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1.
Abstract

The non-exchangeable and imino proton NMR resonances of the non self-complementary decadeoxyribonucleotide d-[(GATCCGTATG) · (GATACGGATC)] as well as those of the 1:1 complex of the monocatonic bis-imidazole lexitropsin 1 to this sequence have been assigned by using a combination of NOE difference, COSY and NOESY techniques. Confirmation of complete annealing of the two non self-complementary decamer strands to give the duplex decadeoxyribonucleotide is obtained by the detection of ten imino protons. It is established that the sugar-base orientations of all the bases in the duplex decamer are anti. From NOE studies, it is concluded that the duplex oligomer is right-handed and adopts a conformation in solution that belongs to the B family. A population analysis reveals that the sugar moieties exist predominantly in the S-form (2′-endo-3′-exo). Addition of 1 to the DNA solution leads to doubling of the resonances for CH6(4,5), GH8(6), TH6(7) and T-CH3(7). The base, anomeric H1′ and imino proton signals for the base sequence 5′-CCGT undergo the most marked drug-induced chemical shift changes. These results provide evidence that the lexitropsin is bound to the sequence 5′-CCGT in the minor groove of the DNA NOE measurements between the amide protons (NH1 and NH4) and the imino proton (IV and V) signals confirmed the location and orientation of 1 in the 1:1 complex, with the amino terminus oriented to C(4). The specific binding of 1 to the sequence 5′-CCGT-3′ deduced in this study is in agreement with the footprinting data obtained using the Hind III/Nci I fragment from pBR322 DNA [Kissinger et al. 1987 (13)]. Intramolecular NOEs observed between H4 and H9 of the lexitropsin suggest that the molecule is not planar, but subjected to propeller twisting, in both the free and bound forms. Furthermore, NOE measurements permit assignment of the DNA duplex in the 1:1 complex to the B-form, which is similar to that of the free DNA The [(T7A8T9)· (A12T13A14)] segment of the DNA shows better stacking, by propeller twisting, compared to the rest of the molecule in the free as well as the complex forms. The intermolecular rate of exchange of 1 between the equivalent 5′-CCGT sites, at a concentration of 12 mM, is estimated to be ~88s?1 at 308°K with ΔG≠ of 63±5 K.J mol?1.  相似文献   

2.
The location, orientation and dynamics of a thiazole-containing analogue of distamycin 1 bound to the decadeoxyribonucleotide d-[CGCAATTGCG]2 have been studied by non-exchangable and imino proton NMR resonances of the 1:1 complex. Using NOE difference, COSY and NOESY experiments, lexitropsin (1) was located in the minor groove of DNA at 5'-CAAT sequence. This was concluded by an intermolecular NOE between the ligand and a minor groove A4H2 proton. The NOE cross-correlations in the NOESY map confirmed that the DNA decamer duplex in the 1:1 complex remains in a right-handed B-conformation similar to that in the free decamer. Experiments on non-exchangeable and exchangeable proton NMR resonances placed the N-formylamino terminus of drug 1 on the 5'-C3 nucleotide, while the rest of the molecule extends onto the 5'-AAT sequence. The structural evidence for sequence preferential binding at 5'CAAT rather than 5'AATT suggests this reflects an attempt on the part of the sterically demanding inward directed sulfur of the thiazole to minimize compression by moving part of the molecule to the somewhat wider CG base site. The lack of evidence for a 2:1 drug:DNA complex, in contrast to distamycin, is in accord with this interpretation. The lexitropsin 1 was found to be in an exchange between the equivalent 5'-CAAT sites at a rate of approximately 35S-1 with a delta G degree of 65 +/- 5 kJ mol-1 at 303 K. The experimental data suggests a slide-swing mechanism for this exchange process.  相似文献   

