||||||||
| Total | 101 | 100 | |||||||||||
| A-like | (æ−)-β-g+ | −114 | +168±14.5 | +79±10.4 | −131±18.4 | +37 | +69 | +83 | +75 | 35 | 33.0 | ||
| (æ-trans)-β-g+ | −163 | −139±14.2 | −41±38.7 | +114±43.1 | −14 | +113 | +88 | +129 | 5 | 4.7 | |||
| (æ+)-β-trans | +117 | +116±65.5 | −4±80.0 | −166±84.5 | −41 | +148 | +91 | −143 | 20 | 18.9 | |||
| (æ+)-β-g− | +121 | +150±54.2 | −42±66.0 | −176±69.8 | −36 | −138 | −77 | −118 | 8 | 7.6 | |||
| Unclassified | 38 | 35.8 | |||||||||||
| Total | 106 | 100 | |||||||||||
| Conformational classes were assigned to residue junctions in the database according to the {æ} domain occupied and hierarchic searches of flanking backbone {β, γ, δ} torsion angle domain vector patterns as described in the Computational Methods section. The total number of residue junctions in each class is indicated by n. Analysis of the A-like heteroduplexes was performed on the original NMR solution data. Contiguous bonded atom quartet definitions of α, β, γ, δ, and ɛ dihedral angles (see Fig. 1) are as given previously 45. The (i/i+1) residue junction pseudodihedral angle (æ) is defined as the sum of the ɛ(i) and α(i+1) dihedral angle values. The ν(i) pseudodihedral angle, introduced by Soliva et al. 49 to describe the orientation of the carbonyl group of the PNA backbone secondary amide bond, is defined at the ith residue position by the atom quartet, CF(–NB(i)–C(i)–O(i). |
Electrostatic interaction energies between the backbone-base linker carbonyl group (>CE(i)=OE(i)) at residue position i and the backbone −NH(i+1) group at residue position i+1 were calculated according to the Kabsch and Sander interatomic distance formula 50. An interresidue hydrogen bond was considered to exist when the interaction energy was <−0.5kcal mol−1. The generous cutoff proposed by the authors allows for bifurcated hydrogen bonds and errors in coordinates.
Ab initio quantum chemical calculations on model PNA thymine dimers
Hartree-Fock (HF) calculations on a model PNA thymine dimer (pTT) system, with hydrogen atoms in place of the terminal −NH and >CO groups, were performed using GAUSSIAN 94 58 or 98 59. Geometry optimizations were carried out using either the 6-31G* or 3-21G* basis set as reported in Table 3. HF/6-31G* provides a sufficiently high level of quantum chemical theory appropriate for the calculation of hydrogen bonding properties and water binding interaction energies in small stable systems 56,60,61. Input geometries were specified as a Z-matrix, and symmetry restraints applied to chemically equivalent bond lengths, valence bond angles, and dihedral angles in the two residue units. Dimer configurations existing at (local) energy minima in regions of dihedral angle space characteristic of the main P-form and A-like conformer classes were identified from geometry optimizations in the absence of constraints using starting internal coordinate values obtained from analyses of experimental structures. Other conformers in a given class were then generated by the application of a constraint to fix α to a selected value within the experimentally observed range. Initial values for ɛ were calculated from the æ coupling constant. In cases such as the (æ−)-P reference model 1.2 (Table 3), where it was necessary to ensure the integrity of α/ɛ dihedral angle coupling, a constraint was applied on the pseudodihedral angle ν without directly fixing either α or ɛ. High-occupancy solvent binding in the minor groove was modeled in the dimer systems by optimization of the interaction of a water molecule with the O2 thymine base atom of the first (N-terminal) residue unit. In these calculations, water molecule (HW-OW) bond length and (HW-OW-HW) bond angle values were constrained to TIP3P reference values 62. Dimer geometries were either held fixed to their optimized configurations, determined in the absence of the water molecule, or a full optimization of the dimer-water complex performed (with dimer symmetry restraints) to explore induced conformational changes. Default convergence criteria were satisfied in all cases. All reported energy differences were determined from (Møller-Plesset) MP2 single point energy calculations on the HF/6-31G* optimized geometries, allowing for a more accurate treatment of electron correlation effects than afforded by Hartree-Fock theory.
