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

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.     

Complete 1H assignments of the non-exchangeable protons of the non self-complementary heptadeoxyribonucleotide d[(GTCGTCA).(TGACGAC)] and its component strands by high field NMR

The non self complementary heptadeoxyribonucleotides d(GTCGTCA) and d(TGACGAC) were synthesized by the phosphotriester method. While complete 1H-NMR assignments of the former were obtained by a combination of one and two-dimensional techniques at room temperature, extensive stacking of the latter under these conditions dictated analysis at 50 degrees C when the lines were sharply resolved. The duplex form of the annealed strands under the conditions of the 1H-NMR experiment was established independently of the NMR evidence by 32P end labeling with T4 polynucleotide kinase followed by butt end joining using the absolute specificity of T4 ligase for double strand DNA. Analysis of the resulting ladder of polymers was performed using gel electrophoresis and autoradiography. Complete 1H-NMR assignments of the non-exchangeable protons in the self complementary heptamer was achieved. The assignments were confirmed using NOE differences, and two-dimensional COSY, and HH-INADEQUATE experiments at 400 and 500 MHz. The assignments are in accord with a conformation for the heptamer belonging to the B family of structures.     

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.     

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.     

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.     

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-NMR study of the OR1 operon in bacteriophage lambda. I. Assignment of signals from imino protons and adenine C2 protons

An oligodeoxyribonucleotide composed of 17 residues, d(TATCACCGCCAGAGGTA), and a complementary chain were synthesized. Their duplex was identical with the operator OR1, the binding site for bacteriophage lambda cro and c1 repressors. The 1H NMR spectra (500 MHz) of the duplex imino and aromatic protons were studied at 10, 20 and 25 degrees C. Signals from the imino protons of complementary base pairs and from the C2 protons of adenine (with the exception of the duplex terminal nucleotides) were assigned using the NOE technique and the known characteristics of short DNA fragment melting. No signals from the imino protons of the terminal base pairs were detected even at 10 degrees C due to fraying which increased as the temperature was raised. The assignment of signals can be used to identify centers of interaction between the operator OR1 and repressors, as well as to study possible local changes in DNA geometry.     

High field 1H-NMR analysis of the 1:1 intercalation complex of the antitumor agent mitoxantrone and the DNA duplex [d(CpGpCpG)

Complete 1H-nmr assignment has been achieved of the stoichiometric 1:1 complex of the antitumor agent mitoxantrone with the duplex oligomer [d(CpGpCpG)]2. The techniques used included 2D-COSY, 1D-NOE and 2D-HH-INADEQUATE. Comparisons of 1H and 13C chemical shift changes upon addition of drug suggest symmetrical intercalative binding to the center of the tetramer. NOE difference measurements and 31P studies suggest binding of the terminal OH groups of the side chains to the central phosphate groups such that the methylene groups are proximate to C(3)6, C(3)6 and G(4)8 base protons all in the major groove. The data suggest that the side chains bind to the neighboring base pairs from the intercalation site. This is in accord with independent evidence of G,C base preference for binding from spectroscopic and electron microscopy studies.     

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.     

1H-NMR study of the lambda operator site OL1: assignment of the imino and adenine H2 resonances.

One- and two-dimensional proton NMR methods are being used to study the synthetic lambda operator site O-L1, a 17 base-pair DNA duplex recognized by lambda repressor and Cro protein. The complete assignment of the 17 imino protons, which participate in Watson-Crick hydrogen bonding, and of the eight adenine H2 protons, which lie in the minor groove of the double helix, is presented.     

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 and conformation of the branch core triribonucleotide containing 2'-5' and 3'-5' phosphodiester linkages (A 2'p5'G 3'p5'C) i solution, essential for yeast mRNA splicing, deduced from 1H-NMR

The non-exchangeable 1H-NMR signals of the branch core trinucleotide of the lariat branch site (A2'p5'G3'p5'C, 1) and its derivatives 2 and 3 are completely assigned using one- and two-dimensional NMR techniques including NOE, COSY, NOESY, 1H-1H INADEQUATE and 2D-J-resolved spectroscopy. From the vicinal coupling constants in the individual ribose rings, NOE data and T1 measurements, the following properties of the trimers are deduced. (i) The unique stacking behavior of the trimers is S2'N3'N, and the sugar rings exist predominantly in the N-conformation (3'-endo-2'-exo). (ii) The sugar-base orientations appear to be anti. (iii) The branched trimers exist in solution as single-stranded right-handed conformations resembling A-RNA with stacking between the adenine and guanine residues in aqueous solution at 21 degrees C and pH 7.2. (iv) The calculated values for the torsion angles epsilon t and gamma+ for the trimers are 201-203 degrees and 71-86%, respectively, while the percent beta t values are higher for the guanine (87-92%) than the cytosine residues (73-77%). The computer generated depiction of the triribonucleotide 1 is also shown. These subtle structural features may act as recognition signals for this critical lariat branch site which is essential for the second step in yeast mRNA splicing.     

