Parallel stranded DNA.

A series of four hairpin deoxyoligonucleotides was synthesized with a four-nucleotide central loop (either C or G) flanked by the complementary sequences d(T)10 and d(A)10. Two of the molecules contain either a 3'-p-3' or 5'-p-5' linkage in the loop, so that the strands in the stem have the same, that is, parallel (ps) polarity. The pair of reference oligonucleotides have normal phosphodiester linkages throughout and antiparallel (aps) stem regions. All the molecules adopt a duplex helical structure in that (i) the electrophoretic mobilities in polyacrylamide gels of the ps and aps oligomers are similar. (ii) The ps hairpins are substrates for T4 polynucleotide kinase, T4 DNA ligase, and Escherichia coli exonuclease III. (iii) Salt-dependent thermal transitions are observed for all hairpins, but the ps molecules denature 10 degrees C lower than the corresponding aps oligomers. (iv) The ultraviolet absorption and circular dichroism spectra are indicative of a base-paired duplex in the stems of the ps hairpins but differ systematically from those of the aps counterparts. (v) The bis-benzimidazole drug Hoechst-33258, which binds in the minor groove of B-DNA, exhibits very little fluorescence in the presence of the ps hairpins but a normal, enhanced emission with the aps oligonucleotides. In contrast, the intercalator ethidium bromide forms a strongly fluorescent complex with all hairpins, the intensity of which is even higher for the ps species. (vi) The pattern of chemical methylation is the same for both the ps and aps hairpins. The combined results are consistent with the prediction from force field analysis of a parallel stranded right-handed helical form of d(A)n.d(T)n with a secondary structure involving reverse Watson-Crick base pairs and a stability not significantly different from that of the B-DNA double helix. Models of the various hairpins optimized with force field calculations are described.

Primer-mediated enzymatic amplification of cytomegalovirus (CMV) DNA. Application to the early diagnosis of CMV infection in marrow transplant recipients.

A nucleic acid amplification procedure, the polymerase chain reaction (PCR), has been used to establish a diagnostic assay for the identification of cytomegalovirus (CMV) immediate-early sequences in clinical specimens. Preliminary testing against virus-infected cell cultures indicated that the PCR assay was highly CMV-specific, recognizing both wild-type and laboratory strains of CMV. There was no cross-reactivity with human DNA or with DNA from other herpes viruses. The sensitivity of the assay, using cloned CMV AD169 Eco RI fragment-J as template, was 1 viral genome per 40,000 cells. In a prospective study of CMV infection in bone marrow transplant recipients, the PCR assay correctly identified four patients with confirmed CMV infection. In three of these patients who were followed longitudinally, correlation of DNA reactivity with CMV culture and CMV antibody status over time indicated that DNA was the most sensitive marker for the diagnosis of CMV infection.

Solution structure of an arabinonucleic acid (ANA)/RNA duplex in a chimeric hairpin: comparison with 2'-fluoro-ANA/RNA and DNA/RNA hybrids.

Hybrids of RNA and arabinonucleic acid (ANA) as well as the 2'-fluoro-ANA analog (2'F-ANA) were recently shown to be substrates of the enzyme RNase H. Although RNase H binds to double-stranded RNA, no cleavage occurs with such duplexes. Therefore, knowledge of the structure of ANA/RNA hybrids may prove helpful in the design of future antisense oligonucleotide analogs. In this study, we have determined the NMR solution structures of ANA/RNA and DNA/RNA hairpin duplexes and compared them to the recently published structure of a 2'F-ANA/RNA hairpin duplex. We demonstrate here that the sugars of RNA nucleotides of the ANA/RNA hairpin stem adopt the C3'-endo (north, A-form) conformation, whereas those of the ANA strand adopt a 'rigid' O4'-endo (east) sugar pucker. The DNA strand of the DNA/RNA hairpin stem is flexible, but the average DNA/RNA hairpin structural parameters are close to the ANA/RNA and 2'F-ANA/RNA hairpin parameters. The minor groove width of ANA/RNA, 2'F-ANA/RNA and DNA/RNA helices is 9.0 +/- 0.5 A, a value that is intermediate between that of A- and B-form duplexes. These results rationalize the ability of ANA/RNA and 2'F-ANA/RNA hybrids to elicit RNase H activity.

