aflatoxin b1 ( afb1 , 1 , scheme 1 ) is a mycotoxin that is isolated from aspergillus 
 flavus and which contaminates agricultural 
 products . dietary exposures to afb1 are high in areas of asia and sub - saharan africa . epidemiological studies 
 suggest that chronic exposure to afb1 is a contributing 
 factor in the etiology of hepatitis b virus ( hbv ) associated hepatocellular 
 carcinomas ( hcc ) . effective biomarkers allowing quantitation of human 
 dietary exposures to afb1 have been identified , and there have been efforts to 
 develop chemopreventive interventions to chronic exposures . the efficiency of 
 adduction at n7-dg by this epoxide is attributed to its intercalation on the 5-face 
 of guanine . this cationic adduct depurinates to release afb1guanine , or alternatively , undergoes base - catalyzed opening 
 of the imidazole ring forming the trans-8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl - formamido)-9-hydroxy 
 aflatoxin b1 ( afb1fapy ) adduct . the afb1fapy adduct ( scheme 1 ) is of interest because 
 the genotoxicity 
 of afb1 is ascribed primarily to this adduct . it induces 
 g  t transitions associated with 
 afb1 mutagenesis in bacteria and mammalian 
 cells . the cationic n7-dg afb1 adduct exhibits a similar spectrum of mutations 
 but at lower levels . moreover , the afb1fapy adduct is persistent in vivo . it may be linked to g  t transversions 
 in the tumor suppression gene p53 and ras proto - oncogenes . smela et al . have demonstrated that 
 the afb1fapy adduct equilibrates between two species , 
 one of which is mutagenic whereas the other blocks dna replication . the  anomer is favored , but in single - strand 
 dna , the equilibrium shifts to favor the  anomer . at the nucleoside level , this has 
 not been observed in duplex dna , probably because both deoxyribose 
 anomers of the adduct intercalate with the afb1 moiety 
 on the 5 face of the adducted nucleotide , similar to 
 the n7-dg cationic adduct . additionally , the afb1fapy adduct exhibits geometrical isomerism involving the 
 formamide moiety . in the 5-xa-3 
 sequence , the potential for a hydrogen bond between the formyl 
 oxygen and the n exocyclic amino group 
 of the 3-neighbor adenine has been recognized , and this has 
 been proposed to explain the preference of the e isomer 
 as opposed to the z isomer of the formamide moiety . this suggests that the orientation of the formamide moiety is controlled 
 by differential hydrogen bonding opportunities in the dna major groove , 
 and the equilibrium between e and z isomers of the formamide moiety is sequence dependent . to 
 characterize how dna sequence modulates the conformation of 
 the afb1fapy formamide moiety , four oligodeoxynucleotide 
 duplexes containing the 5-xa-3 , 5-xc-3 , 
 5-xt-3 , and 5-xy-3 sequences ( x - afb1fapy ; y = 7-deaza - dg ) ( chart 1 ) have been constructed , in which the 3-neighbor base with 
 respect to the adducted guanine has been changed from adenine to thymine , 
 cytosine , and 7-deaza - dg . nmr analyses demonstrate that the equilibrium 
 between e and z isomers of the formamide 
 moiety is controlled by major groove hydrogen bonding interactions . 
 when the 3 neighbor is adenine , the e isomer 
 is favored . in contrast , when the 3 
 neighbor is either thymine or 7-deaza - dg , the z isomer 
 predominates . structures of each of these duplexes have been refined 
 from nmr data using molecular dynamics calculations restrained by 
 noe distances . x = afb1fapy ; y = 7-deaza - dg . in each 
 instance , the 3-neighbor base pair is shown in red . x = afb1fapy ; y = 7-deaza - dg . in each 
 instance , the 3-neighbor base pair is shown in red . unadducted 
 oligodeoxynucleotides were purchased 
 from the midland certified reagent co. ( midland , tx ) . afb1-exo-8,9-epoxide was prepared by oxidizing 
 afb1 in the presence of dimethyldioxirane , as described . crystalline afb1is hazardous due to its electrostatic nature 
 and manipulations should be carried out in 
 a well - ventilated hood with suitable containment procedures . the oligodeoxynucleotide 5-d(ctaagattca)-3 , containing the targeted n7-dg alkylation site ( underlined ) was annealed with 5-d(atctt)-3 
 to form a partially double - stranded scaffold in 200 l of 100 
 mm na2hpo4 ( ph 6.5 ) . the aqueous phase was dissolved in 100 mm na2co3 ( ph 10 ) for 2 h at room temperature to form the afb1fapy adduct . the oligodeoxynucleotide containing the afb1fapy adduct 5-d(ctaaxattca)-3 ( x = afb1fapy ) was recovered 
 from the aqueous phase using reverse - phase hplc ( gemini c18 250 mm 
  10 mm column , phenomenix , inc . , torrance , ca ) at a flow rate 
 of 2 ml / min , with a linear 30 min gradient of 530% ch3cn in 0.1 m ammonium formate ( ph 6.5 ) . the adducted oligodexoynucleotide 
 was lyophilized and characterized by maldi - tof mass spectrometry . each purified afb1fapy modified oligodeoxynucleotide 
 was annealed at room temperature with the complementary strand in 
 buffer consisting of 10 mm nah2po4 , 0.1 m nacl , 
 and 50 m na2edta ( ph 7.0 ) , and the annealed duplex 
