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.

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.

Properties of arabinonucleic acids (ANA & 20'F-ANA): implications for the design of antisense therapeutics that invoke RNase H cleavage of RNA.

Inversion of configuration of the C2' position of RNA leads to a very unique nucleic acid structure: arabinonucleic acid (ANA). ANA, and its 2'-fluoro derivative (2'F-ANA) from hybrids with RNA that are capable of activating RNase H, resulting in cleavage of the RNA strand. In this paper, we review the properties of duplexes formed between ANA (or 2'F-ANA) and its RNA complement. These studies support the notion that RNase H is sensitive to the minor groove dimensions of the hybrid substrate.

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.

NMR solution structure of an oligonucleotide hairpin with a 2'F-ANA/RNA stem: implications for RNase H specificity toward DNA/RNA hybrid duplexes.

The first structure of a 2'-deoxy-2'-fluoro-D-arabinose nucleic acid (2'F-ANA)/RNA duplex is presented. We report the structural characterization by NMR spectroscopy of a small hybrid hairpin, r(GGAC)d(TTCG)2'F-a(GTCC), containing a 2'F-ANA/RNA stem and a four-residue DNA loop. Complete (1)H, (13)C, (19)F, and (31)P resonance assignments, scalar coupling constants, and NOE constraints were obtained from homonuclear and heteronuclear 2D spectra. In the chimeric duplex, the RNA strand adopts a classic A-form structure having C3' endo sugar puckers. The 2'F-ANA strand is neither A-form nor B-form and contains O4' endo sugar puckers. This contrasts strongly with the dynamic sugar conformations previously observed in the DNA strands of DNA/RNA hybrid duplexes. Structural parameters for the duplex, such as minor groove width, x-displacement, and inclination, were intermediate between those of A-form and B-form duplexes and similar to those of DNA/RNA duplexes. These results rationalize the enhanced stability of 2'F-ANA/RNA duplexes and their ability to elicit RNase H activity. The results are relevant for the design of new antisense drugs based on sugar-modified nucleic acids.

Solid-phase supports for oligonucleotide synthesis.

This unit begins with a discussion of the advantages and disadvantages of oligonucleotide synthesis using solid supports. The physical and chemical properties of solid-phase supports are discussed in terms of their suitability for oligonucleotide synthesis. In addition, the unit outlines the properties of linkers used for transient or permanent attachment of properly protected nucleosides to the derivatized support, as well as strategies for coupling nucleosides to linkers and conditions for the release of synthetic oligonucleotides from specific supports.

Attachment of nucleosides to solid-phase supports.

Specific step-by-step instructions are given for coupling nucleosides to LCAA-CPG supports (supports consisting of a long-chain alkylamine linked to controlled-pore glass). Protocols are given for a succinic acid linker and a hydroquinone-O,O'-diacetic acid linker. The former is the most widely used linker arm, and the starting materials are widely available. The latter offers greater compatibility with base-sensitive sequence modifications and great synthetic throughput because it can be cleaved under milder and faster conditions. Additional guidelines are given for selecting a linker arm and coupling protocol. Almost any application requiring synthetic oligonucletodies can be satisfied using one of these linker arms.

Solid-phase oligonucleotide synthesis and flow cytometric analysis with microspheres encoded with covalently attached fluorophores.

A novel combinatorial approach to synthesize oligonucleotides on fluorescently encoded microspheres based on flow sorting and segmental solid-phase synthesis is described. BODIPY dyes were covalently attached to polystyrene (8.8 microm, 55% DVB) microsphere particles to generate four fluorescently encoded sets. 20-mer oligonucleotide sequences can be synthesized on these microspheres with yields comparable to conventional CPG supports (80% overall yield, average stepwise yield = 99%). The concept of segmental solid-phase synthesis by flow sorting was demonstrated by synthesizing unique 20-mer oligonucleotide sequences on each of four fluorescently encoded microsphere sets by including a flow sorting step (after first eight base additions) and flow cytometric detection of sequences synthesized on each microsphere set by hybridization with fluorescently labeled complementary sequence.

Rapid esterification of nucleosides to solid-phase supports for oligonucleotide synthesis using uronium and phosphonium coupling reagents.