3.
Structural and dynamic properties of the self-complementary decadeoxyribonucleotide d(CGCAATTGCG)2 and the interaction between a prototype lexitropsin, or information-reading oligopeptide, and the decadeoxyribonucleotide are deduced by using high-resolution 1H NMR techniques. The nonexchangeable and imino proton resonances of d(CGCAATTGCG)2 have been completely assigned by two-dimensional NMR studies. The decadeoxyribonucleotide exists as a right-handed B-DNA. In the 1H NMR spectrum of the 1:1 complex, the selective chemical shifts and removal of degeneracy of AH2(4), AH2(5), T-CH3(6), and T-CH3(7) due to the anisotropy effects of the heterocyclic moieties of the ligand, and with lesser effects at the flanking base sites C(3) and G(8), locate the drug centrally in the decadeoxyribonucleotide. This conclusion is supported by plots of individual chemical shift changes across the decadeoxyribonucleotide. Similarly, imino protons IV and V experience larger shifts and II and III smaller shifts in accord with this conclusion while drug complexation permits the detection of imino proton I. Strong nuclear Overhauser effects (NOEs) between pyrrole H5 and AH2(5), and weaker NOEs to AH1'(5), TH3'(6), and AH2'(5), firmly locate the ligand in the minor groove. Intraligand NOEs between the adjacent heterocyclic moieties indicate that the lexitropsin is subject to propeller twisting about the N6-C9 bond in both the bound and free forms. Nuclear Overhauser effect spectroscopy (NOESY) and correlated spectroscopy (COSY) experiments also indicate that the removal of degeneracy of the C16 methylene protons upon complexation may arise from restricted rotation about the C15-N9, C15-C16, and C16-C17 bonds. Specific hydrogen bonds between amide NH groups on the concave face of the ligand (N4H, N6H, N9H) and adenine N3 or thymine O2 on the floor of the minor groove are in accord with displacement of the hydration shell by the drug. NOE measurements on the decadeoxyribonucleotide in the 1:1 complex confirm it exists as a right-handed helix and belongs to the B family. Exchange NMR effects permit an estimate of a rate of approximately equal to 44 s-1 for the two-site exchange of the lexitropsin between two equivalent sites on the decamer with delta G++ approximately equal to 70 +/- 5 kJ mol-1 at 294 K. Alternative mechanisms for this exchange process are considered.  相似文献   

4.
The structural and dynamic aspects of the interaction of the thiazole containing lexitropsin (1) with an oligodeoxyribonucleotide were studied by high field 1H-NMR spectroscopy. Complete assignment of the 1H-NMR resonances of lexitropsin 1 was accomplished by 2D-NMR techniques. The complexation-induced chemical shifts and NOE cross peaks in the NOESY map of the 1:1 complex of lexitropsin (1) and d-[CGCAATTGCG]2 reveal that the thiazole ring of the lexitropsin (1) intercalates between dA4.A5 bases and the rest of the ligand resides in the minor groove of the AT rich core of decamer, thus occupying the 5'-AATT sequence on the DNA. Intercalation of the thiazole moiety of the drug has been detected by the presence of intermolecular NOEs both in the major and the minor groove of the decamer helix. The absence of intranucleotide NOEs between base protons and H1'/H2' protons suggested local unwinding of the binding site on the DNA. From COSY and NOESY methods of 2D-NMR, it was established that the N-formyl (amino) terminus of the thiazole lexitropsin (1) is projecting into the major groove towards A5H8 while the amidinium terminus lies in the minor groove towards the T7G8 base pairs of the opposite strand. The expected intranucleotide NOEs confirmed that the decadeoxyribonucleotide in the 1:1 complex exists in a right handed B-conformation. The presence of exchange signals along the binding site 5'-AATT indicated an exchange of the bound drug process wherein the rate of exchange between the two equivalent sites was estimated to be congruent to 130 s-1 at 30 degrees C and with delta G degrees of 62.4 kJ mol-1. Force field and Pi calculations permitted a rationalization of the experimentally observed binding mode in terms of preferred conformation of the ligand and repeat length in lexitropsins compared with the DNA receptor.  相似文献   

5.
High field 1H-NMR techniques have been used to examine the sequence dependent binding of a lexitropsin, the bis-imidazole analogue of netropsin 1, to the decadeoxyribonucleotide d-[CGCAATTGCG]2. The non-exchangeable and imino protons of the 1:1 lexitropsin:DNA complex are assigned by 1D-NOE difference and COSY methods. Addition of 1 to the DNA resulted in marked drug induced chemical shift changes of both the non-exchangeable and imino protons of A(4,5) and T(6,7). These results suggest that the lexitropsin is located in the minor groove along A(4) to T(7) of the DNA. Weaker chemical shift changes are observed for C(3) and G(8) which suggest that the bisimidazole moiety of 1 can also accept G.C sites. Specific NOEs seen between the lexitropsin (H2, H14 and H15) and DNA (AH2(4) and AH2(5] confirmed that the N to C-terminii of 1 is, on average, bound centrally to the sequence in the direction 5'-AATT-3'. However, netropsin 2 is shown to bind tightly only to the AATT sequence. Exchange NMR effects permit the estimate of the rate of exchange of the lexitropsin 1 between the two equivalent sites on the DNA to be approximately 160s and 24s for netropsin under comparable conditions. Several factors contributing to the sequence specificity of lexitropsin binding are discussed.  相似文献   