 oligodeoxynucleotide was eluted from dna grade biogel hydroxylapatite 
 using a gradient of 10200 mm nah2po4 ( ph 7.0 ) . the melting temperatures 
 were measured in 10 mm na2hpo4 , 0.1 m nacl , 
 and 50 m na2edta ( ph 7.0 ) . the strand concentration 
 was 1.6 m . the temperature was increased at a rate of 1 c / min 
 from 10 to 80 c . the melting points ( tm values ) of the unmodified and modified oligodeoxynucleotides were 
 obtained by determining the inflection points of the absorbance vs 
 temperature curves from the first - order derivatives . spectra were recorded at h frequencies 
 of 600 , 800 , and 900 mhz using cryogenic probes ( bruker biospin , inc . samples were prepared in d2o , containing 
 0.1 m nacl , 10 mm nah2po4 , and 50 m na2edta ( ph 7.0 ) . chemical 
 shifts were referenced to the chemical shift of the water resonance 
 at the corresponding temperature , with respect to 4,4-dimethyl-4-silapentane-1-sulfonic 
 acid ( dss ) . hmqc spectra were recorded with 
 96 real data in the t1 dimension and 1024 real data in 
 the t2 dimension . cosy and noesy spectra were recorded 
 with 512 real data in the t1 dimension and 2048 real data 
 in the t2 dimension . in the noesy experiment for exchangeable 
 protons , the samples were prepared in 9:1 h2o : d2o and the water signal was suppressed by using the watergate pulse 
 sequence . resonance assignment and peak 
 integration footprints 
 were drawn around 
 the noe cross - peaks obtained at noesy mixing times of 150 , 200 , and 
 250 ms using the program sparky . the intensities of the cross - peaks were combined 
 with the intensities generated from complete relaxation matrix analysis 
 of a starting dna structure to generate a hybrid intensity matrix . the program mardigras , using the randmardi algorithm , was used to refine the hybrid matrix by iteration between 
 the calculated and experimental noe intensities . the calculations 
 were initiated using isotropic correlation times of 2 , 3 , and 4 ns . restrained molecular 
 dynamics ( rmd ) calculations for the oligodeoxynucleotide duplexes 
 utilized a simulated annealing approach . the afb1fapy modified oligonucleotides were constructed 
 using the builder module of insight ii ( accelrys , inc . , san diego , 
 ca ) . the partial charges on the afb1fapy nucleotide 
 were obtained from density function theory ( dft ) calculations using 
 a neutral total charge , utilizing the b3lyp/6 - 31 g * basis set and the 
 program gaussian . to obtain the starting 
 structures used for rmd calculations , the afb1 fapy - modified 
 duplex was energy minimized using 200 iterations with the conjugate 
 gradients algorithm . the rmd calculations were conducted with amber using the parm99 force field . the generalized born ( gb ) model with parameters developed by tsui and case the program corma was utilized to calculate the noe intensities from the structures 
 emergent from rmd calculations . the average 
 structure converged from the series of simulated annealing rmd calculations 
 was used as the starting structure . this was placed in an truncated 
 octahedral tip3p water box with periodic boundaries at a distance 
 of 8.0  from the solute . the necessary 
 na ions were added to neutralize the duplex using restraints 
 having a lower bound of 3.0  and an upper bound of 8.0  . 
 the system was subjected to 1000 iterations of potential energy minimization 
 using steepest descents . the solvent was brought to thermal equilibrium 
 by a rmd simulation at constant volume for 10000 iterations with an 
 integrator time of 1 fs , at 300 k. the experimental distance and torsion 
 angle restraints and empirical restraints were increased linearly 
 during the heating . rmd calculations were performed at constant pressure 
 for 1 ns with an integrator time of 1 fs . the particle mesh ewald ( pme ) method was used to approximate nonbonded 
 interactions . the 10-mers 
 5-d(ctaagattca)-3 , 5-d(ctaagcttca)-3 , 5-d(ctaagtttca)-3 , and 5-d(ctaagyttca)-3 
 were designed to contain a single guanine , the targeted n7-dg alkylation site ( underlined ) . these were individually annealed 
 with 5-mers , 5-d(atctt)-3 , 5-d(aactt)-3 , 
 containing a c : a mismatch , 5-d(aactt)-3 , or 5-d(acctt)-3 , 
 respectively , to form double - stranded scaffolds extending two base 
 pairs in either direction from the targeted n7-dg 
 alkylation site . the scaffold facilitated the reaction with afb1-exo - epoxide , by allowing intercalation above 
 the 5-face of the targeted guanine , and directing alkylation 
 at the n7-dg position . the resulting 
 10-mers containing trans-8,9-dihydro-8-(n7-guanyl)-9-hudroxy - afb1 adducts were separated from the scaffolds using reverse - phase hplc . under 
 basic conditions , over a period of several hours , the 10-mers spontaneously 
 rearranged to the corresponding afb1fapy adducts . the utilization of 7-deaza - dg in the 5-xy-3 sequence 
 directed the alkylation to the single dg ; it also facilitated the 
 subsequent isolation of the afb1fapy modified oligodeoxynucleotide 
 using reverse - phase hplc . the 7-deaza - dg : c base pair was not anticipated 