Nucleosides can be esterified to solid-phase supports using uronium or phosphonium coupling reagents and a coupling additive, such as 1-hydroxybenzotriazole (HOBT), 7-aza-1-hydroxybenzotriazole (HOAT), N-methylimidazole (NMI), or 4-(dimethylamino)pyridine (DMAP). However, DMAP was far superior to other additives and high nucleoside loadings (up to 60 micromol/g) and rapid coupling reactions (< or = 10 min) were possible. Hydroxyl-derivatized CPG was attached to nucleosides with 3'-succinyl or 3'-hydroquinone-O, O'-diacetic acid (HQDA or Q-Linker) carboxyl groups through a primary ester linkage. Alternatively, supports derivatized with succinic acid or the Q-Linker were attached directly to the 3'-OH group of nucleosides through a secondary ester linkage. Uronium reagents (HATU or HBTU) gave the best results with the HQDA linker arm, while the bromophosphonium (BrOP or PyBrOP) reagents were best with the succinyl linker arm. In all cases, the coupling reactions were much faster than previous methods using carbodiimide coupling reagents. The ease and speed of the reaction make this support derivatization procedure suitable for automated in situ couplings on DNA synthesizers.

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.

Cytarabine-induced destabilization and bending of a model Okazaki fragment.

The effects of cytarabine on the structural and thermodynamic properties of an Okazaki fragment were investigated using UV hyperchromicity and 2D 1H NMR. Cytarabine significantly decreased the stability of this model Okazaki fragment, decreasing the melting temperature from 46.8 degrees C to 42.4 degrees C at 1.33 x 10(-5) M. Cytarabine also markedly increased the bend angle of the Okazaki fragment duplex from 20 degrees to 42 degrees. Changes to the structures and stabilities of Okazaki fragments may cause the biological effects of cytarabine.

Cytotoxicity and in-vivo tolerance of FdUMP[10]: a novel pro-drug of the TS inhibitory nucleotide FdUMP.

The cytotoxicity of the 10mer ODN FdUMP[10] towards human colorectal tumor cells was evaluated using a clonogenic assay. FdUMP[10] was more than 100-fold more active than 5-FU at inhibiting colony formation of H630 cells. FdUMP[10] was also evaluated for cytotoxicity in the NCI 60 cell line screen, and showed markedly improved activity relative to 5-FU against numerous tumor cell lines. The in-vivo tolerance of FdUMP[10] is more than three-fold greater per mole fluorinated pyrimidine, than 5-FU.

Multiple oligodeoxyribonucleotide syntheseson a reusable solid-phase CPG support via the hydroquinone-O,O'-diacetic acid (Q-Linker) linker arm.

A strategy for oligodeoxyribonucleotide synthesis on a reusable CPG solid-phase support, derivatized with hydroxyl groups instead of amino groups, has been developed. Ester linkages, through a base labile hydroquinone- O, O '-diacetic acid ( Q-Linker ) linker arm, were used to couple the first nucleoside to the hydroxyl groups on the support. This coupling was rapidly accomplished (10 min) using O -benzotriazol-1-yl- N, N, N ', N '-tetramethyluronium hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole as the activating reagents. Oligodeoxyribonucleotide synthesis was performed using existing procedures and reagents, except a more labile capping reagent, such as chloro-acetic anhydride, methoxyacetic anhydride or t-butylphenoxyacetic anhydride, was used instead of acetic anhydride. After each oligodeoxyribonucleotide synthesis, the product was cleaved from the support with ammonium hydroxide (3 min) and deprotected as usual. Residual linker arms or capping groups were removed by treatment with ammonium hydroxide/methylamine reagent and the regenerated support was capable of reuse. Up to six different oligodeoxyribonucleotide syntheses or up to 25 cycles of nucleoside derivatization and cleavage were consecutively performed on the reusable support. This method may provide a significant cost advantage over conventional single-use solid supports currently used for the manufacture of antisense oligodeoxyribonucleotides.

Cytarabine-induced destabilization of a model Okazaki fragment.

