CHARMM additive and polarizable force fields for biophysics and computer-aided drug design.

BACKGROUND: Molecular Mechanics (MM) is the method of choice for computational studies of biomolecular systems owing to its modest computational cost, which makes it possible to routinely perform molecular dynamics (MD) simulations on chemical systems of biophysical and biomedical relevance. SCOPE OF REVIEW: As one of the main factors limiting the accuracy of MD results is the empirical force field used, the present paper offers a review of recent developments in the CHARMM additive force field, one of the most popular biomolecular force fields. Additionally, we present a detailed discussion of the CHARMM Drude polarizable force field, anticipating a growth in the importance and utilization of polarizable force fields in the near future. Throughout the discussion emphasis is placed on the force fields' parametrization philosophy and methodology. MAJOR CONCLUSIONS: Recent improvements in the CHARMM additive force field are mostly related to newly found weaknesses in the previous generation of additive force fields. Beyond the additive approximation is the newly available CHARMM Drude polarizable force field, which allows for MD simulations of up to 1µs on proteins, DNA, lipids and carbohydrates. GENERAL SIGNIFICANCE: Addressing the limitations ensures the reliability of the new CHARMM36 additive force field for the types of calculations that are presently coming into routine computational reach while the availability of the Drude polarizable force fields offers an inherently more accurate model of the underlying physical forces driving macromolecular structures and dynamics. This article is part of a Special Issue entitled "Recent developments of molecular dynamics".

Automation of the CHARMM General Force Field (CGenFF) I: bond perception and atom typing.

Molecular mechanics force fields are widely used in computer-aided drug design for the study of drug-like molecules alone or interacting with biological systems. In simulations involving biological macromolecules, the biological part is typically represented by a specialized biomolecular force field, while the drug is represented by a matching general (organic) force field. In order to apply these general force fields to an arbitrary drug-like molecule, functionality for assignment of atom types, parameters, and charges is required. In the present article, which is part I of a series of two, we present the algorithms for bond perception and atom typing for the CHARMM General Force Field (CGenFF). The CGenFF atom typer first associates attributes to the atoms and bonds in a molecule, such as valence, bond order, and ring membership among others. Of note are a number of features that are specifically required for CGenFF. This information is then used by the atom typing routine to assign CGenFF atom types based on a programmable decision tree. This allows for straightforward implementation of CGenFF's complicated atom typing rules and for equally straightforward updating of the atom typing scheme as the force field grows. The presented atom typer was validated by assigning correct atom types on 477 model compounds including in the training set as well as 126 test-set molecules that were constructed to specifically verify its different components. The program may be utilized via an online implementation at https://www.paramchem.org/ .

Automation of the CHARMM General Force Field (CGenFF) II: assignment of bonded parameters and partial atomic charges.

Molecular mechanics force fields are widely used in computer-aided drug design for the study of drug candidates interacting with biological systems. In these simulations, the biological part is typically represented by a specialized biomolecular force field, while the drug is represented by a matching general (organic) force field. In order to apply these general force fields to an arbitrary drug-like molecule, functionality for assignment of atom types, parameters, and partial atomic charges is required. In the present article, algorithms for the assignment of parameters and charges for the CHARMM General Force Field (CGenFF) are presented. These algorithms rely on the existing parameters and charges that were determined as part of the parametrization of the force field. Bonded parameters are assigned based on the similarity between the atom types that define said parameters, while charges are determined using an extended bond-charge increment scheme. Charge increments were optimized to reproduce the charges on model compounds that were part of the parametrization of the force field. A "penalty score" is returned for every bonded parameter and charge, allowing the user to quickly and conveniently assess the quality of the force field representation of different parts of the compound of interest. Case studies are presented to clarify the functioning of the algorithms and the significance of their output data.

Targeting of an interrupted polypurine:polypyrimidine sequence in mammalian cells by a triplex-forming oligonucleotide containing a novel base analogue.