 to disrupt the helical structure of the duplex . the 10-mers containing the afb1fapy 
 adducts were characterized by maldi - tof mass spectrometry : for 5-xa-3 , 
 calcd 3357.1 , found 3356.5 ; for 5-xc-3 , calcd 3333.1 , 
 found 3333.2 ; for 5-xt-3 , calcd 3348.1 , found 3348.0 ; 
 for 5-xy-3 , calcd 3372.1 , found 3372.8 . for each of 
 the four duplexes , one set of resonances of the x afb1 moiety was observed . figures s1 , s2 , and s3 in the supporting information show 
 the corresponding spectra for the 5-xa-3 , 5-xt-3 , 
 and 5-xy-3 duplexes . the afb1 h5 , h6a , 
 h8 , h9 , h9a , and och3 resonances were assigned 
 similar to previous studies , consistent with the notion that in each 
 instance , the afb1 moiety was intercalated above the 5-face 
 of the modified guanine and the each adduct maintained a similar ra axial conformation for the c5n bond linking the pyrimidine ring to the formamido 
 nitrogen . the assignments 
 of the afb1 protons are tabulated in tables s1s4 of the supporting information . noesy spectrum 
 showing the assignments of the afb1 protons 
 of the 5-xc-3 duplex . the cross peaks are assigned 
 as ( a ) x afb1 och3  x afb1 h5 ; ( b ) x afb1 h9a 
  x afb1 cho ; ( c ) x afb1 h9a x afb1 h6a ; ( d ) x afb1 h9a  x afb1 h9 ; ( e ) x afb1 h9  x afb1 cho ; ( f ) x afb1 h9  x afb1 h6a ; ( g ) x afb1 h8 
  x afb1 cho , ( h ) x afb1 h8  x afb1 h6a , ( i ) x afb1 h8  x afb1 h9 , ( j ) x afb1 h5  x afb1 h6a , ( k ) x afb1 h6a  x afb1 cho . the 900 mhz spectrum with a mixing time 
 of 250 ms was collected at 283 k. corresponding spectra for the 5-xa-3 , 
 5-xt-3 , and 5-xy-3 duplexes may be 
 found in figures s1 , s2 , and s3 of the supporting 
 information . a series of hmqc spectra allowed the characterization 
 of the e : z equilibrium of the afb1fapy formyl group in the 5-xa-3 , 5-xc-3 , 
 5-xt-3 , and 5-xy-3 duplexes ( figure 2 ) . for the 5-xa-3 duplex , the carbonyl c resonance was observed at 171 ppm , coupled to a proton 
 resonance at 8.43 ppm . for the 5-xc-3 
 duplex , two cross - peaks were observed in this region of the hmqc spectrum , 
 suggesting that for this duplex , two species were present in equilibrium . 
 one c carbonyl resonance at 167 ppm was coupled to a 
 proton resonance at 8.33 ppm . the other c carbonyl resonance 
 at 168 ppm was coupled to a proton resonance at 7.47 ppm , assigned 
 as the z isomer of the formyl group . for the 5-xt-3 
 duplex , the carbonyl c resonance was observed at 168 
 ppm , coupled to a proton resonance at 7.71 ppm . for the 5-xy-3 
 duplex , the carbonyl c resonance was observed at 169 
 ppm , coupled to a proton resonance at 7.71 ppm . for each of the four 
 duplexes , the assignments of the formyl proton resonances were supported 
 by noes to the deoxyribose h1 and afb1 h6a protons . 
 additionally , different noes were observed between the formyl protons 
 and afb1 h8 . for the 5-xa-3 and the major 
 isomer of the 5-xc-3 duplexes , strong noes were observed 
 between the x formyl protons and the afb1 h8 
 proton , which were observed at  8.3 ppm and  5.7 ppm , 
 respectively . for the 5-xt-3 and 5-xy-3 
 duplexes , weak noes were observed between the x formyl 
 protons and the afb1 h8 protons , which were observed at 
  7.7 ppm and  6.1 ppm , respectively . nmr analysis of the formyl 
 group proton resonances for the four 
 afb1fapy modified duplexes . the spectra show cross - peaks 
 between the carbonyl c resonance and the aldhyde proton 
 resonances , for each of the four sequences . the hmqc spectra were 
 collected at 283 k at a h frequency of 600 mhz . nmr spectra 
 of the nonexchangeable protons of the afb1fapy 
 modified 5-xa-3 , 5-xc-3 , 5-xt-3 , 
 and 5-xy-3 duplexes were assigned using the sequential 
 noe connectivity of the base proton h6 or h8 dipolar couplings with 
 h1 deoxyribose protons . for each of the duplexes , 
 the deoxyribose spin systems were assigned from the assignments of 
 the h1 protons . the assignments of the deoxyribose h2 
 and h2 resonances were determined from the relative cross 
 peak intensities in the noesy spectrum between the h2 and 
 h2 resonances and the h3 resonances . these measurements 
 were obtained at a noe mixing time of 60 ms , which minimized spin 
 diffusion . the anomeric configurations at x c1 
 were determined by analyzing the noes between the h2 and h2 
 protons and the h1 protons . figure 3 shows the results for the e and z geometrical isomers of 5-xc-3duplex . in each instance , 
 the intensity of the x h1 to x h2 
 noe was less than the x h1 to x h2 
 noe , confirming the  configurations of the glycosyl bonds . figures s4 and s5 of the supporting information show the corresponding data for the 5-xa-3-and 5-xt-3-duplexes . comparison 
 of noe intensities for the cross - peaks arising between 
 the x h1 and h3 deoxyribose protons to 
 the x h2 and h2 deoxyribose protons for 
 the e and z geometrical isomers 
 of the 5-xc-3 duplex . ( b ) the z isomer . the 900 mhz noesy spectrum with 
 a mixing time of 60 ms was collected at 283 k. corresponding spectra 
 of the 5-xa-3 and 5-xt-3 duplexes 