| Table 3 Structural analysis of geometry-optimized quantum chemical PNA thymine dimer models |
| Model | R* | Basis set | H2O† | æ (°) | α (°) | ɛ (°) | ν (°) | ω (°) | β (°) | γ (°) | δ (°) | χ1 (°) | χ2 (°) | χ3 (°) | κ1 (°) | ||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1.1 | −H | 6-31G* | − | −116.4 | −97.5 | −18.8 | 108.7‡ | −166.6 | 71.3 | 72.4 | 108.1 | −2.3 | −164.7 | 78.5 | – | ||
| 1.2 | −H | 6-31G* | + | −117.5 | −99.3 | −18.2 | 108.7‡ | −167.0 | 71.8 | 73.6 | 101.4 | 3.3 | −174.2 | 85.4 | – | ||
| 1.3 | −H | 6-31G* | + | −89.6 | −110.8 | 21.2 | 158.7 | −178.9 | 58.0 | 71.6 | 72.1 | 2.9 | −179.3 | 85.5 | – | ||
| 1.4 | −H | 6-31G* | − | −79.4 | −104.6 | 25.2 | 173.9 | 177.5 | 58.5 | 76.4 | 82.8 | −2.2 | −166.5 | 74.7 | – | ||
| 2.1 | −H | 6-31G* | − | −108.2 | 91.7 | 160.1 | −57.9 | 172.9 | 60.8 | 76.0 | 85.6 | 8.6 | 179.0 | 80.0 | – | ||
| 2.2 | −H | 6-31G* | + | −105.0 | 101.7 | 153.3 | −59.1 | 176.4 | 60.3 | 76.4 | 101.4 | 3.9 | −178.7 | 82.9 | – | ||
| 2.3 | −H | 6-31G* | + | −99.8 | 107.2 | 153.0 | −60.7 | 176.8 | 57.7 | 77.5 | 92.5 | 5.3 | 179.6 | 80.7 | – | ||
| 2.4 | −H | 6-31G* | − | −106.6 | 101.8 | 151.6 | −61.6 | 178.3 | 61.0 | 77.8 | 93.6 | 7.0 | 179.2 | 84.3 | |||
| 3.1 | −H | 6-31G* | − | −113.3 | 145.0‡ | 101.7 | −100.6 | −174.9 | 64.8 | 77.0 | 83.2 | 4.3 | −179.4 | 81.4 | – | ||
| 3.2 | −H | 6-31G* | − | −111.3 | 155.9 | 92.8 | −106.7 | −174.4 | 63.6 | 76.5 | 81.9 | 5.0 | −179.9 | 82.1 | – | ||
| 3.3 | −H | 6-31G* | + | −115.0 | 155.9‡ | 89.1 | −113.1 | −169.7 | 62.8 | 75.0 | 77.0 | 11.1 | 173.6 | 88.5 | – | ||
| 3.4 | −H | 6-31G* | − | −107.4 | 168.2‡ | 84.4 | −113.1 | −175.0 | 61.8 | 75.8 | 80.9 | 5.6 | 179.3 | 82.1 | – | ||
| 3.5 | −H | 6-31G* | + | −109.0 | 168.2‡ | 82.8 | −119.7 | −170.6 | 57.7 | 71.2 | 73.0 | 13.3 | 171.8 | 86.3 | – | ||
| 3.6 | −H | 6-31G* | − | −102.4 | 180.0‡ | 77.6 | −119.2 | −176.0 | 59.9 | 75.1 | 80.6 | 5.4 | 178.9 | 81.2 | – | ||
| 3.7 | −H | 6-31G* | − | −96.6 | −167.0‡ | 70.4 | −126.6 | −177.4 | 58.4 | 74.7 | 81.2 | 4.5 | 179.4 | 80.3 | – | ||
| 3.8 | −H | 6-31G* | − | −91.2 | −154.0‡ | 62.8 | −136.2 | −178.6 | 58.2 | 74.9 | 82.6 | 2.7 | 178.7 | 79.2 | – | ||
| 4.1 |  | 3-21G* | − | −131.2 | −111.3 | −19.9 | 119.4 | −161.5 | 82.9 | 100.2 | 91.7 | 20.2 | −134.5 | 60.3 | 59.8 | ||
| 4.2 | | 3-21G* | + | −136.2 | −115.0 | −21.2 | 120.9 | −163.8 | 86.5 | 91.2 | 102.6 | 5.9 | −177.8 | 82.8 | 58.5 | ||
| 4.3 | | 3-21G* | − | −113.8 | 168.2‡ | 78.0 | −104.7 | −163.4 | 70.5 | 86.8 | 86.1 | −13.9 | 178.1 | 69.7 | 53.3 | ||
| 5.1 |  | 3-21G* | − | −102.5 | −99.9 | −2.6 | 157.5 | −167.8 | 69.5 | 92.3 | 102.3 | −9.1 | −160.5 | 65.9 | 65.1 | ||
| 5.2 | | 3-21G* | + | −125.4 | −94.4 | −30.1 | 113.1 | −168.4 | 80.4 | 83.7 | 103.4 | 9.4 | −174.0 | 81.8 | 62.5 | ||
| 5.3 | | 3-21G* | − | −108.3 | −103.1 | −5.1 | 151.9 | −168.2 | 71.8 | 81.2 | 114.4 | −16.5 | −158.9 | 65.2 | 168.1 | ||
| 5.4 | | 3-21G* | + | −124.2 | −97.2 | −27.0 | 119.0 | −169.0 | 80.0 | 73.2 | 113.4 | 0.1 | −174.0 | 75.0 | 175.1 | ||