The DNA triple helix consists of a third strand of nucleic acid lying in the major groove of an intact DNA duplex. The most stable triplexes form on polypurine:polypyrimidine sequences, and pyrimidine interruptions in the purine strand are destabilizing. Sequence stringency is imparted by specific Hoogsteen hydrogen bonds between third strand bases and the purine bases in the duplex. Appropriate base and sugar modifications of triple helix-forming oligonucleotides (TFOs) confer chromosome targeting activity in living cells. However, broad utilization of TFOs as gene targeting reagents in mammalian cells has been limited by the requirement for homopurine target sequences. Although there have been a number of base analogues described that appear to be promising as candidates for triplex target expansion, none has been examined in a biological system. We have employed a postsynthetic strategy to prepare a collection of TFOs with base analogues at a defined position. Following assessment of affinity for a triplex target with a single C:G inversion, TFOs with a second generation of analogues were synthesized. One of these, TFO-5a, with 2'-OMe-guanidinylethyl-5-methylcytosine at the position corresponding to the C:G interruption in the target sequence, was further modified to confer bioactivity. The activity of this TFO, linked to psoralen, was measured in a mammalian cell line that was engineered by directed sequence conversion to carry a triplex target with a single C:G interruption. TFO-5a was active against this target and inactive against the corresponding target with an uninterrupted polypurine:polypyrimidine sequence.

CHARMM general force field: A force field for drug-like molecules compatible with the CHARMM all-atom additive biological force fields.

The widely used CHARMM additive all-atom force field includes parameters for proteins, nucleic acids, lipids, and carbohydrates. In the present article, an extension of the CHARMM force field to drug-like molecules is presented. The resulting CHARMM General Force Field (CGenFF) covers a wide range of chemical groups present in biomolecules and drug-like molecules, including a large number of heterocyclic scaffolds. The parametrization philosophy behind the force field focuses on quality at the expense of transferability, with the implementation concentrating on an extensible force field. Statistics related to the quality of the parametrization with a focus on experimental validation are presented. Additionally, the parametrization procedure, described fully in the present article in the context of the model systems, pyrrolidine, and 3-phenoxymethylpyrrolidine will allow users to readily extend the force field to chemical groups that are not explicitly covered in the force field as well as add functional groups to and link together molecules already available in the force field. CGenFF thus makes it possible to perform "all-CHARMM" simulations on drug-target interactions thereby extending the utility of CHARMM force fields to medicinally relevant systems.

CHARMM: the biomolecular simulation program.

CHARMM (Chemistry at HARvard Molecular Mechanics) is a highly versatile and widely used molecular simulation program. It has been developed over the last three decades with a primary focus on molecules of biological interest, including proteins, peptides, lipids, nucleic acids, carbohydrates, and small molecule ligands, as they occur in solution, crystals, and membrane environments. For the study of such systems, the program provides a large suite of computational tools that include numerous conformational and path sampling methods, free energy estimators, molecular minimization, dynamics, and analysis techniques, and model-building capabilities. The CHARMM program is applicable to problems involving a much broader class of many-particle systems. Calculations with CHARMM can be performed using a number of different energy functions and models, from mixed quantum mechanical-molecular mechanical force fields, to all-atom classical potential energy functions with explicit solvent and various boundary conditions, to implicit solvent and membrane models. The program has been ported to numerous platforms in both serial and parallel architectures. This article provides an overview of the program as it exists today with an emphasis on developments since the publication of the original CHARMM article in 1983.

Re-examination of the intrinsic, dynamic and hydration properties of phosphoramidate DNA.

Intrinsic energetic and solvation factors contributing to the unusual structural and biochemical properties of N3'-phosphoramidate DNA analogs have been re-examined using a combination of quantum mechanical and molecular dynamics methods. Evaluation of the impact of the N3'-H substitution was performed via comparison of N3'-phosphoramidate DNA starting from both A- and B-form structures, B-form DNA and A-form RNA. The N3'-H group is shown to be flexible, undergoing reversible inversion transitions associated with motion of the hydrogen atom attached to the N3' atom. The inversion process is correlated with both sugar pucker characteristics as well as other local backbone torsional dynamics, yielding increased dihedral flexibility over DNA. Solvation of N3'-phosphoramidate DNA is shown to be similar to RNA, consistent with thermodynamic data on the two species. A previously unobserved intrinsic conformational perturbation caused by the N5'-phosphoramidate substitution is identified and suggested to be linked to the differences in the properties of N3'- and N5'-phosphoramidate oligonucleotide analogs.

New insights into the structure of abasic DNA from molecular dynamics simulations.