6.
MPE-Fe(EDTA) footprinting of a novel monocationic bis-furan lexitropsin 6 on a HindIII/EcoRI restriction fragment of pBR322 DNA revealed a series of four-base binding sites (all 5'----3') of (primary) TGTA, TGAA, AAAT, ACAA, TTAT, and (secondary) CTAA, TCGT, TGTA, GTCA, and GGTT. Thus 6 can accept a GC pair at positions 1, 2 or 3 of the binding site with a strict 3' (4 position) AT requirement. Marked enhancement of cleavage, particularly at GC rich sequences, is observed at regions flanking or even up to 18 base pairs remote from a given binding site. The non-exchangeable and imino 1H NMR resonances of the 1:1 complex and d-[CATGGCCATG]2 were assigned using a combination of NOE differences, NOESY and COSY techniques. 1H NMR studies (ligand induced chemical shifts and NOE differences) of Lexitropsin 6 with d-[CATGGCCATG]2 show unambiguously the location and orientation of the N to C termini of 6 on the sequence 5'-G5C6C7A8-3', with the C terminus oriented to A8. This orientation of 6 in the minor groove of 5'-GCCA is confirmed by an NOE observed between H1 2a of 6 and AH8(8). This preference for binding of 6 to the sequence 5'-GCCA when challenged with d-[CATGGCCATG]2 is in accord with the conclusions of the footprinting experiments wherein GC base pairs can be accepted in the first three positions and with a strict 3' terminus AT reading requirement. Collectively the data support the inference of a GC recognizing capacity for a 2,5-substituted furan moiety within a lexitropsin. The 1H NMR data indicate that the decadeoxyribonucleotide duplex exists in the B conformation in both the 1:1 complex and the free form. The apparent binding constant of 6 to calf thymus DNA is 1.68 X 10(5) M-1 whereas netropsin under similar conditions gives a value of 1.85 X 10(7) M-1. This suggests that if advantage is to be taken of the GC recognizing property of a 2,5-substituted furan in longer lexitropsins it should be flanked by more strongly bound moieties.  相似文献   

7.
Nuclear magnetic resonance (NMR) has been used to monitor the conformation and dynamics of the d(C1-G2-A3-T4-T5-A6-T6-A5-A4-T3-C2-G1) self-complementary dodecanucleotide duplex (henceforth called Pribnow 12-mer), which contains a TATAAT Pribnow box and a central core of eight dA X dT base pairs. The exchangeable imino and nonexchangeable base protons have been assigned from one-dimensional intra and inter base pair nuclear Overhauser effect (NOE) measurements. Premelting conformational changes are observed at all the dA X dT base pairs in the central octanucleotide core in the Pribnow 12-mer duplex with the duplex to strand transition occurring at 55 degrees C in 0.1 M phosphate solution. The magnitude of the NOE measurements between minor groove H-2 protons of adjacent adenosines demonstrates that the base pairs are propeller twisted with the same handedness as observed in the crystalline state. The thymidine imino proton hydrogen exchange at the dA X dT base pairs has been measured from saturation recovery measurements as a function of temperature. The exchange rates and activation barriers show small variations among the four different dA X dT base pairs in the Pribnow 12-mer duplex.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

8.
High-resolution two-dimensional NMR studies have been completed on the self-complementary d(C-G-C-G-A-G-C-T-T-G-C-G) duplex (designated G.T 12-mer) and the self-complementary d(C-G-C-G-A-G-C-T-O4meT-G-C-G) duplex (designated G.O4meT 12-mer) containing G.T and G.O4meT pairs at identical positions four base pairs in from either end of the duplex. The exchangeable and nonexchangeable proton resonances have been assigned from an analysis of two-dimensional nuclear Overhauser enhancement (NOESY) spectra for the G.T 12-mer and G.O4meT 12-mer duplexes in H2O and D2O solution. The guanosine and thymidine imino protons in the G.T mismatch resonate at 10.57 and 11.98 ppm, respectively, and exhibit a strong NOE between themselves and to imino protons of flanking base pairs in the G.T 12-mer duplex. These results are consistent with wobble pairing at the G.T mismatch site involving two imino proton-carbonyl hydrogen bonds as reported previously [Hare, D. R., Shapiro, L., & Patel, D. J. (1986) Biochemistry 25, 7445-7456]. In contrast, the guanosine imino proton in the G.O4meT pair resonates at 8.67 ppm. The large upfield chemical shift of this proton relative to that of the imino proton resonance of G in the G.T mismatch or in G.C base pairs indicates that hydrogen bonding to O4meT is either very weak or absent. This guanosine imino proton has an NOE to the OCH3 group of O4meT across the pair and NOEs to the imino protons of flanking base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