| 5.5 | | 6-31G* | + | −130.8 | −103.7 | −27.0 | 115.8 | −164.0 | 81.9 | 78.1 | 110.3 | 4.3 | −171.5 | 79.6 | 170.7 | ||
| 6.1 | −CH2(CH3)2NH+ | 3-21G* | + | −125.7 | −97.3 | −28.4 | 114.4 | −167.8 | 80.0 | 84.9 | 100.8 | 10.3 | −174.7 | 83.3 | 58.9 | ||
| 6.2 | −CH2(CH3)2NH+ | 3-21G* | + | −125.0 | −98.2 | −26.8 | 119.5 | −168.7 | 80.8 | 73.6 | 113.3 | 0.3 | −174.4 | 75.2 | 177.3 | ||
| Geometry optimizations of symmetry-restrained pTT dimer systems were performed using the indicated basis set as described in the Computational Methods section. Atom quartet definitions of (α, β, γ, δ, ɛ, and ω) backbone and (χ1, χ2, and χ3) backbone-base linker dihedral angles (see Fig. 1) are those given previously 45. Pseudodihedral angles (æ) and ν are defined in the legend to Table 2. The κ1 side-chain dihedral angle refers to rotation around the CA – Cβ bond in cationic D-amino acid-based chiral pTT dimer analogues, and is defined by the atom quartet NB(i) – CA(i) – Cβ(i) – Xγ(i), where X is carbon in analogues in which the prototype glycine moiety is replaced by D-lysine, and nitrogen in analogues based on 3-amino-D-alanine or 4-aza-D-leucine. |
| * pro-R substituent at the CA atom (see Fig. 1). † Geometry optimizations carried out in the presence (+) or absence (−) of a water molecule targeted to the O2atom of the N-terminal thymine base. ‡ Values of α or ν held fixed during geometry optimization. |
Comparison of base stacking patterns in modeled residue junctions and experimental structures
Intrastrand base stacking patterns in four representative PNA-containing experimental structures, the standard A80 and B80 DNA fiber conformations, and the pTT model dimers were compared in a pairwise manner using (averaged) root mean square distance (RMSD) values, calculated over the 12 heavy ring atoms of stacked py/py bases (see Supplementary Material , Table S1). A total of 20 stacked py/py base atom sets were culled from (Watson-Crick) PNA strands in the 176D (chain A, 10 models) Protein Data Bank (PDB) structure, and 16 from each from the 1PDT (chain B, 8 models) and 1PNN (chains A and C) coordinate sets. None of the four experimental PNA-PNA homoduplex structures contains stacked py/py bases, and 10 stacked py/py base atom sets were generated by reconstruction of chains A and B in the 1PUP PDB structure as polypyrimidine strands using JUMNA 63,64. Helicoidal parameters were calculated using a CURVES 65,66 analysis of the original coordinates. Canonical A80 and B80 AT duplex structures were generated using JUMNA and the helicoidal parameter sets tabulated by Lavery et al. 64. Base stacking patterns were compared using principal coordinates analysis (classical scaling) techniques 67. RMSD values were loaded into a (n×n) distance (dissimilarity) matrix, and the elements normalized in a driver routine before analysis using the CMDS Fortran77 subroutine (http://astro.u-strasbg.fr/(fmurtagh//mda-sw/). The program outputs the (n−1) eigenvalues, and projections of the base stacking pattern on the first seven principal components for plotting.