Abasic (AP) sites constitute a common form of DNA damage, arising from the spontaneous or enzymatic breakage of the N-glycosyl bond and the loss of a nucleotide base. To examine the effects of such damage on DNA structure, especially in the vicinity of the abasic sugar, four 1.5 ns molecular dynamics simulations of double-helical DNA dodecamers with and without a single abasic (tetrahydrofuran, X) lesion in a 5'-d(CXT) context have been performed and analyzed. The results indicate that the abasic site does not maintain a hole or gap in the DNA, but instead perturbs the canonical structure and induces additional flexibility close to the abasic site. In the apurinic simulations (i.e., when a pyrimidine is opposite the AP site), the abasic sugar flipped in and out of the minor groove, and the gap was water filled, except during the occurrence of a novel non-Watson-Crick C-T base pair across the abasic site. The apyrimidinic gap was not penetrated by water until the abasic sugar flipped out and remained extrahelical. Both AP helices showed kinks of 20-30 degrees at the abasic site. The Watson-Crick hydrogen bonds are more transient throughout the DNA double helices containing an abasic site. The abasic sugar displayed an unusually broad range of sugar puckers centered around the northern pucker. The increased motion of the bases and backbone near the abasic site appear to correlate with sequence-dependent helical stability. The data indicate that abasic DNA contorts more easily and in specific ways relative to unmodified DNA, an aspect likely to be important in abasic site recognition and hydrolysis.

Comparative molecular field analysis-based predictive model of structure-function relationships of polyamine transport inhibitors in L1210 cells.

Maintenance of intracellular polyamine concentrations necessary for cell growth and proliferation is regulated in part by an energy-dependent polyamine uptake system. To obtain information on the characteristics of the polyamine uptake system in L1210 leukemia cells, we have applied computational chemistry techniques to the study of relationships between structure and function of 57 polyamine analogues. Ki values of polyamine analogues, derived from competitive inhibition of [3H]spermidine transport into L1210 cells, were chosen as the measure of biological activity. Using comparative molecular field analysis (CoMFA), a model was constructed to relate molecular structure with biological activity. The model was based on 4 monocationic, 8 dicationic, 14 tricationic, and 20 tetracationic polyamine analogues with a range of Ki values for the inhibition of [3H]spermidine uptake of 0.97-521 microM. The CoMFA model successfully predicted the inhibitory potency of 11 polyamines that had not previously been tested for polyamine uptake inhibitory activity. The 11 values predicted were within 33 +/- 62% of the actual Ki values. The test group included aziridinyl diamines, acetylated spermidines, two new oxazolidinonyl spermidines, monoaziridinyl spermidines, and a diaziridinyl spermine. Several of the compounds from this test group have been shown to have anticancer activity in mice. Consistent with the CoMFA model, certain basic functional groups, such as aziridines that have pKa values in the range of 6-7, seem to interact with the polyamine transporter in a cationic form. The results suggest that the CoMFA model is useful in drug design strategies as a predictive tool for the discovery of new anticancer agents that utilize a polyamine transporter for cellular uptake.

Chemical modification of human aldehyde dehydrogenase by physiological substrate.

Employing 3,4-dihydroxyphenylacetaldehyde (dopal) as a substrate for human aldehyde dehydrogenase (aldehyde:NAD+ oxidoreductase, EC 1.2.1.3) in anaerobic conditions, inactivation of both cytoplasmic E1 and mitochondrial E2 isozymes during catalysis has been observed. Incorporation of 14C-labelled dopal has been demonstrated by retention of label following denaturation and exhaustive dialysis and by peptide mapping following tryptic digestion. Incorporation of label gave linear plots vs. activity remaining with up to two molecules incorporated per molecule of enzyme and 30% activity remaining. Further incorporation (up to 16 molecules) occurred, but was non-linear when plotted vs. activity remaining. Protection against activity loss during incorporation of the first two molecules was afforded by NAD, NADH, chloral, and by chloral and NAD together, the last being the most effective. Saturation kinetics gave y-axis intercepts, suggesting interaction at a specific point on the enzyme surface. The Ki value from saturation kinetics was the same as that from the slope replot in catalytic reaction. Peptide mapping of tryptic digests showed that a single peptide was labelled, confirming specificity of interaction. Even in the absence of complete inactivation, the results suggest that reaction with the first two molecules occurs at some point on the enzyme surface important for enzyme activity. The possibility of such a reaction occurring in vivo is discussed.

Thermodynamic analysis of the equilibrium, association and dissociation of 2'GMP and 3'GMP with ribonuclease T1 at pH 5.3.

