Structure and Conformation of the Duplex Consensus Acceptor Exon: Intron Junction d [(CpTpApCpApGpGpT) (ApCpCpTpGpTpApG)] deduced from High-Field 1H-NMR of Non-Exchangeable and Imino Protons

Abstract

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°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 nonexchangeable 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.     

1H and 31P-NMR assignments of the non-exchangeable protons of the consensus acceptor exon:intron junction d(CpTpApCpApGpGpT)

The consensus acceptor exon:intron junction d(CpTpApCpApGpGpT) has been synthesized by a modified phosphotriester method. The non-self complementary octamer exists in the single strand 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 the double quantum technique 1H-1H-INADEQUATE as well as inversion recovery T1 experiments. The new technique of 31P-1H shift correlation is particularly valuable in removing certain ambiguities in the sugar proton assignments. Characteristic chemical shifts for the base protons which are determined by their immediate molecular environments are also useful in assignments. The consensus acceptor exon:intron junction adopts a random coil conformation in solution under the experimental conditions employed.     

1H and 31P-NMR Assignments of the Non-exchangeable Protons of the Consensus Acceptor Exon: Intron Junction d(CpTpApCpApGpGpT)

Abstract

The consensus acceptor exon: intron junction d(CpTpApCpApGpGpT) has been synthesized by a modified phosphotriester method. The non-self complementary octamer exists in the single strand form in aqueous buffer at 20°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 the double quantum technique ¹H-¹H-INADEQUATE as well as inversion recovery T1 experiments. The new technique of ³¹P-¹H shift correlation is particularly valuable in removing certain ambiguities in the sugar proton assignments. Characteristic chemical shifts for the base protons which are determined by their immediate molecular environments are also useful in assignments. The consensus acceptor exon: intron junction adopts a random coil conformation in solution under the experimental conditions employed.     

1H-NMR assignments of the non-exchangeable protons of the consensus donor exon:intron junctioń d(CpApGpGpTpApApGpT)

The consensus donor exon:intron junction d(CpApGpGpTpApApGpT) has been synthesized by a modified phosphotriester method. The non-self-complementary nonamer has, in principle, only two G,C or four A,T points of self-recognition. The inference that it exists in the single strand form at 20 degrees C was confirmed by temperature variable 1H-NMR and NOE measurements. The proton assignments were secured using two-dimensional COSY which provided intra-nucleotide correlations, then NOE difference measurements as well as inversion recovery T1 experiments. Systematic procedures were developed for the assignment of the individual bases and their component protons based on the effects of molecular environment on chemical shifts. These latter procedures should be useful for the assignment of other random-coil single strand oligodeoxyribonucleotides.     

1H-NMR Assignments of the Non-exchangeable Protons of the Consensus Donor Exon:Intron Junction d(CpApGpGpTpApApGpT)

Abstract

The consensus donor exon:intron junction d(CpApGpGpTpApApGpT) has been synthesized by a modified phosphotriester method. The non-self-complementary nonamer has, in principle, only two G,C or four A,T points of self-recognition. The inference that it exists in the single strand form at 20°C was confirmed by temperature variable ¹H-NMR and NOE measurements. The proton assignments were secured using two-dimensional COSY which provided intra-nucleotide correlations, then NOE difference measurements as well as inversion recovery T₁ experiments. Systematic procedures were developed for the assignment of the individual bases and their component protons based on the effects of molecular environment on chemical shifts. These latter procedures should be useful for the assignment of other random-coil single strand oligodeoxyribonucleotides.     

alpha-DNA-V. Parallel annealing, handedness and conformation of the duplex of the unnatural alpha-hexadeoxyribonucleotide alpha-[d(CpApTpGpCpG)] with its beta-complement beta-[d(GpTpApCpGpC)] deduced from high field 1H-NMR.

The beta-complementary hexamer, beta-d[GTACGC], to the alpha-sequence, alpha-d[CATGCG], was synthesized by the phosphotriester method. The non-exchangeable proton assignments were obtained using 1D- and 2D-NMR techniques, including NOE, COSY and NOESY. The beta-strand exists as a random coil at 21 degrees C; however, at 4 degrees C, it forms an antiparallel self-recognition duplex annealing at positions 1-4. The beta-strand was annealed to the alpha-strand, and confirmation of complete annealing was obtained by detection and assignment of the six base pair imino protons in H2O/D2O solution at 21 degrees C. 1D-NOE experiments of the alpha, beta duplex d[alpha-(CATGCG) X beta-(GTACGC)] reveal that (i) it exists in aqueous solution in a conformation that belongs to the B family, (ii) it is 70 +/- 10% right-handed, (iii) the sugar-base orientations of the beta-strand are anti, and the deoxyribose units exist predominantly in the 2'-endo-3'-exo conformation. NOE measurements of the imino proton signals in the alpha, beta duplex reveal that the duplex exhibits parallel polarity.     