Inhibition of c-myc expression by phosphorothioate antisense oligonucleotide identifies a critical role for c-myc in the growth of human breast cancer.

In search of critical genes in the mechanism of estrogen action in human breast cancer, we previously showed that estrogen stimulates transcription of the c-myc gene in estrogen-dependent (MCF-7) cells. We have now examined the role of c-myc in estrogen-stimulated growth of MCF-7 cells through the use of a synthetic antisense c-myc phosphorothioate oligonucleotide to specifically inhibit expression of the c-myc protein. Estrogen induces a 5-fold increase in c-myc protein expression within 90 min in steroid-deprived cells, as detected by Western blot. Prior exposure of MCF-7 cells to 10 microM c-myc antisense oligonucleotide results in up to 95% inhibition of the c-myc protein expression induced by estrogen. Antisense-myc oligonucleotide inhibits estrogen-stimulated cell growth by up to 75% over 9 days and also exerts a cytostatic effect on the growth of estrogen-independent MDA-MB-231 cells which show relatively high, constitutive expression of c-myc. Sense-myc and antisense-pS2 oligonucleotides have no effect on c-myc protein level or growth in either cell line. These results demonstrate both the specific and durable effects of antisense phosphorothioate oligonucleotides. Furthermore, these results indicate a critical role for c-myc in the growth of breast cancer cells and support the hypothesis that loss of estrogen regulation of this gene may be an important factor in the progression of breast cancer.

Large-scale synthesis of "Cpep" RNA monomers and their application in automated RNA synthesis.

Small interfering RNAs (siRNA) are the latest candidates for oligonucleotide-based therapeutics. Should siRNA be successful in clinical trials, a huge demand for synthetic RNA is anticipated. We believe that 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep) is an ideal 2'-protecting group for large-scale syntheses. Unlike 2'-silyl groups, mild acid hydrolysis instead of fluoride ion is used for the 2'-deprotection. The syntheses of 2'-Cpep protected nucleosides (A, C, G, and U) has been accomplished on a 0.5 Kg scale. The 2'-Cpep monomers were transformed into 3'-O-phosphoramidites for conventional automated solid-phase synthesis. Cost-effective processes for large-scale synthesis of Cpep monomers and initial automated solid-phase synthesis are demonstrated.

Derivatization of controlled pore glass beads for solid phase oligonucleotide synthesis.

An improved and simplified procedure for the attachment of nucleosides onto long chain alkylamine controlled pore glass beads (LCAA-CPG) is presented. This procedure uses 1-(3-dimethylamino-propyl)-3-ethylcarbodiimide (DEC) to couple nucleoside 3'-succinates directly to the LCAA-CPG. The preparation of nucleoside 3'-succinate anhydrides, p-nitrophenyl, or pentachlorophenyl esters and the use of highly toxic dicyclohexylcarbodiimide (DCC) is no longer required. Procedures involving acidic activation of the LCAA-CPG before derivatization and a pre-synthesis capping are also described, which prevent the formation of oligonucleotides linked by 3'-phosphates to the LCAA-CPG. Evidence is presented indicating that this type of linkage is responsible for the apparently greater than 100% coupling yields observed for the first coupling cycle.

A survey of nucleic acid services in core laboratories.

Core facility services related to DNA synthesis and sequencing were surveyed by the Association of Biomolecular Resource Facilities. Responses from 85 facilities offering DNA synthesis and 37 facilities offering DNA sequencing were obtained. Data on instrumentation, volume, number of users, cost, methodology and a number of other criteria were obtained. The volume of work performed by these centralized core facilities was quite substantial (combined synthesis output of 4 million bases per year and a combined sequencing output of 35 million bases per year). The large number of users supported by these facilities and the high sample throughput make these core resource facilities good indicators of technological trends.