Fluorescence titrations and temperature-jump relaxation experiments were performed as a function of temperature on ribonuclease T1 with the inhibitors 2'GMP and 3'GMP to obtain information on the energetics and molecular events controlling the binding of those inhibitors. Results from the titration and temperature-jump experiments were in agreement concerning the equilibrium constant. The larger equilibrium constant for 2'GMP is enthalpic in origin and is due to both a higher on rate and a lower off rate as compared to 3'GMP. On rates for both inhibitors appear to be below the diffusion controlled limit, apparently due to conformational changes in the portion of the active site responsible for recognition of the guanine base. Comparison of the measured enthalpic and entropic terms associated with the equilibrium constant determined from the fluorescence titrations are in disagreement with those calculated from the on and off rates indicating the presence of an induced conformational change in the 2'GMP-enzyme complex. This second conformational change appears to be due to additional interactions between 2'GMP and the catalytic portion of the active site, which may also be responsible for the differences in the binding constant, the on rate and the off rate between 2'GMP and 3'GMP.

pH dependence of binding reactions from free energy simulations and macroscopic continuum electrostatic calculations: application to 2'GMP/3'GMP binding to ribonuclease T1 and implications for catalysis.

An approach is described for extending free energy calculations to take into account the pH dependence of the relative binding of ligands to an enzyme or other receptor protein. The method is based on the calculation of the free energy difference for a single protonation state via the thermodynamic cycle simulation approach followed by inclusion of all possible protonation states of the enzyme and the inhibitor by use of a macroscopic continuum dielectric (Poisson-Boltzmann) model. A detailed formulation of the combined model is presented. It involves solution of the multiple equilibrium problem and makes use of the calculated pKa values of all titrating groups on both enzyme and ligand. The method is illustrated by calculations of the pH dependence of the differential binding of the inhibitors 2'GMP and 3'GMP to ribonuclease T1. A free energy simulation of the differential binding is made for a given protonation state of the enzyme and inhibitor. Although only qualitative agreement with experiment is obtained, the results provide insights concerning the interactions involved. The pH dependence of the binding is calculated by using the protonation state of the residues from the free energy simulation as the standard state for a Poisson-Boltzmann calculation. Information is obtained concerning the pKa values of the titrating amino acids in the free, 2'GMP and 3'GMP bound enzyme forms of RNase T1 and the difference in the pH dependence of the binding of 2'GMP and 3'GMP to RNase T1. The contributions of different types of interactions (e.g. protein residues versus solvent) to the free energy differences are examined. A free energy simulation of the pKa shift of Glu58 shows that it is important to consider both carboxyl oxygen atoms as possible protonation sites since they may behave very differently in a protein. It is found in the protein that the interactions with the solvent favor the neutral (protonated) state of Glu58. This contrasts sharply with the solution behavior, where the solvent favors the charged state. Analysis of the results shows that the interactions of bound water with other protein residues leads to the observed effect. Comparisons are made with a continuum calculation that uses the charged state employed in the free energy simulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Human mitochondrial aldehyde dehydrogenase inhibition by diethyldithiocarbamic acid methanethiol mixed disulfide: a derivative of disulfiram.

A derivative and possible physiological metabolite of disulfiram, diethyldithiocarbamic acid methanethiol mixed disulfide, is shown here for the first time to inactivate the mitochondrial low-Km isozyme of human aldehyde dehydrogenase (EC 1.2.1.3). By comparing inactivating effects of diethyldithiocarbamic acid mixed disulfides with thiols of increasing chain length evidence is provided that steric hindrance is the reason for lack of inhibition of the mitochondrial enzyme by disulfiram in vitro. Since methanethiol is a normal metabolite [(1983) Annu. Rev. Biochem. 52, 187-222] the results also suggest a mechanism by which aldehyde dehydrogenase is inhibited by disulfiram and diethyldithiocarbamic acid in vivo.

Molecular modeling and dynamics of biologically active peptides: application to neuropeptide Y.