Complete assignment of signals in 1D and 2D H-NMR spectra of a 17-member oligonucleotide, a model symmetrical analog of lambda operators

A synthetic analog of lambda phage operators, a symmetric duplex of oligonucleotides, was studied by 1H NMR at 400 MHz. Signals in the spectrum of imino protons of the duplex in H2O were assigned based on the results of NOE experiments and temperature dependences. Resonance assignment of the non-exchangeable protons of bases and deoxyribose was performed by analysing the NOESY spectrum obtained in the single experiment. The results indicate that the major part of the duplex has a conformation similar to the B-form of DNA, and the region of the central non-complementary base pair exhibits deviations from the regular structure.     

Two-dimensional nuclear Overhauser enhancement investigation of the solution structure and dynamics of the DNA octamer [d(GGTATACC)]2

The resonances of nearly all 70 of the non-exchangeable protons of the duplex [d(GGTATACC)]2 in aqueous solution are assigned by proton two-dimensional nuclear Overhauser enhancement (2D NOE) spectra obtained in pure absorption phase at 500 MHz. Experimental and theoretical 2D NOE spectra are compared at each mixing time (100, 175, 250 and 400 ms) using two B-DNA structures: a standard B-form and an energy-minimized form. The GG and CC ends of the octamer duplex are well represented by the regular B-DNA structure. But large discrepancies from these models are observed for the 'TATA' box. All 2D NOE data are consistent with nanosecond correlation times, as indicated by non-selective proton spin-lattice relaxation times, but small variations in the correlation time are observed, suggesting that there are some local differences in mobility within the octamer duplex structure in solution.     

NMR solution structure of a DNA dodecamer containing a transplatin interstrand GN7-CN3 cross-link.

The DNA duplex d(CTCTCG*AGTCTC).d(GAGAC-TC*GAGAG) containing a single trans- diammine-dichloroplatinum(II) interstrand cross-link (where G* and C* represent the platinated bases) has been studied by two-dimensional NMR. All the exchangeable and non-exchangeable proton resonance lines were assigned (except H5'/H5") and the NOE intensities were transformed into distances via the RELAZ program. By combining the NOESY and COSY data (330 constraints) and NMR-constrained molecular mechanics using JUMNA, a solution structure of the cross-linked duplex has been determined. The duplex is distorted over two base pairs on each side of the interstrand cross-link and exhibits a slight bending of its axis ( approximately 20 degrees ) towards the minor groove. The platinated guanine G* adopts a syn conformation. The rotation results in a Hoogsteen-type pairing between the complementary G(6)* and C(19)* residues which is mediated by the platinum moiety and is stabilized by a hydrogen bond between O6(G(6)*) and N4H(C(19)*). The rise between the cross-linked residues and the adjacent residues is increased owing to the interaction between these adjacent residues and the ammine groups of the platinum moiety. These results are discussed in relation to the slow rate of closure of the monofunctional adducts into interstrand cross-links.     

Conformational changes in the oligonucleotide duplex d(GCGTTGCG) x d(CGCAACGC) induced by formation of a cis-syn thymine dimer. A two-dimensional NMR study

In order to obtain insight into the repair mechanism of DNA containing thymine photo-dimer, the conformation of the duplex d(GCGTTGCG) x d(CGCAACGC) with a thymine dimer incorporated has been studied by proton NMR and the results are compared with NMR data of the parent octamer. Two-dimensional nuclear Overhauser enhancement (2D NOE) spectroscopy and two-dimensional homonuclear Hartmann-Hahn spectroscopy have been applied to assign all the non-exchangeable base protons and most of the deoxyribose protons of both duplexes. From these experiments it is clear that indeed a cis-syn cyclobutane-type thymine photodimer is formed by the irradiation of this oligonucleotide with ultraviolet light. Comparison of 2D NOE spectra and the 1H chemical shifts of the damaged and the intact DNA duplexes reveals that formation of a thymine dimer induces small distortions of the B-DNA structure, the main conformational change occurring at the site of the thymine dimer.     

alpha-DNA-III. Characterization by high field 1H-NMR, anti-parallel self-recognition and conformation of the unnatural hexadeoxyribonucleotides alpha-[d(CpApTpGpCpG)] and alpha-[d(CpGpCpApTpG)]. Alpha-oligodeoxynucleotides as potential cellular probes for gene control.