Quantum chemical modeling of interactions between Hoogsteen strand PNA analog derivative model compounds and pyrimidine bases
Geometric interactions of proposed functional groups with DNA pyrimidine bases in the major groove (see Fig. 2) were investigated using quantum chemical techniques and spatial constraints derived from the 1PNN (py·pu-py) PNA·DNA-PNA triplex x-ray crystal structure 39. Calculations were performed using the (May, 2004) GAMESS 68 implementation of the B3LYP hybrid Hartree-Fock/density functional method and the 6–311++G(d,p) basis set. The model systems (Fig. 9) comprised a pyrimidine base and N,N-dimethyl substituted alkyl amide derivatives of the functional group: (A) isopropyl (−CH(CH3)2) to target the thymine 5-methyl atom, and (B) imidazolyl (attached at the N1 position) or (C) isoxazolyl (attached at the C5 position) to hydrogen bond with cytosine. Six spatial constraints (three dihedral angles, two angles, and one distance) were imposed between the C5 and C6 atoms of the target pyrimidine base and atom positions in the model compounds analogous to the PNA backbone CA, NB, and CG atoms (Fig. 2). Pyrimidine base coordinates were obtained by conversion of the 1PNN polypurine DNA chains using JUMNA 63,64. Helicoidal parameters were calculated from a CURVES 65,66 analysis of the heterotriplex. Spatial constraints were derived from centrally positioned representative pT20·dA5 and pT18·dA3 PNA·DNA duplets (chains C and D), remote from the out-swinging Hoogsteen pC16 base in PNA chain A of the other triplex in the asymmetric unit. To take co-ordinate error into account, the CA, NB, and CG atom positions were not taken directly from the original 1PNN data, but from coordinates obtained by superposition of all heavy atoms in a HF/6-31G* geometry-optimized N,N-dimethyl thymine-1-acetamide model structure, built in the same conformation as the experimental PNA residue unit using dihedral angle constraints.
Figure 2
Proposed tandem use of conformationally restricted PNA and small Hoogsteen strand functionalities to target DNA pyrimidine bases via triplex formation. (A) Solvent shielding of thymine 5-methyl group by an isopropyl group. (B) Major groove recognition of cytosine by an isoxazole base. Watson-Crick PNA strand chiral PNA analogs based on 3-amino-D-alanine (A) or 4-aza-D-leucine (B) may promote P-form helix formation.Strain energy in the model compounds in the pyrimidine base complexes was calculated from energy differences with respect to the fully geometry-optimized conformation of the isolated molecule in the absence of constraints. Calculations of atomic solvent-accessible surface area were carried out using NACCESS version 2.1.1 69, a probe size of 1.4Å, and the program default van der Waals radii of Chothia and co-workers for common chemical atom types. Basepair propeller and buckle parameters in the complexes of the azolyl derivatives with cytosine were calculated using CURVES 65,66 with a guanine base in place of the azole bases. Superposition was performed using the following ring atom mappings to guanine N9, C4, C5, N7, and C8 positions: N1, C2, N3, C4, C5 (imidazole); C5, O1, N2, C3, C4 (isoxazole).
Results
Influence of α/ɛ dihedral angle coupling on PNA backbone carbonyl group orientation
Our previous work drew attention to the existence of coupled rotations around the two bonds flanking the backbone secondary amide at junctions connecting residues at positions (i) and (i+1) in the PNA chain, described by the ɛ(i) and α(i+1) dihedral angles 45. Coupling of these dihedral angles operates over relatively short ranges of values characteristic of the conformer class. Fig. 3 shows that values of the pseudodihedral angle ν(i), describing the orientation of the carbonyl group of the PNA backbone secondary amide bond at residue positions (i), correlate closely with values of α(i+1) in the seven conformational classes identified in experimental structures. The correlations indicate that coupled change in α(i+1) and ɛ(i) exerts a direct effect on the orientation of the secondary amide carbonyl group.