9.
All 1H-NMR resonances of d-[CATGGCCATG]2 and the 1:1 complex of lexitropsin 1 and the DNA were assigned by the NOE difference, COSY and NOESY methods. Addition of 1 causes the base and imino protons for the sequence 5'-CCAT to undergo the most marked drug-induced chemical shift changes, thereby indicating that 1 is located in this base pair sequence. NOEs confirmed the location and orientation of the drug in the 1:1 complex, with the amino terminus oriented to C(6). The van der Waals interaction between H12a,b of 1 and AH2(8) may be responsible for reading of the 3' A.T base pair in the 5'-CCAT sequence. Exchange NMR effects allow an estimate of approximately equal to 62 s-1 for the intramolecular "slide-swing" exchange of the lexitropsin between two equivalent binding sites with delta G = 58 +/- 5 kJ mol-1 at 301 degrees K.  相似文献   

10.
1H NMR has been used to study the interactions of ellipticine and the ellipticine analogues 2-3-dimethyl-6-(2-dimethylaminoethyl)6H-indolo-[2,3-b]quinoxaline and 6-(2-dimethylaminoethyl)6H-indolo-[2,3-b]quinoxaline with the self-complementary decadeoxyribonucleotide d(CGCGATCGCG)2. The Watson-Crick H-bonded imino proton resonances were studied. The drugs were shown to bind to the duplex by intercalation involving slow exchange kinetics for the imino proton resonances on the NMR time scale (500 MHz). Ellipticine and the 2,3-dimethyl analogue were found not to show strong base preferences, while the other analogue was found to have a preferred primary binding site between the A.T base pairs with a probable minor secondary binding site between the A.T and adjacent G.C base pairs. The new drug-shifted imino proton resonances were assigned through saturation transfer experiments. The base-specific interactions were accompanied by drug-induced non-uniform broadening of the resonances (due to intermediate chemical exchange kinetics), in the spectral region of the non-exchangeable aromatic and sugar H1' proton resonances of the oligonucleotide at 25 degrees C.  相似文献   

11.
The complementary consensus acceptor exon:intron junction d(ApCpCpTpGpTpApG) has been synthesized by a modified phosphotriester method. The non self-complementary octamer exists in the random coil form in aqueous buffer at 20 degrees C as evidenced by temperature variable 1H-NMR and NOE measurements. The non-exchangeable proton assignments were secured using a combination of techniques including two-dimensional COSY, NOESY and 1H-1H-INADEQUATE. The octamer was annealed with the primary consensus sequence d(CpTpApCpApGpGpT). Confirmation of complete duplex formation was confirmed by detection and assignment of imino protons in D2O:H2O mixtures. Assignment of the non-exchangeable proton signals in the duplex consensus junction was then secured by a combination of two-dimensional COSY correlations, NOESY and NOE experiments. Determination of individual vicinal coupling constants in the component deoxyribose moieties permitted deduction of the population of S conformations in this sequence. It is concluded that the consensus acceptor junction exists in solution in a conformation belonging to the B family, and that the bases are oriented anti. In addition the deoxyribose moieties in the 5' regions exist predominantly in the S form (2'endo-3'exo) whereas those residues on or adjacent to the junction on the primary strand show more N character (2'exo-3'endo). The contiguous bases A5-G6 (adjacent to the junction) and A15-G16 are stacked more closely than the other neighbor bases in this duplex sequence. These subtle structural and conformational differences in the exon:intron junction may serve as recognition signals for these critical sites in the genome.  相似文献   