Multi-facility survey of oligonucleotide synthesis and an examination of the performance of unpurified primers in automated DNA sequencing.

The purity of 208 crude synthetic 25- and 50-base oligonucleotides synthesized in 71 DNA core facilities was assessed by capillary electrophoresis (CE), and the average coupling efficiency of each synthesis was determined. The median average coupling efficiencies of the 25-mers and 50-mers were 98.9% and 98.7%, respectively, and 85% of the samples exceeded the minimum industry standard of 98% average coupling efficiency. The overall yields estimated by on-line trityl monitors showed poor agreement with the empirically determined yield, and accuracy of the monitors decreased as synthesis efficiency decreased. The performance of the unpurified 25-base oligonucleotides, ranging in purity from 14% to 94%, as primers for automated DNA sequencing was evaluated. Over 85% of these oligonucleotides exhibited an unedited sequencing accuracy of > 97.5% over the 400-base test sequence. Surprisingly, sequencing performance was not strictly related to primer purity, though a marked loss of performance was observed for primers < or = 70% pure (< or = 98.5% coupling efficiency). Thus, the vast majority of the oligonucleotides synthesized by the 71 core facilities participating in this study were of high quality and performed well as sequencing primers without post-synthesis purification or desalting. Finally, our results suggest that an increase in the standard minimum performance specifications of DNA synthesis instruments and reagents from > or = 98% to > or = 98.5% average coupling efficiency, or the development of rapid, inexpensive and efficient methods to detect syntheses below the 98.5% threshold, could obviate post synthesis purification of sequencing primers.

Design strategies and performance of custom DNA sequencing primers.

This study surveyed strategies of sequencing primer selection and evaluated primer performance in automated DNA sequencing. We asked participants to relate their preferred primer design strategies to identify primer characteristics that are considered most important in sequencing primer design. The participants preferred primers of 18-24 nucleotides (nt), 39%-58% G + C, a melting temperature (Tm) of 53 degrees-65 degrees C with a 1-2 nt 3' GC clamp, hairpin stems of less than 2-3 bp, homopolymeric runs of less than 4-5 nt, primer dimers of less than 3-4 bp and secondary priming sites of less than 3-4 bp. We provided a 300-bp test sequence and asked participants to submit sequences of 1-3 optimal sequencing primers. Submitted primers ranged from 17-24 nt and largely conformed to the preferred parameters. Submitted primers were distributed across the test sequence, although some sites were disfavored. Surprisingly, approximately 45% of the primers were selected "manually", more than by any software package. Each of 69 submitted and 95 control primers, distributed at 3-bp intervals across the test sequence, were synthesized, purified and tested using a Model 377 PRISM DNA Sequencer with dichlororhodamine dye terminator reagents (dRhodamine dye terminators). Approximately half of the control primers were also tested using rhodamine dye terminator reagents ("old" rhodamine dye terminators). The results indicated that primer physico-chemical characteristics thought to have a strong impact on sequencing performance had surprisingly little effect. Thus, primers with high or low percent G + C or Tm, strong secondary priming scores or long 3' homopolymeric stretches yielded excellent sequences with the dRhodamine dye terminator reagents, although these characteristics had a stronger effect when the old rhodamine reagents were used. The old rhodamine reagents gave sequences with a similar average read length, but the number of errors and ambiguities or "N's" was consistently higher. Moreover, the effects of the primer physico-chemical characteristics were also more evident with the old rhodamine dyes. We conclude that under optimal sequencing conditions with highly pure template and primer, many of the commonly applied primer design parameters are dispensable, particularly when using one of the new generation of sequencing reagents such as the dichlororhodamine dye terminators.

Accuracy of automated DNA sequencing: a multi-laboratory comparison of sequencing results.