Extrahelical adenosine stacks into right-handed DNA: solution conformation of the d(C-G-C-A-G-A-G-C-T-C-G-C-G) duplex deduced from distance geometry analysis of nuclear Overhauser effect spectra

This paper reports on features of the three-dimensional structure of the d(C-G-C-A-G-A-G-C-T-C-G-C-G) self-complementary duplex (designated adenosine 13-mer), which contains symmetrical extrahelical adenosines in the interior of the helix. The majority of the protons have been assigned from two-dimensional nuclear Overhauser effect (NOESY) spectra of the adenosine 13-mer in H2O and D2O solution. The measurement of NOESY cross-peak volume integrals as a function of mixing time has yielded a set of 96 short (less than 4.5-A) proton-proton distances defined by lower and upper bounds, which have served as input parameters for a distance geometry analysis of one symmetric half of the adenosine 13-mer duplex. We demonstrate that the extrahelical adenosine stacks into the duplex for all refined structures without disruption of base pairing on either side of the modification site. The distance geometry refinement yields two classes of conformations consistent with distance measurements but which differ in orientation of the stacked extrahelical adenosine at the modification site.     

Synthesis and characterization of the diastereomers Lambda- and Delta-[Ru(bpy)2(m-bpy-L-Arg-Gly-L-Asn-L-Ala-L-His-L-Glu-L-Arg)]Cl2 1H NMR studies on their interactions with the deoxynucleotide duplex d[(5'-GCGCTTAAGCGC-3')2] and d[(5'-CGCGATCGCG-3')2

The diastereomeric complexes Lambda- and Delta-[Ru(bpy)(2)(m-bpy-7p)]Cl(2), (bpy=2,2'-bipyridine, m-bpy-7p=4-methyl-4'-Arg-Gly-Asn-Ala-His-Glu-Arg-CONH(2)-2,2'-bipyridine) were synthesized and characterized and their binding properties to the deoxynucleotide duplexes d(5'-CGCGATCGCG-3')(2) and d(5'-GCGCTTAAGCGC-3')(2) were studied by means of (1)H NMR spectroscopy. 7p is part of the recognition loop of the restriction endonuclease MunI, a type II restriction enzyme from Mycoplasma unidentified which recognizes the palindromic hexanucleotide sequence C/AATTG and cleaves it as indicated by the slash. The Delta-isomer binds to the terminal CG/GC major groove of d(CGCGATCGCG)(2) decanucleotide, whereas the Lambda-isomer approaches the GCT/CGA sequence. On the other hand, weak binding of the Delta-isomer to the end of d(GCGCTTAAGCGC)(2) into two different orientations is observed. In the case of the Lambda-isomer, the bpy ligand(s) are located into the major groove of the central TT/AA sequence. The role of appended peptide sequences in sequence selectivity binding to DNA is being addressed.     

Sequence specific molecular recognition by a monocationic lexitropsin of the decadeoxyribonucleotide d-[CATGGCCATG]2: structural and dynamic aspects deduced from high field 1H-NMR studies.

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.     

1H-NMR study of the quadruplex [d(TGGGT)]4 containing a modified thymine

A NMR structural study of quadruplex [d(TGGGT)]4 containing a modified thymine is reported. The three dimensional structure of the complex is very similar to those of other parallel stranded quadruplexes. The modified thymines (T*) are able, at least in the minimised structures, to form a tetrad containing extra H-bonds through the hydroxyl groups. Nevertheless, in this new tetrad the modified thymines are slightly open towards the solvent respect to the unmodified T-tetrad.     

alpha-DNA. I. Synthesis, characterization by high field 1H-NMR, and base-pairing properties of the unnatural hexadeoxyribonucleotide alpha-[d(CpCpTpTpCpC)] with its complement beta-[d(GpGpApApGpG)].