High field 2-D-1H-NMR techniques permitted the assignment of all non-exchangeable protons of the unnatural deoxyribonucleotides alpha-[d(CpApTpGpCpG)] and alpha-[d(CpGpCpApTpG)]. 1-D and 2-D NOESY experiments show strong H6H8-H4' dipolar interactions for all nucleotides in both sequences. These data, together with COSY and J-resolved spectra, indicate that these two alpha-oligomers adopt 3'-exo conformations of the sugar moieties in solution with anti conformations of the glycosyl linkages. Both 1H-NMR data, and hypochromocity comparison of alpha-CATGCG and beta-CATGCG, demonstrate a higher degree of base stacking in the case of the alpha-sequence. The UV hyperchromicity at 260 nm, and symmetry considerations in the imino proton NMR experiments reveal antiparallel self-recognition and duplex annealing at positions 1-4 for alpha-[d(CATGCG)] and positions 3-6 for alpha-[d(CGCATG)]. The temperature variation of the imino proton NMR signals suggests that the hydrogen bonding in self-recognition is comparable in strength with that in a beta-DNA duplex, and NOE data are in accord with Watson-Crick rather than Hoogsteen base pairing.     

Structural and dynamic aspects of non-intercalative (1:1) binding of a thiazole-lexitropsin to the decadeoxyribonucleotide d-[CGCAATTGCG]2: An 1H-NMR and molecular modeling study.

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.     

Sequence specific molecular recognition and binding of a monocationic bis-imidazole lexitropsin to the decadeoxyribonucleotide d-[(GATCCGTATG).(CATACGGATC)]: structural and dynamic aspects of intermolecular exchange studied by 1H-NMR

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 imino 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 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 approximately 88s-1 at 308 degrees K with delta G not equal to of 63 +/- 5 KJ mol-1.     

Structure of the anthramycin-d(ATGCAT)2 adduct from one- and two-dimensional proton NMR experiments in solution

One- and two-dimensional 400-MHz proton NMR experiments are used to examine the solution structure of the covalent adduct formed by the interaction of anthramycin methyl ether with the self-complementary deoxyoligonucleotide d(ATGCAT)2. The concentration dependence of chemical shifts and nuclear Overhauser enhancement (NOE) experiments are utilized to assign the adenine H2 protons within the minor groove for both free d(ATGCAT)2 and the adduct. These studies demonstrate that one of the four adenine H2 protons is in close proximity to the bound anthramycin and this results in its upfield shift of 0.3 ppm compared to the adenine H2 protons of the free duplex. Effects of the covalent attachment of anthramycin to the d(ATGCAT)2 duplex result in an increased shielding of selected deoxyribose protons located within the minor groove of the adduct, as demonstrated by two-dimensional autocorrelated (COSY) NMR techniques. Interactions between the protons of the covalently attached anthramycin and the d(ATGCAT)2 duplex are determined by utilizing two-dimensional NOE (NOESY) techniques. Analysis of these data reveals NOE cross-peaks between the anthramycin methyl, H6, and H7 protons with specific deoxyoligonucleotide protons within the minor groove, thus allowing the orientation of the drug within the minor groove to be determined. Nonselective inversion recovery (T1) relaxation experiments are used to probe the structural and dynamic properties of the anthramycin-d(ATGCAT)2 adduct. These data suggest that the binding of anthramycin alters the correlation time of the d(ATGCAT)2 duplex and stabilizes both of the internal A X T base pairs with respect to solvent exchange.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence dependent conformation and local geometry of the conserved branch site sequence element d(TpApCpTpApApC), essential for yeast mRNA splicing, deduced from high resolution 1H-NMR

The conserved sequence element and branch site splice signal d(TpApCpTpApApC) has been synthesized by a solid phase procedure. All the non-exchangeable protons have been assigned using a combination of one-dimensional and two-dimensional 1H-NMR analytical procedures. On the basis of the low NOE intensities in the 1D-NOE and NOESY experiments, the heptamer exists in solution as a random coil. The deoxyribose rings towards the 5' terminus exist predominantly in the S form (2'-endo-3'-exo) while residues on or adjacent to the 2' branch site in the eventual lariat structure [A(6) of TACTAAC] show more N-character (3'endo-2'-exo). In addition unique propeller twisting at contiguous AT base pairs in the consensus 5'-splice site occurs in the region in which there is partial complementarity with the branch splice signal TACTAAC. These subtle structural features, if carried over to the corresponding RNA, may have significance either as a recognition signals or for stereochemical reasons in the formation of the lariat intermediate in the maturation process of mRNA.     