Figure 3
Dependence of PNA polyamide backbone carbonyl group orientation on the ∝ dihedral angle at residue junctions in experimental structures. Plots of the pseudodihedral angle ν(i) at the ith PNA residue unit versus the α(i+1) dihedral angle at the following residue position are shown. Dihedral angle pairs from four conformers identified in PNA strands in A-like aeg PNA-RNA (PDB code 176D, ■) and aeg PNA-DNA (1PDT, □) helical structures are plotted in panels on the left-hand side. Data for three PNA conformer classes in P-form structures are shown in panels on the right-hand side: 1PNN homopyrimidine PNA·DNA-PNA triplex (Watson Crick, ●, and Hoogsteen strands, ○); 1PUP (▵), 1HZS (▴), 1QPY (▾), and 1RRU (∇) self-complementary PNA-PNA homoduplexes; 1NR8 (♦) PNA-DNA heteroduplex comprising a three-residue D-lysine “chiral box”. Further details concerning the origins of the data are given in the Computational Methods section. The “forward” and “backward” pseudodihedral angle domains, describing 180° rotations of the PNA backbone carbonyl oxygen (○) atom around the NB-C vector with respect to the CF atom, defined by Soliva et al. 49, are, respectively, indicated as gray shaded and unshaded areas, centered at values of ν of 120° and −60° (300°). Solid line fits to the angle data were constructed from median estimates of (α(i+1)+ν(i)) given in Table 2. Regression analysis of ν(i) on α(i+1) yielded a r2 (coefficient of determination) value of 0.95 (n=125) for combined (classified) data in the {æ−} domain, and respective r2 values of 0.71 (n=12) and 0.83 (n=28) for data in the {æ-trans} and {æ+} domains. Corresponding r2 values obtained from regression analysis of ɛ(i) on α(i+1) are 0.97 (n=127), 0.43 (n=12), and 0.91 (n=28), respectively.Backbone carbonyl group orientation accommodates minor groove water binding in P-form structures
The (æ−)-P conformer is present in all PNA chains of P-form crystal structures. All the experimental (æ−)-P residue junction ν(i) data fall within the so-called “forward” ν domain (Fig. 3), defined by Soliva et al. 49 for PNA residue units with backbone carbonyl groups pointing toward the helix C terminus, corresponding to values of ν in the range 120±90°. This geometric arrangement allows the backbone −NH group at the next (ith+1) residue position of Watson-Crick PNA chains to interact with pyrimidine O2(i) or purine N3(i) base atoms via the intermediary of a bound water molecule in the minor groove. The modeled (æ−)-P pTT dimer junction (1.2) shown in Figure 4A was minimized in the presence of a bridging water molecule with ν held fixed at the average value of 108.7° (±11.8°) for 16 residue junctions in Watson-Crick PNA chains of the PNA·DNA-PNA triplex x-ray diffraction structure 39. Base stacking in the quantum chemical model is similar to that in the homopyrimidine Watson-Crick chains of the 1PNN coordinate set, with an average RMSD value of 0.35Å for the ring atoms.
Figure 4
HF/6-31G* geometry-optimized aeg pTT dimer models of reference PNA conformers. (A) (æ−)-P model 1.2 in the presence of a water molecule. (B) (æ−)-β-g+ model 3.2. (C) (æ−)-Pminor model 2.1 showing the optimized interaction geometry of a bridging water molecule, the water oxygen lying approximately in the plane of the first thymine base. Detailed structural and energetic analysis of individual models can be found in Table 3,Table 4,Table 5. The N- to C-terminal direction is from right to left. Hydrogen bond interactions are represented as spheres. Stereo graphics images were prepared using SETOR 84.In contrast to the (æ−)-P conformation, the backbone carbonyl group points in the opposite direction in (æ−)-Pminor residue junctions. Experimental ν(i) data for (æ−)-Pminor residue junctions all lie within the “backward” domain, covering the ν angle range −60±90°. This type of P-form residue junction geometry is found in the four PNA-PNA duplexes and the first two junctions at the N-terminus of the mixed-sequence PNA-DNA decamer, carrying a centrally positioned three-residue unit D-lysine “chiral box” 38. The orientation of the carbonyl group in the experimental structures supports minor groove water bridging with pyrimidine O2 or purine N3 base atoms in the same PNA residue. The geometry-optimized reference model (2.1) of an (æ−)-Pminor pTT residue junction (Figure 4C) exists at an energy minimum on the pTT energy surface. The value of ν in the dimer model is −57.9°, in close agreement with an average value of −56° for experimental data. Base stacking in model 2.1 most closely resembles stacking in the 1PUP PNA-PNA data set, the average RMSD being 0.40Å for the ring atoms.