The novel deoxyribonucleotide alpha-[d(CpCpTpTpCpC)] and its complement beta-[d(GpGpApApGpG)] were synthesized by the phosphotriester method. 1H-NMR-NOE examination of the alpha-hexamer revealed that the cytosine and thymine bases appear to adopt anti conformations in this strand. In addition the deoxyribose of the thymidine moieties may adopt average conformations approximating to C3'-endo while the cytidine furanose groups are close to C2'-endo conformations. Both hyperchromicity in thermal melting and detection of base paired imino protons in 1H-NMR studies in H2O provide evidence for the annealing of alpha-d[CCTTCC] with its complement beta-d[GGAAGG] in potassium phosphate buffer pH 7.1 containing 10 mM magnesium chloride. Under these conditions thermal melting begins at 38 degrees C and its complete at approximately 45 degrees C. NOE experiments do not permit a decision on the polarity of annealing (predicted to be parallel) for this particular pair of sequences.     

Wobble dG X dT pairing in right-handed DNA: solution conformation of the d(C-G-T-G-A-A-T-T-C-G-C-G) duplex deduced from distance geometry analysis of nuclear Overhauser effect spectra

We report below on features of the three-dimensional structure of the d(C-G-T-G-A-A-T-T-C-G-C-G) self-complementary duplex (designated 12-mer GT) containing symmetrical G X T mismatches in the interior of the helix. The majority of the base and sugar protons in the 12-mer GT duplex were assigned by two-dimensional nuclear Overhauser effect (NOESY) spectra in H2O and D2O solution. A set of 92 short (less than 4.5-A) proton-proton distances defined by lower and upper bounds for one symmetrical half of the 12-mer GT duplex were estimated from NOESY data sets recorded as a function of mixing time. These experimental distances combined with nucleotide bond length parameters were embedded into Cartesian space; several trial structures were refined to minimize bond geometry and van der Waals and chirality error. Confidence in this approach is based on the similarity of the refined structures for the solution conformation of the 12-mer GT duplex. The G and T bases pair through two imino-carbonyl hydrogen bonds, and stacking is maintained between the G X T wobble pair and adjacent Watson-Crick G X C pairs. The experimental distance information is restricted to base and sugar protons, and hence structural features such as base pair overlap, glycosidic torsion angles, and sugar pucker are well-defined by this combination of NMR and distance geometry methods. By contrast, we are unable to define the torsion angles about the bonds C3'-O3'-P-O5'-C5'-C4' in the backbone of the nucleic acid.     

1H NMR study of the exchangeable protons of the duplex d(GCGTTGCG).d(CGCAACGC) containing a thymine photodimer.

A comparison is presented of the imino proton NMR spectra of the double stranded octamer d(GCGTTGCG).d(CGCAACGC) and the same octamer in which the two central thymine residues occur as a cis-syn thymine dimer. Except for the terminal base pairs all imino protons were detected and assigned in the NMR spectrum. The spectra show that in the thymine dimer duplex, contrary to common belief, all base pairs occur in a hydrogen bonded form, although the hydrogen bonds of the two central AT base pairs are substantially weakened. The melting temperature decreases about 13 degrees C on thymine dimer formation.     

The effect of two antipodal fluorine-induced sugar puckers on the conformation and stability of the Dickerson-Drew dodecamer duplex [d(CGCGAATTCGCG)]2.

UV thermal melting studies, CD and NMR spectroscopies were employed to assess the contribution of antipodal sugar conformations on the stability of the canonical B-DNA conformation of the Dickerson-Drew dodecamer duplex [[d(CGCGAATTCGCG)]2, (ODN 1)]. Different oligodeoxynucleotide versions of ODN 1 were synthesized with modified thymidine units favoring distinct sugar conformations by using a 3'- endo (north) 2'-fluoro-2'-deoxyribofuranosyl thymine (1) or a 2'- endo (south) 2'-fluoro-2'-deoxyarabinofuranosyl thymine (2). The results showed that two south thymidines greatly stabilized the double helix, whereas two north thymidines destabilized it by inducing a more A-like conformation in the middle of the duplex. Use of combinations of north and south thymidine conformers in the same oligo destabilized the double helix even further, but without inducing a conformational change. The critical length for establishing a detectable A-like conformation in the middle of a B-DNA ODN appears to be 4 bp. Our results suggest that manipulation of the conformation of DNA in a sequence-independent manner is possible.