12.
13.
Identification of I:A mismatch base-pairing structure in DNA   总被引:7,自引:0,他引:7  
Deoxyoligonucleotides containing deoxyinosine residues at positions corresponding to ambiguous nucleotides derived from an amino acid sequence have been successfully used as hybridization probes. It is assumed that the hypoxanthine residue can make base pairs with multiple bases. In order to obtain direct evidence for I:A base-pairing, a self-complementary deoxyoligonucleotide, d(G-G-I-A-C-C), was synthesized and its properties were examined by NMR spectroscopy. Three hydrogen-bonded imino proton resonances are observed at low temperatures in H2O suggesting the formation of a self-duplex with complete base pairing. Nuclear Overhauser effect (NOE) experiments showed that a signal at 15.1 ppm originated from the imino proton (H1) of the dI residue (I3) which is hydrogen-bonded to the dA residue (A4). Both the I3 and A4 residues were assumed to have taken an anti glycosidic conformation since irradiating the H1 of I3 gave NOEs both to its own H2 and to that of A4, an NOE also being observed between the H2 protons of I3 and A4. Comparison of the 31P NMR spectra of d(G-G-I-A-C-C) and d(G-G-I-C-C-C) showed the backbone structure of d(G-G-I-A-C-C) to have been disturbed by the presence of purine:purine base pairs in the middle of the hexamer duplex.  相似文献   

14.
Structural features of pyrimidine.pyrimidine mismatches in the interior of oligonucleotide duplexes have been investigated by high resolution two-dimensional proton nuclear magnetic resonance (n.m.r.) spectroscopy. These studies were conducted on the self-complementary d(C-G-C-T-A-G-C-T-T-G-C-G) duplex (designated T.T 12-mer) and the self-complementary d(C-G-C-C-A-G-C-T-C-G-C-G) duplex (designated C.C 12-mer) containing T.T and C.C pairs located at identical positions four base-pairs from either end of the duplex. Proton n.m.r. studies on the T.T 12-mer duplex were undertaken in the neutral pH range, while studies on the C.C 12-mer duplex were recorded at acidic pH. The proton spectra narrowed considerably on lowering the pH below neutrality for the C.C 12-mer duplex. Two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) data sets have been recorded on the T.T 12-mer and C.C 12-mer duplexes in high salt H2O and D2O solution. The magnitude of the NOE crosspeaks and the directionality of the NOE connectivities demonstrate that both duplexes are right-handed with all bases, including those at the mismatch site, adopting an anti configuration about the glycosidic bond. The observed base and sugar proton chemical shifts suggest structural similarities for the trinucleotide segments centered about the T.T and C.C mismatches. A NOE is detected between the resolved imino protons of T4 and T9 at the mismatch site, consistent with formation of a stacked "wobble" T4(anti).T9(anti) pair in the T.T 12-mer duplex. A comparison of the imino proton chemical shift and NOE data suggests that the imino-carbonyl hydrogen bonds in the wobble T.T mismatch are weaker than the corresponding imino-carbonyl hydrogen bonds in the wobble G.T mismatch. The 4-amino protons of C4 and C9 at the mismatch site in the C.C 12-mer duplex do not exhibit the pattern of hydrogen-bonded and exposed protons separated by approximately 1.5 parts per million characteristic of cytidine amino protons involved in Watson-Crick G.C pairing. The experimental data are insufficient to differentiate between wobble C(anti).C+(anti) and other pairing possibilities for the mismatch in the C.C 12-mer duplex at acidic pH.  相似文献   