Nuclear magnetic resonance studies of complex formation between the oligonucleotide d(TATC) covalently linked to an acridine derivative and its complementary sequence d(GATA)

The oligodeoxynucleotide d(TATC) was covalently attached to the 9-amino group of 2-methoxy-6-chloro-9-aminoacridine (Acr) through its 3'-phosphate via a pentamethylene linker (m5). Complex formation between d(TATC)m5Acr and the complementary strand d(GATA) in aqueous solution was investigated by nuclear magnetic resonance. The COSY and NOESY connectivities allowed us to assign all the proton resonances of the bases, the sugars (except the overlapping 5'/5' resonances), the acridine, and the pentamethylene chain. Structural informations derived from relative intensities of COSY and NOESY maps revealed that the duplex d(TATC)-d(GATA) adopts a B-type conformation and that the deoxyriboses preferentially adopt a 2'-endo conformation. The NOE connectivities observed between the protons of the bases or of the sugars and the protons of the dye and of the pentamethylene chain led us to propose a model involving an equilibrium between two families of configurations. In the first family, the acridine derivative is intercalated between base pairs C4-G4 and T3-A3. In the second family, the acridine derivative is sandwiched between two aggregated duplexes. The structure of the intercalated complex as well as that of the aggregated species is discussed.     

Sequential resonance assignments in 1H NMR spectra of oligonucleotides by two-dimensional NMR spectroscopy

A sequential assignment procedure is outlined, based on two-dimensional NOE ( NOESY ) and two-dimensional J-correlated spectroscopy ( COSY ), for assigning the nonexchangeable proton resonances in NMR spectra of oligonucleotides. As presented here the method is generally applicable to right-handed helical oligonucleotides of intermediate size. We applied it to a lac operator DNA fragment consisting of d( TGAGCGG ) and d( CCGCTCA ) and obtained complete assignments for the adenine H8, guanine H8, cytosine H6 and H5, thymine H6 and 5-methyl, and the deoxyribose H1', H2', H2", H3', and H4' resonances, as well as some H5', H5" (pairwise) assignments. These assignments are required for the analysis of two-dimensional NOE and J-coupling data in terms of the solution structure of oligonucleotides.     

Conformational transitions in cytidine bulge-containing deoxytridecanucleotide duplexes: extra cytidine equilibrates between looped out (low temperature) and stacked (elevated temperature) conformations in solution

High-resolution homonuclear and heteronuclear two-dimensional NMR studies have been carried out on the self-complementary d(C-C-G-C-G-A-A-T-T-C-C-G-G) duplex (designated GCG 13-mer) in aqueous solution. This sequence contains an extra cytidine located between residues G3 and G4 on each strand of the duplex. The exchangeable and nonexchangeable proton resonances have been assigned from an analysis of two-dimensional nuclear Overhauser enhancement (NOESY) and correlated (COSY and relay COSY) spectra for the GCG 13-mer duplex in H2O and D2O solution. The extra cytidine at the bulge site (designated CX) results in more pronounced changes in the NOE distance connectivities for the G3-CX-G4 segment centered about the CX residue compared to the C9-C10 segment on the partner strand opposite the CX residue for the GCG 13-mer duplex at 25 degrees C. The cross-peak intensities in the short mixing time NOESY spectrum also establish that all glycosidic torsion angles including that of CX are anti in the GCG 13-mer duplex at 25 degrees C. The observed chemical shift changes for the CX base protons and the G3pCX phosphorus resonance with temperature between 0 and 40 degrees C demonstrate a temperature-dependent conformational equilibrium in the premelting transition region. The NOE and chemical shift parameters establish that the predominant conformation at low temperature (0 degree C) has the extra cytidine looped out of the helix with the flanking G3.C10 and G4.C9 base pairs stacked on each other. These results support conclusions based on earlier one-dimensional NMR studies of extra cytidine containing complementary duplexes in aqueous solution [Morden, K. M., Chu, Y. G., Martin, F. H., & Tinoco, I., Jr. (1983) Biochemistry 22, 5557-5563. Woodson, S. A., & Crothers, D. M. (1987) Biochemistry 26, 904-912]. By contrast, the chemical shift and NOE parameters demonstrate that the conformational equilibrium shifts toward a structure with a stacked extra cytidine on raising the temperature to 40 degrees C prior to the helix-coil melting transition. The most downfield shifted phosphorus resonance in the GCG 13-mer duplex has been assigned to the phosphate in the C2-G3 step, and this observation demonstrates that the perturbation in the phosphodiester backbone extends to regions removed from the (G3-CX-G4).(C9-C10) bulge site.