 may be found in figures s4 and s5 of the supporting 
 information . for 
 each of the 5-xa-3 , 5-xc-3 , 5-xt-3 , 
 and 5-xy-3 duplexes , the resonances of the nucleobase 
 imino protons were assigned on the basis of sequential connectivity 
 between adjacent base pairs in noesy spectra . these assignments were 
 supported by noes to the amino protons of watson  crick base 
 pairs . in each duplex , there was an interruption 
 of the sequential imino - to - imino proton noes between the t n3h proton at base pair a : t and the x n1h proton at base pair x : c , consistent 
 with the 5-intercalation of the afb1 moiety . in 
 each duplex , at the x : t base pair , the observation 
 of strong interstrand cross peaks from x n1h to the cn h1 and cn h2 amino protons indicated that watson crick 
 hydrogen bonding between x and c was intact 
 ( figure 4 ) . for the x n1h imino 
 proton in the 5-xc-3 duplex , two sets of resonances 
 were observed at 278 k. the ratio between them was 1:2.8 . compared 
 to the unmodified duplex , the cn h1 non - hydrogen bonded exocyclic amine proton shifted downfield 
 0.21 ppm at 278 k ( figure 4 ) . the assignments 
 of the exchangeable protons are tabulated in tables 
 s5s8 of the supporting information . comparison of the an h1 
 and n h2 amino proton resonances of the 
 unmodified 5-ga-3 duplex with those of the 5-xa-3 
 duplex . the cross peaks are 
 assigned as ( a ) an h1  
 t n3h ; ( b ) an h2  t n3h ; ( c ) a h2  t n3h ; ( d ) c h5  g n1h ; ( e ) 
 cn h1  g n1h ; ( f ) cn h2  
 g n1h . the cross - peaks 
 are assigned as ( g ) an h1 
  t n3h ; ( h ) an h2  t n3h ; ( i ) a h2  
 t n3h ; ( j ) c h5  x n1h ; 
 ( k ) cn h1  x n1h ; ( l ) cn h2 
  x n1h . comparison of the cn h1 and nh2 amino 
 proton resonances of the unmodified 5-gc-3 duplex 
 with those of the 5-xc-3 duplex . the cross - peaks are assigned as ( a ) cn h1  g n1h ; ( b ) cn h2  g n1h ; ( c ) 
 cn h1  g n1h ; ( d ) cn h2  
 g n1h . g n1h ; 
 ( f ) cn h1  g n1h ; ( g ) cn h2 
  g n1h ; ( h ) c h5  x n1h ; ( i ) cn h1 
  x n1h ; ( j ) cn h2  x n1h . the noesy spectra were collected 
 at 800 mhz with a mixing time of 250 ms . the temperature was 278 k. for each of the 
 5-xa-3 , 5-xc-3 , 5-xt-3 , 
 and 5-xy-3 duplexes , in the modified strand , the sequential 
 noe connectivity between nucleobase purine h8 or pyrimidine h6 protons 
 and deoxyribose h1 protons was observed from c to a. in each instance , the sequential connectivity 
 exhibited an interruption between a h1 and x due to the loss of the guanine h8 proton because of the opening 
 of the guanine imidazole ring ( figure 5 ) . the 
 connectivity resumed starting from an intranucleotide noe between 
 x h1 and the formyl proton of the fapy base and 
 continued to the 3-terminus . for the complementary strand , 
 in all instances , an interruption of the sequential noes was observed 
 between c h1 and t h8 . for the 5-xc-3 
 duplex , two sets of resonances for several protons were observed for 
 the base pairs x : c , c : g , and t : a , in both cosy and noesy experiments , 
 indicating the existence of two species . the assignments of the nonexchangeable 
 protons are tabulated in tables s9s12 
 of the supporting information . sequential noe connectivity of base h8 
 or h6 protons with deoxyribose 
 h1 protons for the four afb1fapy modified 
 duplexes . the arrows 
 indicate the noes between the formyl protons and the afb1 h8 protons . the 900 mhz noesy spectra with a mixing time of 250 
 ms were collected at 283 k. for the 
 5-ga-3 , 5-gc-3 , 5-gt-3 , 
 and 5-gy-3 unmodified duplexes , the tm values , as monitored by uv absorbance at 260 nm , were 
 31 , 37 , 32 , and 35 c , respectively . the higher values of tm for the 5-gc-3 and 5-gy-3 
 duplexes were attributed to the presence of the 3-neighbor 
 c : g or 7-deaza - dg : c base pairs vs 3-neighbor a : t or t : a base 
 pairs , respectively . as anticipated , the presence of the afb1fapy adducts 
 increased the thermal stabilities of each of the four duplexes . the 
 melting points for the 5-xa-3 , 5-xc-3 , 
 5-xt-3 , and 5-xy-3 duplexes were 41 , 
 45 , 41 , and 45 c , respectively . the higher values of tm for the 5-xc-3 and 5-xy-3 
 duplexes were also attributed to the presence of the 3-neighbor 
 c : g or 7-deaza - dg : c base pairs , respectively . the thermal melting 
 of the 5-xa-3 , 5-xc-3 , 5-xt-3 , 
 and 5-xy-3 duplexes was also examined by monitoring 
 nmr spectra of the imino protons as a function of temperature ; the 
 imino proton resonances were assigned using standard methods . for each duplex , at the x : c base pair the x n1h imino proton resonance and 
 the 5-neighbor a : t base pair t n3h imino proton resonance were observed at 40 c ( figure 6 ) . increased stabilization of the 3-neighbor 
 base pairs was also observed . for the 5-xa-3 duplex the t n3h imino proton resonance was observed . for the 
 5-xc-3 duplex , the g n1h imino proton 
 resonance was observed . at 308 k , the additional 
 imino resonances at 308 k were also observed for the g n1h resonance ( figure 6 ) . for the 5-xy-3 
 duplex , the y n1h imino proton resonance was observed . 