15.
M W Kalnik  B F Li  P F Swann  D J Patel 《Biochemistry》1989,28(15):6170-6181
High-resolution two-dimensional NMR studies are reported on the self-complementary d-(C1-G2-C3-O6etG4-A5-G6-C7-T8-T9-G10-C11-G12) duplex (designated O6etG.T 12-mer) containing two symmetrically related O6etG.T lesion sites located four base pairs in from either end of the duplex. Parallel studies were undertaken on a related sequence containing O6meG.T lesion sites (designated O6meG.T 12-mer) in order to evaluate the influence of the size of the alkyl substituent on the structure of the duplex and were undertaken on a related sequence containing G.T mismatch sites (designated G.T 12-mer duplex), which served as the control duplex. The exchangeable and nonexchangeable proton and the phosphorus nuclei have been assigned from an analysis of two-dimensional nuclear Overhauser enhancement (NOE) and correlated spectra of the O6etG.T 12-mer, O6meG.T 12-mer, and G.T 12-mer duplexes in H2O and D2O solutions. The distance connectivities observed in the NOESY spectra of the O6alkG.T 12-mer duplexes establish that the helix is right-handed and all of the bases adopt an anti conformation of the glycosidic torsion angle including the O6alkG4 and T9 bases at the lesion site. The imino proton of T9 at the O6alkG.T lesion sites resonates at 8.85 ppm in the O6etG.T 12-mer duplex and at 9.47 ppm in the O6meG.T 12-mer duplex. The large upfield shift of the T9 imino proton resonance at the O6alkG4.T9 lesion site relative to that of the same proton in the G4.T9 wobble pair (11.99 ppm) and the A4.T9 Watson-Crick pair (13.95 ppm) in related sequences establishes that the hydrogen bonding of the imino proton of T9 to O6alkG4 is either very weak or absent. The imino proton of T9 develops NOEs to the CH3 protons of the O6etG and O6meG alkyl groups across the base pair, as well as to the imino and H5 protons of the flanking C3.G10 base pair and the imino and CH3 protons of the flanking A5.T8 base pair in the O6alkG.T 12-mer duplexes. These observations establish that the O6alkG4 and T9 residues are stacked into the duplex and that the O6CH3 and O6CH2CH3 groups of O6alkG4 adopt a syn orientation with respect to the N1 of the alkylated guanine.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

16.
The non-exchangeable and imino proton NMR resonances have been assigned of the 1:1 complex of an analogue 2 of Hoechst 33258 1 bound to the decadeoxyribonuycleotide d-[CATGGCCATG]2 by a combination of NOE difference, COSY and NOESYPH techniques. In contrast to Hoechst 33258 which recognizes 5'-AATT sequences exclusively, analogue 2 possesses structural features designed to permit the recognition of GC sites. The NOESY and 1D-NOE experiments place the drug in the minor groove and it is located on the 5'-CCAT sequence. The orientation of the drug in the groove is such as to place the N-methylpiperazine terminus at a GC site. Cross-correlation peaks in the NOESY experiment show that the DNA duplex retains its right-handed B form, similar to that in the free decamer. Specific NOEs locate the benzoxazole moiety on the 5'-CCAT and are consistent with the pyridine nitrogen forming a new hydrogen bond to G(4)-2NH2 at 5'-CCAT. The drug appears to undergo rotation around the C9-C10 bond, at a rate comparable with NMR time scale, even after binding. Variable temperature 1H-NMR studies established that the DNA is thermally stabilized as a result of the drug binding. The drug binding is a dynamic process involving exchange between the equivalent 5'-CCAT sites at approximately 60s-1 with delta G degree of 65 kJ mol-1 at 308K. The experimental evidence is in accord with a slide-swing mechanism for this process.  相似文献   

17.
Nuclear magnetic resonance (NMR) has been used to monitor the conformation and dynamics of the d-(C1-G2-A3-G4-A5-A6-T6-T5-C4-G3-C2-G1) self-complementary dodecanucleotide (henceforth called 12-mer GA) that contains a dG X dA purine-purine mismatch at position 3 in the sequence. These results are compared with the corresponding d(C-G-C-G-A-A-T-T-C-G-C-G) dodecamer duplex (henceforth called 12-mer) containing standard Watson-Crick base pairs at position 3 [Patel, D.J., Kozlowski, S.A., Marky, L.A., Broka, C., Rice, J.A., Itakura, K., & Breslauer, K.J. (1982) Biochemistry 21, 428-436]. The dG X dA interaction at position 3 was monitored at the guanosine exchangeable H-1 and nonexchangeable H-8 protons and the nonexchangeable adenosine H-2 proton. We demonstrate base-pair formation between anti orientations of the guanosine and adenosine rings on the basis of nuclear Overhauser effects (NOE) observed between the H-2 proton of adenosine 3 and the imino protons of guanosine 3 (intra base pair) and guanosines 2 and 4 (inter base pair). The dG(anti) X dA(anti) pairing should result in hydrogen-bond formation between the guanosine imino H-1 and carbonyl O-6 groups and the adenosine N-1 and NH2-6 groups, respectively. The base pairing on either side of the dG X dA pair remains intact at low temperature, but these dG X dC pairs at positions 2 and 4 are kinetically destabilized in the 12-mer GA compared to the 12-mer duplex. We have estimated the hydrogen exchange kinetics at positions 4-6 from saturation-recovery measurements on the imino protons of the 12-mer GA duplex between 5 and 40 degrees C. The measured activation energies for imino proton exchange in the 12-mer GA are larger by a factor of approximately 2 compared to the corresponding values in the 12-mer duplex. This implies that hydrogen exchange in the 12-mer GA duplex results from a cooperative transition involving exchange of several base pairs as was previously reported for the 12-mer containing a G X T wobble pair at position 3 [Pardi, A., Morden, K.M., Patel, D.J., & Tinoco, I., Jr. (1982) Biochemistry 21, 6567-6574]. We have assigned the nonexchangeable base protons by intra and inter base pair NOE experiments and monitored these assigned markers through the 12-mer GA duplex to strand transition.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