A methodology is presented to allow results from molecular dynamics simulations to be combined with pharmacological binding and activity measurements in order to study the structure-function relationships of neuropeptide Y. This approach is a general one and should also be applicable to other peptides for which stable structures are known to exist in solution. The basis of the method is the calculation of energetically stable structures via a simulate and test approach. This approach uses molecular dynamics simulations to search conformational space in order to find low potential energy structures. The energetic stability of the structures is then tested via additional simulations. Once energetic stability has been achieved, perturbations of the structure may be performed via molecular modeling. The simulate and test approach is then used to obtain energetically stable structures for the perturbed compound. Comparison between the energetically stable starting and perturbed structures can then be made concerning both structural and dynamic changes. By using an energetically stable structure prior to the perturbation, the assumption can be made that the calculated differences are primarily due to the perturbation rather than to one or both of the structures reaching a more energetically favorable state. It should be emphasized that the calculations are being performed employing a limited physical model such that the influence of that model on the observed results must always be taken into account.

(-)-3 beta-Substituted ecgonine methyl esters as inhibitors for cocaine binding and dopamine uptake.

Ten 3 beta-ecgonine analogues were synthesized and characterized by 1H and 13C NMR, MS, and elemental analysis. The compounds were synthesized as (-)-stereoisomers from (-)-cocaine. These compounds were assessed for their ability to inhibit [3H]cocaine binding to rat striatal tissue and to inhibit [3H]DA uptake into rat striatal synaptosomes. In this series of compounds, the length of the spacer between the aryl group and the tropane skeleton ranged from 1 to 4 bond distances, and conformational flexibility of the linkage and orientation of the aryl ring system were controlled by various types of linkages. The most potent of the analogues was methyl-(1R-2-exo-3-exo)-8-methyl-3-(beta-styrenyl)-8-azabicyclo[3. 2.1] octane-2-carboxylate. One of the less potent compounds was found to inhibit [3H]cocaine binding and [3H]DA uptake with significantly different IC50 values, in contrast to 14 other 3 beta-substituted analogues. Molecular modeling and CoMFA analysis were used to obtain a rigorous structure-function relationship for the studied compounds. The results showed that the potencies of these 3 beta-substituted ecgonine methyl esters were dominated by steric effects and were acutely sensitive to the distance between the aryl ring and the tropane skeleton and to the orientation of the aryl ring system relative to the tropane skeleton. The current study provides a clearer picture of the shape and size of the putative hydrophobic binding pocket for the 3 beta substituent at the cocaine receptor as well as emphasizing the importance of a drug's free energy of solvation in obtaining structure-activity relationships.

Analysis of structure-function relationships of neuropeptide Y using molecular dynamics simulations and pharmacological activity and binding measurements.

Studies on the structure-function relationship of neuropeptide Y (NPY) were undertaken using a combination of in vacuo molecular dynamics (MD) simulations and pharmacological receptor binding and biological activity measurements. Following a conformational search of NPY from which a theoretical structure was determined, a study of the structural and dynamic changes in the region of amino acids 25-36 was performed in a variety of NPY fragments and in the NPY free acid. Results revealed an increased structural change as the fragment size was decreased. Also, the mobility appears to be lowest in the full NPY vs the NPY fragments. Pharmacological measurements showed a decreased receptor binding and biological activity as fragment size decreased. Combination of the two approaches suggests a model where conformational maintenance and low configurational entropy of the 25-36 region of NPY favors both receptor binding and biological activity. Furthermore, the possibility of two receptor interaction modes is suggested. Analysis of the NPY structure suggests the direct importance of the amidated C-terminus, Gln34 and His26, an indirect importance of the Tyr1 sidechain as well as the potential importance of an apparent electric 'dipole' in NPY for receptor binding and biological activity.

All-atom empirical potential for molecular modeling and dynamics studies of proteins.