 for the 5-xy-3 duplex , a single resonance was observed 
 at both 278 and 308 k for the x n1h imino proton ( figure 6 ) . for the 5-xc-3 duplex , the presence 
 of two species in equilibrium was evident , with doubling of the imino 
 proton resonances being observed for the x : c and 3-neighboring c : g base pairs . the imino proton resonance for the 5-neighbor a : t base pair exhibited line broadening . the ratio of 
 the two species was approximately 3:1 at 10 c , decreasing to 
 2:1 at 40 c . crick 
 base paired imino proton 
 region for the four afb1fapy modified duplexes 
 as a function of temperature . the arrows indicate 
 imino proton resonances assigned to the z geometrical 
 isomers in equilibrium with the e isomers for the 
 5-xa-3 and 5-xc-3 duplexes . x = afb1fapy , y = 7-deaza - dg . the spectra were collected at 
 ah frequency of 800 mhz . for each of the 5-xa-3 , 5-xc-3 , 5-xt-3 , 
 and 5-xy-3 duplexes , the protons of the two afb1-fused furan rings showed noes to major groove and imino protons 
 of the dna ; most of these were to the 5 neighboring base - pair 
 a : t . thus , h6a and h9a , which are located 
 on the same face of the afb1 moiety , both exhibited noes 
 to a h8 . a weaker noe was observed for afb1 h9 . the afb1 h5 and och3 protons exhibited 
 noes with minor groove and imino dna protons . these were primarily 
 to base a : t in the 5 direction and 
 to the modified nucleotide x. these included noes between 
 afb1 och3 and a h1 , 
 a h2 , a h2 , a h2 , 
 t n3h , x h1 , and x n1h . the cyclopentenone ring h2 and h2 produced noes with 
 c h1 , h2 , and h2 , and t h1 , and h3 of the complementary strand . a 
 series of rmd calculations , using a simulated annealing protocol , 
 were performed from initial a- and b - form dna starting structures 
 for each of the afb1fapy modified duplexes . for 
 the 5-xc-3 duplex , only the structure of the e isomer was refined ; the z isomer did 
 not exhibit sufficient noe cross - peaks to allow structural refinement . emergent structures , five each from a- and b - dna starting structures , 
 with lowest energies , were obtained for each duplex and subjected 
 to potential energy minimization . for each duplex , all structures 
 converged as indicated by pairwise rmsd comparisons ( table 1 , and figures s6 , s7 , s8 , and 
 s9 in the supporting information ) . the accuracies of the emergent 
 structures were evaluated by comparison of theoretical noe intensities 
 calculated by corma for the refined structure 
 to the experimental noe intensities to yield sixth root residuals 
 ( r1 ) . the r1 values for overall residuals , as well as the residuals 
 for intra- or internucleotide noes , were consistently less than 0.1 
 ( table 1 ) and for each nucleotide were less 
 than 0.15 , suggesting that the refined structures for each duplex 
 were in good agreement with the noesy data . figures 
 s10 , s11 , s12 , and s13 in the supporting information show the r1 values for individual 
 nucleotides . expanded views of the structures are shown in figure 7 . in each instance , the afb1 moiety intercalated 
 above the 5-face of the modified nucleotide x and 
 between base pairs a : t and x : c , causing the rise between these base pairs to increase to 
 7  . the adduct - induced unwinding was localized to the adducted 
 base pairs x : c and their 5- and 3-neighbor 
 base pairs a : t and a : t . crick 
 hydrogen bonded exocyclic amine proton was within hydrogen bonding 
 distance of the x formyl oxygen ; this positioned the formamide 
 moiety in the e configuration . for the 5-xc-3 
 duplex , only the structure of the e isomer was refined ; 
 the z isomer did not exhibit sufficient noe cross - peaks 
 to allow structural refinement . the cn h1 non - watson  crick hydrogen bonded exocyclic 
 amine proton was within hydrogen bonding distance of the x formyl oxygen ; this positioned the formamide in the e configuration . the structure of the 5-xt-3 duplex 
 positioned the formamide in the z configuration . the structure suggested that the x formyl oxygen was within 
 hydrogen bonding distance of the x n9h exocyclic amine 
 proton . x = afb1--fapy , y = 7-deaza - dg . for 
 each of the 5-xa-3 , 
 5-xc-3 , 5-xt-3 , and 5-xy-3 
 duplexes , 1 ns of equilibrium rmd calculation was performed in explicit 
 water at constant pressure at 300 k to examine the dynamics of the 
 refined structure and to analyze hydrogen bond occupancies involving 
 the formyl oxygen atom of the afb1fapy adduct . hydrogen bond occupancies were calculated using a distance 