18.
The structures of d(GCAATTGC)2 and its complex with berenil in solution were analyzed by two-dimensional 1H NMR spectroscopy. Intra- and internucleotide nuclear Overhauser effect (NOE) connectivities demonstrate that the octanucleotide duplex is primarily in the B conformation. Binding with berenil stabilizes the duplex with respect to thermal denaturation by about 10 degrees C, based on the appearance of the imino proton signals. The berenil-d(GCAATTGC)2 system is in fast exchange on the NMR time scale. The two-dimensional NMR data reveal that berenil binds in the minor groove of d(GCAATTGC)2. The aromatic drug protons are placed within 5 A of the H2 proton of both adenines, the H1', H5', and H5" of both thymidines, and the H4', H5', and H5" of the internal guanosine. The amidine protons on berenil are also close to the H2 proton of both adenines. The duplex retains an overall B conformation in the complex with berenil. At 18 degrees C, NOE contacts at longer mixing times indicate the presence of end-to-end association both in the duplex alone and also in its complex with berenil. These intermolecular contacts either vanished or diminished substantially at 45 degrees C. Two molecular models are proposed for the berenil-(GCAATTGC)2 complex; one has hydrogen bonds between the berenil amidine protons and the carbonyl oxygen, O2, of the external thymines, and the other has hydrogen bonds between the drug amidine protons and the purine nitrogen, N3, of the internal adenines. Quantitative analysis of the NOE data favors the second model.  相似文献   

19.
X L Gao  D J Patel 《Biochemistry》1988,27(5):1744-1751
We report on two-dimensional proton NMR studies of echinomycin complexes with the self-complementary d(A1-C2-G3-T4) and d(T1-C2-G3-A4) duplexes in aqueous solution. The exchangeable and nonexchangeable antibiotic and nucleic acid protons in the 1 echinomycin per tetranucleotide duplex complexes have been assigned from analyses of scalar coupling and distance connectivities in two-dimensional data sets recorded in H2O and D2O solution. An analysis of the intermolecular NOE patterns for both complexes combined with large upfield imino proton and large downfield phosphorus complexation chemical shift changes demonstrates that the two quinoxaline chromophores of echinomycin bisintercalate into the minor groove surrounding the dC-dG step of each tetranucleotide duplex. Further, the quinoxaline rings selectively stack between A1 and C2 bases in the d(ACGT) complex and between T1 and C2 bases in the d(TCGA) complex. The intermolecular NOE patterns and the base and sugar proton chemical shifts for residues C2 and G3 are virtually identical for the d(ACGT) and d(TCGA) complexes. A change in sugar pucker from the C2'-endo range to the C3'-endo range is detected at C2 on formation of the d(ACGT) and d(TCGA) complexes. In addition, the sugar ring protons of C2 exhibit upfield shifts and a large 1 ppm separation between the H2' and H2" protons for both complexes. The L-Ala amide protons undergo large downfield complexation shifts consistent with their participation in intermolecular hydrogen bonds for both tetranucleotide complexes.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

20.
S H Chou  D R Hare  D E Wemmer  B R Reid 《Biochemistry》1983,22(13):3037-3041
Using solid-phase phosphite triester methods, we have synthesized both strands of the phage lambda OR3 DNA sequence, reannealed them, and studied the native operator duplex by high-resolution NMR at 500 MHz. At 7 degrees C the imino protons of the two terminal base pairs at each end have disappeared from the spectrum by exchange broadening. The 13 detectable imino resonances have been assigned to their respective base pairs in the duplex by using sequential nearest-neighbor NOE connectivity methods described previously. In cases where two imino protons overlap in the spectrum, spin diffusion was used to drive the cross-saturation further afield in order to produce second-order next-nearest-neighbor effects. The results show that the imino connectivity method can be used to unambiguously assign the imino proton spectrum of operator DNAs containing one to two full turns of the helix.  相似文献   

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