Cytarabine is a potent anticancer drug that interferes with elongation of the lagging strand at the replication fork during DNA synthesis. The effects of cytarabine substitution on the structural and thermodynamic properties of a model Okazaki fragment were investigated using UV hyperchromicity and 1H NMR spectroscopy to determine how cytarabine alters the physicochemical properties of Okazaki fragments that are intermediates during DNA replication. Two model Okazaki fragments were prepared corresponding to a primary initiation site for DNA replication in the SV40 viral genome. One model Okazaki fragment consisted of five ribo- and seven deoxyribonucleotides on the hybrid strand, together with its complementary (DNA) strand. The second model Okazaki fragment was identical to the first with the exception of cytarabine substitution for deoxycytidine at the third DNA nucleotide of the hybrid strand. Thermodynamic parameters for the duplex to single strand transition for each model Okazaki fragment were calculated from the concentration dependence of the T m at 260 nm. Cytarabine significantly decreased the stability of this model Okazaki fragment, decreasing the melting temperature from 46.8 to 42.4 degrees C at a concentration of 1.33 x 10(-5) M. The free energy for the duplex to single strand transition was 1.2 kcal/mol less favorable for the cytarabine-substituted Okazaki fragment relative to the control at 37 degrees C. Analysis of the temperature dependence of the imino1H resonances for the two duplexes demonstrated that cytarabine specifically destabilized the DNA:DNA duplex portion of the model Okazaki fragment. These results are consistent with inhibition of lagging strand DNA synthesis by cytarabine substitution resulting from destabilization of the DNA:DNA duplex portion of Okazaki fragments in vivo .

Hydroquinone-O,O'-diacetic acid ('Q-linker') as a replacement for succinyl and oxalyl linker arms in solid phase oligonucleotide synthesis.

When hydroquinone-O,Ooffiacetic acid is used as a linker arm in solid phase oligonucleotide synthesis, the time for NH4OH cleavage of oligodeoxy- or oligoribonucleotides is reduced to only 2 min. This allows increased productivity on automated DNA synthesizers without requiring any other modifications to existing reagents or synthesis and deprotection methods. The Q-linker may also be rapidly cleaved by milder reagents such as 5% NH4OH, potassium carbonate, anhydrous ammonia, t-butylamine or fluoride ion. However, the Q-linker is sufficiently stable for long-term storage at room temperature without degradation and no loss of material occurs during synthesis. The linker is also reasonably resistant to 20% piperidine/DMF, 0.5 M DBU/pyridine and 1:1 triethylamine/ethanol. The Q-linker can therefore serve as a general replacement for both succinyl and oxalyl linker arms.

Solution structure studies of the cobalt complex of a bleomycin functional model bound to d(CGCAATTGCG)2 by two-dimensional nuclear magnetic resonance methods and restrained molecular dynamics simulation.

The interaction between the cobalt(III) complex of a bleomycin functional model (AMPHIS-NET) and the oligonucleotide d(CGCAATTGCG)2 and the structural features of the 1:1 ligand-DNA complex have been determined by high-resolution two-dimensional nuclear magnetic resonance methods and restrained molecular dynamics calculations. The intermolecular nuclear Overhauser effect (NOE) cross-peaks between ligand protons and the DNA minor groove protons suggest that the cobalt(III) complex of AMPHIS-NET binds in the minor groove of DNA at the central AATT site. The NOE connectivities also clearly indicate that the H8 pyridine proton and the H2 imidazole proton in the metal-binding domain interact with the H4' sugar proton of C19 and the H4' sugar proton of A5, respectively, which defines a structure where the metal binding moiety of Co(III).AMPHIS-NET participates in binding to the DNA and extends into the region two base pairs beyond the central AATT site in the minor groove. This binding model is in accord with the consistently observed nondiffusion DNA cleavage in locations two to three residues beyond the end of AT-rich binding sites induced by the corresponding iron(II) complexes of AMPHIS-NET and other AMPHIS-lexitropsin hybrids of the bleomycin functional model compounds.

Solution structures of 5-fluorouracil-substituted DNA and RNA decamer duplexes.