A double-stranded (ds)DNA template of "unknown" sequence was distributed to approximately 80 core DNA sequencing laboratories by the Association of Biomolecular Resource Facilities (ABRF) for automated DNA sequence analysis. Forty-four different facilities responded with 83 usable sequence submissions. These sequences were grouped by both sequencing protocol (dye-primer or dye-terminator) and whether manually edited or not. The sequences were aligned with the known sequence, and the number of correct base calls, insertions, deletions, no-calls and miscalls were determined for each group. The dye-primer sequencing protocol provided the longest and most accurate sequence. The edited dye-primer data were > 95% accurate out to 400-450 bp, while the edited dye-terminator data could call only 300-350 bases at this accuracy. However, 75% of the laboratories in this sampling preferred the dye-terminator protocol, presumably because of its versatility and convenience. Laboratories that manually edited the automatically called data were able to obtain an additional 100 bases of good sequence when the dye-primer protocol was used. Surprisingly though, editing of dye-terminator results did not increase the amount of good sequence, although the dye-terminator protocol had a superior base-calling ability within the first 100 bases of called sequence.

Structural and dynamic aspects of the sequence specific binding of netropsin and its bis-imidazole analogue on the decadeoxyribonucleotide d-[CGCAATTGCG]2.

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.

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.

Conformational characteristics of high affinity Sp1 binding enhancer elements of HIV-LTR by high resolution 2D-NMR.

The understanding of early events in the expression of genes has vastly improved in recent years with the identification of a variety of gene- and sequence-specific DNA binding transcription factors. One such protein, Sp1, has been implicated in activating transcription of various cellular and viral genes including those of HIV, and SIV types of retroviruses. The basic recognition site for Sp1 has been identified as variants of a 10 base-pairs long GC-rich DNA, often containing a hexanucleotide segment 5'-GGGCGG (termed GC-box). However, variations in both the relative protein-DNA binding affinity and the nature of binding sequences have been noted. Two-dimensional 1H-NMR experiments (500 MHz) were employed for conformational studies of two decadeoxyribonucleotide duplexes, d(GAGGCGTGGC).d(GCCACGCCTC), termed Sp1-III, and d(GGGAGTGGCG).d(CGCCACTCCC), termed Sp1-I. These are two of the highest affinity Sp1 binding sites and consist of diverse positioning of the tri- and tetranucleotide segments GAG, GTG, GCG, GGCG, GTGG and GGAG, that occur frequently in other Sp1 binding sites as well, and may form specific contacts with the protein. Phase-sensitive nuclear Overhauser enhancement (2D-NOESY and MINSY) and correlation (COSY) spectra were obtained for the assignment of the exchangeable and nonexchangeable protons in a sequence-specific fashion. As a prelude to determination of the detailed solution structures of the selected sequences, numerous structural constraints were obtained from angle-dependent coupling constants and relative intensities of distance-dependent intra- and internucleotide NOEs. Overall, each duplex adopts a structure similar to B-DNA with predominantly C2'-endo/S-type sugar conformation and anti-glycosidic torsion angles. A selective disruption of sequential NOE connectivities at the GAG.CAC and GTG.CAC steps, irrespective of the flanking sequence, suggests that conformational changes at these sites may act as unique determinants of sequence specific recognition/binding of Sp1. Implications for a specific inhibition of Sp1-mediated transcription by minor groove binding class of drugs, designed to recognize GC-rich sequences, are also briefly discussed.

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.

High resolution 2D-NMR studies indicating complete assignments and conformational characteristics of the NF-kappa B binding enhancer element of HIV-LTR.