Backbone carbonyl group orientation in A-like heteroduplexes associated with nonideal interresidue hydrogen bonding
The (æ−)-β-g+ class is the most populated of the four conformational classes observed in A-like PNA-DNA and PNA-RNA heteroduplex helices studied by NMR. Average values of ν(i), α(i+1), and ɛ(i) for this class are ∼90° out of phase with respect to corresponding values in (æ−)-P and (æ−)-Pminor residue junctions. The range of values of ν(i) in the experimental structures (200–260°) straddles the boundary at 210° (−150°) separating the “forward” and “backward” ν domains. The backbone carbonyl group in these conformers points toward the solvent, allowing the backbone −NH group of the next residue unit in the chain to interact with the backbone-base carbonyl (OE) oxygen, as exemplified in the geometry-optimized model (3.2) of an (æ−)-β-g+ junction shown in Figure 4B. The dimer exists in a local energy minimum, with an α-value of 156° and a ν-value of −107° (253°), as compared to respective average α(i+1) and ν(i) values of 168° and −131° (229°) for (æ−)-β-g+ conformers in A-like experimental structures. The OE(i)–HN(i+1) distance (2.59Å) and OE(i)–HN(i+1)–N(i+1) angle (116.0°) in this model are close to respective average experimental values of 2.81±0.39Å and 127±9° for (æ−)-β-g+ conformers. While base stacking in model 3.2 is not particularly A-like, it being most similar to that of the 1PNN structure with an average RMSD value of 0.41Å, a shift away from the 1PUP PNA-PNA homoduplex stacking pattern, relative to stacking in the 1.2 and 2.1 P-form reference model residue junctions, is nevertheless evident from the principal coordinates analysis in Figure 5A.
Figure 5
Principal coordinates analysis of base stacking patterns in modeled and experimental PNA residue junctions. Scatter plots show projections of the first two principal components (1 and 2) for py/py base-step RMSD data in Table S1: Panel A shows structural shifts in aeg pTT dimer stacking patterns (open symbols) toward (solid arrows) and away from (dashed arrows) experimental stacking patterns (■) induced by water molecule binding to the O2atom of the N-terminal thymine base (solid symbols); (B) Imposition of a pseudodihedral angle constraint to clamp the orientation of the backbone secondary amide groups in the aeg pTT dimer results in reorganization of base-step stacking in the presence of a backbone-base bridging water (solid arrow) closer to patterns in the P-form 1PNN triplex crystal structure and in fully geometry-optimized cationic D-amino acid-based chiral pTT dimer analogs. The first two dimensions account for 48.2% of the total variation in panel A and 52.4% in panel B. Data for standard A80 and B80 DNA fiber conformations (□) are included for reference. Geometric descriptions of annotated symmetry-restrained quantum chemical dimer models are given in Table 3.The chemical shifts and solvent exchange properties of HN(i+1) amide protons in the PNA-RNA 27 and PNA-DNA 44 duplexes appear to contradict the existence of interresidue hydrogen bonds 70. Indeed, respective average OE(i)–HN(i+1) distances of 2.81 (±0.39) Å and 2.69 (±0.51) Å for (æ−)-β-g+ and pooled (æ+)-β-trans and (æ+)-β-g− residue junctions in the NMR solution structures are longer than the 2.5-Å limit often employed in standard definitions of hydrogen bonding 71,72. An analysis of the dependence of the electrostatic interaction energy (