 cutoff of 3.5  and an angle cutoff of 120. using these 
 criteria , for the 5-xa-3 duplex , the hydrogen bond 
 between the formyl oxygen and the a n - da exocyclic 
 amino group was satisfied for > 95% of the trajectory . using the 
 same 
 criteria , for the e configuration of the 5-xc-3 
 duplex , this hydrogen bond was satisfied for 93% of the trajectory . in contrast , using these criteria , for the 5-xt-3 , 
 and 5-xy-3 duplexes the rmd trajectories in explicit 
 solvent suggested that the x formyl oxygen was within 
 hydrogen bonding distance of the x n9h exocyclic amine 
 proton ; this positioned the formamide in the z configuration . this orientation was satisfied for approximately 75% of the trajectory 
 for both the 5-xt-3 , and 5-xy-3 duplexes . the structural 
 coordinates for the 5-xa-3 duplex , the e isomer of the 5-xc-3 duplex , the 5-xt-3 
 duplex , and the 5-xy-3 duplex have been deposited 
 in the protein data bank . pdb i d codes : 2mmf for the 5-xa-3 duplex , 2mmr for the e isomer of the 5-xc-3 duplex , 2mmq for the 5-xt-3 
 duplex , and 2mms for the 5-xy-3 duplex . the 
 structures of afb1fapy adducts in dna and in template : primer complexes with dna damage bypass polymerases are of interest due to their potential to equilibrate 
 between  and  deoxyribose anomers , atropisomers of the 
 c5n bond linking the pyrimidine 
 ring to the formamide nitrogen , and geometrical isomers of the formamide 
 moiety . in duplex dna , in differing sequence 
 contexts , these equilibria are anticipated to modulate the biological 
 processing and genotoxicity of afb1fapy adducts . the afb1fapy adduct may be linked 
 to site - specific g  t transversions in the tumor suppression 
 gene p53 and ras proto - oncogenes . in the 5-xa-3 duplex , the e configuration 
 of the formamide group ( figure 2 ) allows formation 
 of a hydrogen bond between the formyl oxygen of the afb1fapy adduct and the n - da non - watson crick 
 hydrogen bonded exocyclic amino proton of the 3-neighbor a ( figure 7 ) . several features of the 
 nmr spectrum provide evidence for the existence of this hydrogen bond , 
 which had been predicted from studies by mao et al . and the downfield 
 chemical shifts of the formyl proton ( figure 2 ) and the a non - watson crick exocyclic amino proton 
 ( figure 4 ) are consistent with the electronic 
 deshielding of both protons . the appearance of an additional nmr resonance 
 at 308 k , assigned to the z configuration ( figure 6 ) , suggests that the z configuration 
 is favored in the absence of a thermally stable dna duplex , as is 
 observed at the nucleoside level . the 
 slow interconversion of e and z isomers 
 ( figure 6 ) is consistent with a significant 
 lifetime for this hydrogen bond on the nmr time scale . it suggests 
 that the interconversion between the two configurations within the 
 major groove is sterically hindered in duplex dna . in support of this 
 conclusion , the rmd trajectories calculated in the presence of explicit 
 solvent over a 1 ns time period predict a significant occupancy of 
 this hydrogen bond . in the 5-xc-3 duplex , a 
 mixture of e and z isomers is observed 
 ( figure 2 ) . the intensity of the noe between 
 the formyl proton and afb1 h8 proton establishes the major 
 isomer as the e configuration ( figure 5 ) . this allows for the formation of a hydrogen bond between 
 the formyl oxygen and the cn - dc non - watson the distance between the formyl 
 oxygen and cn h1 is 2.7 
  , and the calculated angle ohn is 
 110 ( figure 7 ) . as compared to the 5-xa-3 
 duplex , the cn - dc non - watson crick 
 amino proton is less deshielded in the 5-xc-3 duplex 
 ( figure 4 ) . the coexistence 
 of  and  anomers is ruled out by comparison of the noes 
 intensities between the x h1 to x h2 
 and h2 , which indicates that both e and z geometrical isomers maintain the  configuration 
 of the deoxyribose ( figure 3 ) . moreover , only 
 one set of nmr resonances is observed for the x afb1 moiety ( figure 1 ) and the coexistence 
 of both  and  anomers would have been predicted to result 
 in distinguishable subspectra for each isomer . similar to the 5-xa-3 duplex , as the temperature 
 is increased from 278 to 308 k , the equilibrium shifts toward the z isomer ( figure 6 ) . this again suggests 
 that the z configuration is favored in single - strand 
 dna , but that in duplex dna , the major 
 groove hydrogen bonding favors the e configuration . 