The structures in solution of eight oligonucleotide duplexes each containing either zero, one, or two 5-fluorodeoxyuridine (FdUrd) or 5-fluorouridine (FUrd) nucleosides were determined by the combined use of NMR spectroscopy, restrained molecular dynamics, and full relaxation matrix refinement to determine how FdUrd and FUrd substitution affects the structure of duplex DNA and RNA and to establish whether structural differences due to FdUrd and FUrd substitution in nucleic acids may be responsible, in part, for the biological effects of the anticancer drug 5-fluorouracil (FUra). The nucleic acid directed effects of FUra include induction of single-strand breaks in duplex DNA and altered processing of pre-mRNA and rRNA. Four self-complementary oligodeoxyribonucleotide sequences were prepared and studied as duplexes in aqueous solution: (5' dGCGAAUUCGC)2, (5' dGCGAAUFCGC)2, (5' dGCGAAFUCGC)2, and (5' dGCGAAFFCGC)2. The corresponding oligoribonucleotide sequences (5' rGCGAAUUCGC)2, (5' rGCGAAUFCGC)2, (5' rGCGAAFUCGC)2, and (5' rGCGAAFFCGC)2 were also prepared and studied. The helical parameters for the structures of these eight duplexes were analyzed to determine how substitution of FdUrd and FUrd affects the three-dimensional structures of duplex DNA and RNA. FdUrd substitution affects the base roll angle at the site of FdUrd substitution, causing the helical axis of FdUrd-substituted DNA duplexes to be bent compared to the nonsubstituted duplex. A-FUrd base pairs show substantial RMS deviations from A-Urd base pairs in all three of the RNA duplexes substituted with FUrd. Bending of the helical axis due to FdUrd substitution may contribute to the occurrence of single-strand breaks in duplex DNA while the altered structures of A-FUrd base pairs may affect RNA-RNA and RNA-protein recognition.

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.

Structure of a DNA duplex that contains alpha-anomeric nucleotides and 3'-3' and 5'-5' phosphodiester linkages: coexistence of parallel and antiparallel DNA.

We report a comparative spectroscopic study of a novel self-complementary duplex decamer, d(GCGAAT-3'-3'-(alpha T)-5'-5'-CGC)2, in which an alpha-anomeric nucleotide has been inserted into the sequence in a parallel orientation via 3'-3' and 5'-5' phosphodiester bonds, and its unmodified B-DNA analog, d(GCGAATTCGC)2. Plots of the hyperchromicity and circular dichroism of these oligonucleotides are virtually identical, indicating that the overall base stacking and handedness are preserved in the alpha duplex. Thermodynamic parameters extracted from UV melting experiments show that the alpha duplex is only slightly less stable than the control. A near complete set of 1H and 31P nuclear magnetic resonance (NMR) assignments were obtained for both duplexes using classical one- and two-dimensional approaches. Several lines of evidence, in particular, imino 1H, 31P, nuclear Overhauser enhancement, and deoxyribose ring proton spin-spin coupling data, convincingly demonstrate that the overall structural integrity of the alpha and control duplexes are quite comparable, with any perturbations in the former localized to the regions of the construct encompassing the alpha-nucleotide and the unique backbone linkages. Specifically, the alpha duplex exhibits normal Watson-Crick type base pairing, it remains antiparallel except at the inverted nucleotide, all bases are in the anti orientation, and the sugar ring puckering is predominantly "S"-type. However, the J-coupling information for the alpha-nucleotide and the neighboring (3') cytidine are notably different, and reflect a decrease in the amplitude of the sugar pucker in alpha T7, and a significant shift in the conformational equilibrium of the furanose ring in C8 toward the "N"-type pucker. The feasibility of synthesizing oligodeoxynucleotides containing a combination of alpha sugars and short parallel stranded segments, their propensity for forming stable duplexes, and the structural insights into such complexes reported here are of potential importance in the area of antisense therapy.

Studies on cooperative binding of an extended distamycin A analogue in the minor groove of DNA by NMR spectroscopy.

An analogue of the DNA minor groove binding antibiotic distamycin A, P4+, was found to bind in a 2:1 cooperative manner into the narrow groove of oligodeoxybibonucleotide d(CGTATATACG)2 by 1D-NMR titration. The resulting 2:1 ligand-DNA complex was characterized by 2D NMR experiments including NOESY, COSY, and TOCSY. Two molecules are oriented side by side opposite to each other; each has the N terminus to C terminus direction parallel to the 5' to 3' direction of its adjacent DNA strand. This study corroborates our previous CD and ethidium fluorometry results and provides new support for the generality of the antiparallel side by side binding motif first observed with distamycin A.