New protein parameters are reported for the all-atom empirical energy function in the CHARMM program. The parameter evaluation was based on a self-consistent approach designed to achieve a balance between the internal (bonding) and interaction (nonbonding) terms of the force field and among the solvent-solvent, solvent-solute, and solute-solute interactions. Optimization of the internal parameters used experimental gas-phase geometries, vibrational spectra, and torsional energy surfaces supplemented with ab initio results. The peptide backbone bonding parameters were optimized with respect to data for N-methylacetamide and the alanine dipeptide. The interaction parameters, particularly the atomic charges, were determined by fitting ab initio interaction energies and geometries of complexes between water and model compounds that represented the backbone and the various side chains. In addition, dipole moments, experimental heats and free energies of vaporization, solvation and sublimation, molecular volumes, and crystal pressures and structures were used in the optimization. The resulting protein parameters were tested by applying them to noncyclic tripeptide crystals, cyclic peptide crystals, and the proteins crambin, bovine pancreatic trypsin inhibitor, and carbonmonoxy myoglobin in vacuo and in crystals. A detailed analysis of the relationship between the alanine dipeptide potential energy surface and calculated protein φ, χ angles was made and used in optimizing the peptide group torsional parameters. The results demonstrate that use of ab initio structural and energetic data by themselves are not sufficient to obtain an adequate backbone representation for peptides and proteins in solution and in crystals. Extensive comparisons between molecular dynamics simulations and experimental data for polypeptides and proteins were performed for both structural and dynamic properties. Energy minimization and dynamics simulations for crystals demonstrate that the latter are needed to obtain meaningful comparisons with experimental crystal structures. The presented parameters, in combination with the previously published CHARMM all-atom parameters for nucleic acids and lipids, provide a consistent set for condensed-phase simulations of a wide variety of molecules of biological interest.

Protein dynamics. A time-resolved fluorescence, energetic and molecular dynamics study of ribonuclease T1.

Studies using time-resolved fluorescence depolarization were performed on the internal motion of Trp 59 of ribonuclease T1 (EC 3.1.27.3) in the free enzyme, 2'-GMP-enzyme complex and 3'-GMP-enzyme complex. The Trp 59 motion was also studied in the free enzyme using molecular dynamics simulations. Energetic analysis of activation barriers to the Trp 59 motion was performed using both the transition state theory and Kramers' theory. The activation parameters showed a dependence on solvent viscosity indicating the transition state approach in aqueous solution to be inadequate. When taking solvent viscosity contributions into account agreement between the transition state and Kramers' theories was obtained. The results indicate the three enzyme forms to have different conformations with the free enzyme and 3'-GMP-enzyme complex being similar. Comparison of the experimental and theoretical results showed a good agreement on the Trp 59 motion in the free enzyme. Trp 59 appears to vibrate rapidly, with a relaxation time of the order of 1 ps, within free space in the protein matrix and to have a slower motion, with a relaxation time of the order of 100 ps, which is related to breathing of the surrounding protein matrix. Molecular dynamics results indicate high mobility in regions of the enzyme involved in the interaction with the guanine base of the inhibitor or substrate while much lower mobility occurred in residues involved in the catalytic mechanism of ribonuclease T1.

TIT for TAT: the properties of inosine and adenosine in TATA box DNA.

The sequence dependent conformation, flexibility and hydration properties of DNA molecules constitute selectivity determinants in the formation of protein-DNA complexes. TATA boxes in which AT basepairs (bp) have been substituted by IC bp (TITI box) allow for probing these selectivity determinants for the complexation with the TATA box-binding protein (TBP) with different sequences but identical chemical surfaces. The reference promoter Adenovirus 2 Major Late Promoter (mlp) is formed by the apposition of two sequences with very different dynamic properties: an alternating TATA sequence and an A-tract. For a comparative study, we carried out molecular dynamics simulations of two DNA oligomers, one containing the mlp sequence (2 ns), and the other an analog where AT basepairs were substituted by IC basepairs (1 ns). The simulations, carried out with explicit solvent and counterinons, yield straight purine tracts, the A-tract being stiffer than the I-tract, an alternating structure for the YRYR tracts, and hydration patterns that differ between the purine tracts and the alternating sequence tracts. A detailed analysis of the proposed interactions responsible for the stiffness of the purine tracts indicates that the stacking between the bases bears the strongest correlation to stiffness. The hydration properties of the minor groove in the two oligomers are distinctly different. Such differences are likely to be responsible for the stronger binding of TBP to mlp over the inosine-substituted variant. The calculations were made possible by the development, described here, of a new set of forcefield parameters for inosine that complement the published CHARMM all-hydrogen nucleic acid parametrization.

Inhibition of (cytosine C5)-methyltransferase by oligonucleotides containing flexible (cyclopentane) and conformationally constrained (bicyclo[3.1.0]hexane) abasic sites.

Pseudorotationally locked sugar analogues based on bicyclo[3.1.0]-hexane templates were placed in DNA duplexes as abasic target sites in the M. HhaI recognition sequence. The binding affinity of the enzyme increases when the abasic site is constrained to the South conformation and decreases when it is constrained to the North conformation. A structural understanding of these differences is provided.