 as for the 5-xa-3 duplex , slow interconversion of e and z isomers ( figure 6 ) suggests a significant lifetime for this hydrogen bond and 
 that interconversion between the two geometrical isomers within the 
 major groove is hindered in duplex dna . the rmd trajectories calculated 
 in the presence of explicit solvent over a 1 ns time period are consistent 
 with this conclusion and predict significant occupancy of this hydrogen 
 bond . in the 5-xt-3 and 5-xy-3 
 duplexes , 
 there is no 3-neighbor exocyclic amino proton in the major 
 groove for either t or 7-deaza - dg , so a major groove hydrogen bond 
 can not form . consequently , the formamide moiety favors the z geometrical isomer ( figure 2 ) , 
 as is observed at the nucleoside level . for both duplexes , the preference of the z isomer 
 is confirmed by the less intense noe between the formyl proton and 
 afb1 h8 proton , as compared to the 5-xa-3 
 duplex , indicating a greater distance between the two protons in the z isomer ( figure 5 ) . for the 5-xt-3 
 duplex , the refined structure predicts that the distance between the 
 formyl oxygen and x n9h is 3.1  , and the calculated 
 angle ohn is 138 ( figure 7 ) . for the 5-xy-3 duplex , the corresponding 
 predicted distance is 2.7  and 147 ( figure 7 ) . these results suggest that the z isomer 
 could be stabilized by a hydrogen bond between the formyl oxygen of 
 the afb1fapy adduct and the x n9h exocyclic 
 amine proton . however , the rmd trajectories calculated in explicit 
 solvent are equivocal . they suggest 76% and 81% occupancies for this 
 hydrogen bond in the 5-xt-3 and 5-xy-3 
 duplexes , respectively . the observation of a single nmr resonance 
 for the x n1h imino proton at both 278 and 308 k for both 
 the 5-xt-3 and 5-xy-3 duplexes ( figure 6 ) is consistent with the notion that the z isomer is favored at all temperatures . likewise , a single resonance is observed for the imino 
 protons of the 3-neighbors , t n3h and 7-deaza - dg n1h in the 5-xt-3 and 5-xy-3 
 duplexes , respectively , at 308 k ( figure 6 ) . the 
 significant structural differences between the four afb1fapy adducts studied here , under equilibrium conditions , 
 are with regard to the geometrical isomerization of the formamide 
 moiety in the major groove ( figure 7 ) . for 
 each of the four duplexes , only one set of nmr resonances is observed 
 for the x afb1 moiety ( figure 1 ) , which indicates that the equilibrium between e and z isomers induces a localized perturbation 
 to the duplex . spectroscopic differences between the e and z isomers are confined to base pairs x : c , c : g , and t : a , and the modest chemical shift perturbations of corresponding 
 nucleobase and deoxyribose h1 protons of the e and z isomers become smaller proceeding from the 
 modified base pair x : c and proceeding in 
 the 3-direction toward base pairs c : g , and t : a ( figure 5 ) . the equilibria between  and  anomers and the atropisomerism about the c5n bond do not exhibit 
 sequence dependence . for all four duplexes , the afb1 moiety 
 intercalates above the 5-face of the damaged base , the ra axial conformation about the c5n bond is maintained , and the  anomer 
 of the deoxyribose is favored , similar to studies of the  anomer 
 of the afb1fapy adduct . a study of the  anomer of the afb1fapy 
 adduct concluded that the afb1 moiety also intercalates on the 5 face of the damaged base , 
 also maintaining the ra axial conformation 
 about the c5n bond . however , 
 perturbations of the  and  backbone torsion angles were 
 observed , and the base stacking of the duplex was perturbed , which 
 correlates with the observation that the  anomer of the afb1fapy adduct blocks dna replication . one 
 notable feature of 
 the  anomer of the afb1fapy adduct is its 
 thermal stabilization of the dna duplex . it has been proposed that hydrogen bonding between the formyl oxygen 
 of the afb1fapy adduct and the n non - watson  crick hydrogen bonded exocyclic amino 
 proton of the 3-neighbor a provides a potential 
 contribution to this stabilization , but the present 
 results suggests that this is not the major contributing factor . although 
 in the 5-xa-3 and 5-xt-3 duplexes , 
 the formamide is held in e vs z isomers , 
 the 5-intercalation of the afb1 moiety stabilizes 
 both 5-xa-3 and 5-xt-3 duplexes by 
 a similar 10 c increase in tm , as 
 compared to the unadducted duplexes . therefore , it seems that the 
 hydrogen bond between the afb1 formamide moiety and the 
 3- neighbor base , an intrastrand hydrogen bond , does not contribute 
 to the stability of the afb1fapy modified dna duplex . instead , the thermal stability of the  anomer of the afb1fapy adduct in duplex dna is likely attributable to 
 favorable base stacking interactions . sequence - dependent hydrogen 
 bonding patterns in the dna major groove modulate e vs z geometrical isomerism of the formamide moiety 
 of the afb1fapy adduct . the differential accommodation 
 of these afb1fapy adducts within the active site 
 may , in part , modulate lesion bypass . it will be of interest to determine 