The asymmetrical DNA duplex [5'd(AAGGGACTTTCC)].[5'-d(GGAAAGTCCCTT)] has been studied by one- and two-dimensional NMR techniques. The sequence is comprised of the actual 10 base-pair long binding site for the transcription factor NF-kappa B in the enhancer sequence of the long term repeat (LTR) region of HIV and SIV types of retroviruses associated with the AIDS syndrome. Two additional A.T base-pairs are also included on one end for an added interest in the 12-bp duplex sequence with a pseudo dyad-symmetric disposition of the oligopurine and oligopyrimidine segments, as it appears in the HIV-1 genome. Phase-sensitive two-dimensional spectra (NOESY, ROESY, COSY and TOCSY) were obtained at three different temperatures (5, 15 and 25 degrees C) for a complete assignment of the non-exchangeable protons by tracing through sequence specific intra- and internucleotide connectivities. 2D-NOESY spectra were also acquired in aqueous (90% H2O-D2O) solutions, with two different methods of water signal suppression, to assign the exchangeable protons from specific NOE correlations. Adenine H2 protons were assigned by the use of NOE correlations and from T1 relaxation time measurements. The general spectral features and semi-quantitative interproton distance estimates indicate a B-DNA type conformation. However, some distinctly unusual features associated with the nucleotides at and immediately adjacent to both the 5'-and 3'-ends of AAA/TTT and GGG/CCC segments were noted. The complete assignments, and the observed characteristics, will be of significant value in studying the complexes of this transcriptionally active DNA domain with the protein and other rationally designed DNA binding agents.

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 by a GC recognizing Hoechst 33258 analogue to the decadeoxyribonucleotide d-[CATGGCCATG]2: structural and dynamic aspects deduced from high field 1H-NMR studies.

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.

Structure and conformation of the duplex consensus 5'-splice site d[(CpApGpGpTpApApGpT).(ApCpTpTpApCpCpTpG)] deduced from high field 1H-NMR of the non-exchangeable and imino protons.

The complementary consensus donor exon intron junction d(ApCpTpTpApCpCpTpG) has been synthesized by a solid phase procedure. The non-exchangeable proton assignments were obtained using one- and two-dimensional NMR techniques including NOE, COSY, NOESY and 1H-1H-INADEQUATE. The non self-complementary nonamer exists as a random coil form in aqueous buffer at 21 degrees C as evidenced by the temperature variable 1H-NMR and NOE measurements. The nonamer was annealed to the primary consensus donor junction d(CpApGpGpTpApApGpT) and confirmation of complete annealing was obtained by detection and assignment of base pair imino protons in D2O/H2O mixtures. Application of one- and two-dimensional NMR techniques permitted the complete assignment of all the non-exchangeable protons in the duplex nonamer. These data, together with determination of vicinal coupling constants in the individual deoxyribose moieties, permits the following conclusions on the structure and conformation of the consensus donor junction: (i) it exists in aqueous solution in a conformation that belongs to the B family (ii) the sugar-base orientations are anti (iii) the deoxyribose units exist predominantly in the S conformation (2'-endo-3'-exo) (iv) the contiguous A.T base pairs d[T(5)-A(6)-A(7)].d[T(12)-T(13)-A(14)], two positions removed downstream from the splice site (5'-CAG decreases GTAAGT-3'), are uniquely propeller twisted. The propeller twisting occurs in the region in which there is partial complementarity with the branch site splice signal TACTAAC. The cross-correlation rates were used to derive the interproton distances between adjacent AH2 protons of 4.00 A in the T(5)-A(6).T(13)-A(14) step and of 3.87 A in the A(6)-A(7).T(12)-T(13) step. This structural and conformational feature if carried over into the primary RNA transcripts may serve as a recognition signal for this critical site in the genome.

Molecular recognition and binding of a GC site-avoiding thiazole-lexitropsin to the decadeoxyribonucleotide d-[CGCAATTGCG]2: 1H-NMR evidence for thiazole intercalation.

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.

Multiple oligonucleotide synthesis in tandem on solid-phase supports for small and large scale synthesis.

Multiple oligonucleotides linked end-to-end in tandem can be synthesized by adding a nucleoside to the 5'-OH end of a prior sequence. Nucleosides with 3'-succinyl or Q-Linker arms are coupled with HBTU/DMAP. Alternatively, new phosphoramidite reagents with 3'-ester linkages can be used. Hydroxyl or amino supports can also be used as universal starting materials. Treatment with NH4OH cleaves the 3'-ester to yield only 3'-OH groups and no unwanted 3'-phosphorylated products occur.