 if sequence - dependent structural perturbations , observed here for 
 dna duplexes in vitro and at equilibrium , are translated into differential 
 outcomes during error - prone replication bypass of the afb1fapy adduct . banerjee et al . have obtained structural data for the error - prone bypass of the 
 afb1fapy adduct by the sulfolobus 
 solfataricus p2 dna polymerase iv ( dpo4 ) in the 5-xa-3 sequence context . the dpo4 polymerase conducts 
 error - prone replication past the afb1fapy adduct , 
 including misinsertion of datp , consistent with g  t transversions . in the 5-xa-3 
 sequence , the structure of a ternary ( dpo4dna  datp ) 
 afb1fapy adducted template reveals that the oxygen 
 atom of the fapy formyl group participates in a water - mediated hydrogen 
 bond with polymerase residue arg . in this case , the afb1fapy formamide 
 group exists as the z geometrical isomer . thus , geometrical isomerism of the afb1fapy formamide group is modulated not only by dna sequence 
 but also by specific interactions within the active sites of specific 
 error - prone bypass polymerases . it is also possible that geometrical 
 isomerism of the afb1fapy formamide group modulates 
 the recognition and repair of these adducts in a sequence - specific 
 manner . fapy - type adducts are often substrates for base excision repair 
 ( ber ) , but the afb1fapy 
 adduct is a substrate for nucleotide excision repair ( ner ) in bacterial , yeast , and mammalian 
 cells . these reports are consistent with the notion that 
 ner targets bulky dna lesions . on the other hand , additionally , the data suggest differences 
 in damage recognition between bacterial and mammalian repair systems . 
 croy and wogan noted that the afb1fapy lesion is removed less efficiently in mammalian 
 cells than is the initially formed afb1n7-dg cationic adduct , whereas both lesions are repaired 
 with equal efficiencies in bacteria . more recently , alekseyov et al . confirmed that in escherichia 
 coli afb1fapy lesions are efficiently 
 repaired by ner . while takahashi had 
 reported that xpa - deficient mice exhibited enhanced afb1-induced liver tumorigenesis , mulder et al . did not observe increased repair of afb1fapy 
 adducts in lung or liver extracts of ner - compromised heterozygous 
 p53 knockout mice and they also did not observe changes in xpa or 
 xpb protein levels in the p53 knockout mice . consequently , the precise 
 factors modulating ner recognition and repair of afb1fapy 
 lesions remain incompletely understood . there is precedent for the 
 idea that sequence - specific differences in dna adduct structure may 
 modulate ner efficiencies . geacintov , broyde , and co - workers have investigated the sequence dependence of ner and have noted 
 the potential for sequence specific differences in steric factors 
 involving the minor groove - aligned pah adducts and nearby guanine 
 amino groups ; they conclude that the ner apparatus binds productively 
 or unproductively to the damaged dna duplex depending upon the structural 
 and stereochemical properties of specific dna adducts . thus , it will be of interest to examine dna 
 sequence modulation of ner for afb1fapy adducts .
aflatoxin 
 b1 ( afb1 ) , a mycotoxin produced 
 by aspergillus flavus , is oxidized 
 by cytochrome p450 enzymes to aflatoxin b1 - 8,9-epoxide , 
 which alkylates dna at n7-dg . under basic conditions , 
 this n7-dg adduct rearranges to yield the trans-8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl - formamido)-9-hydroxy 
 aflatoxin b1 ( afb1fapy ) adduct . 
 the 
 afb1fapy adduct exhibits geometrical isomerism 
 involving the formamide moiety . 
 nmr analyses of duplex oligodeoxynucleotides 
 containing the 5-xa-3 , 5-xc-3 , 5-xt-3 , 
 and 5-xy-3 sequences ( x = afb1fapy ; 
 y = 7-deaza - dg ) demonstrate that the equilibrium between e and z isomers is controlled by major groove hydrogen 
 bonding interactions . 
 structural analysis of the adduct in the 5-xa-3 
 sequence indicates the preference of the e isomer 
 of the formamide group , attributed to formation of a hydrogen bond 
 between the formyl oxygen and the n6 exocyclic amino group 
 of the 3-neighbor adenine . while the 5-xa-3 
 sequence exhibits the e isomer , the 5-xc-3 
 sequence exhibits a 7:3 e : z ratio 
 at equilibrium at 283 k. the e isomer is favored 
 by a hydrogen bond between the formyl oxygen and the n4-dc exocyclic amino group of the 3-neighbor cytosine . 
 the 
 5-xt-3 and 5-xy-3 sequences can not 
 form such a hydrogen bond between the formyl oxygen and the 3-neighbor 
 t or y , respectively , and in these sequence contexts the z isomer is favored . 
 additional equilibria between  and  
 anomers and the potential to exhibit atropisomers about the c5n5 bond do not depend upon sequence . in each 
 of the four dna sequences 
 , the afb1fapy adduct 
 maintains the  deoxyribose configuration . 
 each of these four 
 sequences feature the atropisomer of the afb1 moiety that 
 is intercalated above the 5-face of the damaged guanine . 
 this 
 enforces the ra axial conformation for 
 the c5n5 bond .