Synthesis, Thermodynamic Stability and Enzymic Behavior of Oligonucleotides Containing Pyrazole Nucleobase Analogs.

Non-hydrogen bonding nucleosides with iodo, nitro, propynyl and thiazolyl substituted at the 4-position of pyrazole were prepared. These nucleosides were converted to their corresponding nucleoside phosphoramidites and incorporated into a series of complementary oligonucleotides in order to determine the effect that varying size, charge distribution and polarizability has on duplex stability and structure. The self-complementary Dickerson dodecamer sequence 5\u27-CGCXAATTYGCG-3\u27, as well as the non-self complementary sequence 5\u27-CAAAATGGTGGCCAAGT-3 \u27 previously investigated by Brown, were used to determine the duplex stabilization and thermodynamic consequences of placing 5-nitroindole and 4-substituted pyrazole nucleosides across from each natural base. In the pyrazole series, the largest and smallest duplex destabilization in all cases studied was found with cytosine and adenosine, respectively, while the 4-thiazolyl substitution was determined to form the most stable duplex regardless of the complementary base. We investigated the directing ability of the pyrazole analogs for incorporation of dNTP\u27s. The DNA polymerases chosen were Taq and Pfu exo-, which lack 3\u27 --5\u27 proofreading exonuclease activity, and Vent, Deep Vent, Pfu which contain exonuclease activity. The enzymes were able to incorporate natural nucleotides across from our modification as detected using the polymerase chain reaction. The Sanger method of dideoxysequencing was used to determine the natural base incorporated across from the modification. The non-proofreading enzymes mainly incorporated deoxyadenosine across from our modification where as the proofreading enzymes removed our modification but not with total efficiency. Automated DNA synthesis procedures might modifying the 5-nitroindole base via electrophilic aromatic substitution by replacing the nitro group or addition of iodine to the ring system. An alternative oxidation reagent, CSO, was used to prepare a series of sequences for comparison with sequences prepared using standard oxidation conditions. Results from MALDI-MS did not provide conclusive evidence for either substitution or iodination of the bicyclic ring system. Examination of the thermodynamic results led to the preparation of thiazole-C-nucleoside, which historically is more difficult to synthesize. The thiazole ring offers a site for the formation of an N-oxide. It has been suggested this group is responsible for enzyme recognition. We prepared the thiazole and thiazole-N-oxide nucleosides for incorporation into oligonucleotides for thermodynamic and enzymic investigations

Solution structure and dynamics of DNA duplexes containing the universal base analogues 5-nitroindole and 5-nitroindole 3-carboxamide

Universal bases hybridize with all other natural DNA or RNA bases, and have applications in PCR and sequencing. We have analysed by nuclear magnetic resonance spectroscopy the structure and dynamics of three DNA oligonucleotides containing the universal base analogues 5-nitroindole and 5-nitroindole-3-carboxamide. In all systems studied, both the 5-nitroindole nucleotide and the opposing nucleotide adopt a standard anti conformation and are fully stacked within the DNA duplex. The 5-nitroindole bases do not base pair with the nucleotide opposite them, but intercalate between this base and an adjacent Watson–Crick pair. In spite of their smooth accommodation within the DNA double-helix, the 5-nitroindole-containing duplexes exist as a dynamic mixture of two different stacking configurations exchanging fast on the chemical shift timescale. These configurations depend on the relative intercalating positions of the universal base and the opposing base, and their exchange implies nucleotide opening motions on the millisecond time range. The structure of these nitroindole-containing duplexes explains the mechanism by which these artificial moieties behave as universal bases

Hybridization properties and enzymatic replication of oligonucleotides containing the photocleavable 7-nitroindole base analog

Universal DNA base analogs having photocleavable properties would be of great interest for development of new nucleic acid fragmentation tools. The photocleavable 7-nitroindole 2′-deoxyribonucleoside d(7-Ni) was previously shown to furnish a highly efficient approach to photochemically trigger DNA backbone cleavage at preselected position when inserted in a DNA fragment. In the present report, we examine its potential use as universal DNA nucleoside, by analogy with the 5-nitroindole analog that is generally considered as universal base. The d(7-Ni) phosphoramidite was incorporated into oligonucleotides. Hybridization properties of resulting 11mer duplexes indicated a behavior close to that of the 5-nitroindole analog. Enzymatic recognition by Klenow fragment exonuclease-free using 40mers containing the unnatural bases as templates indicated notably a decrease of the polymerase activity with preferential incorporation of dAMP opposite both the 7-Ni and 5-Ni bases. Incorporation of the d(7-Ni) triphosphate was also studied indicating absence of significant differences between the incorporation kinetics opposite each natural base in the template. All the hybridization and enzymatic data indicate that 7-nitroindole can be considered as a cleavable base analog, although not strictly fulfilling, like the 5-nitro isomer, all properties required for a universal base

Use of continuous/contiguous stacking hybridization as a diagnostic tool

A method for detecting disease-associated alleles in patient genetic material is provided whereby a first group of oligonucleotide molecules, synthesized to compliment base sequences of the disease associated alleles is immobilized on a predetermined position on a substrate, and then contacted with patient genetic material to form duplexes. The duplexes are then contacted with a second group of oligonucleotide molecules which are synthesized to extend the predetermined length of the oligonucleotide molecules of the first group, and where each of the oligonucleotide molecules of the second group are tagged and either incorporate universal bases or a mixture of guanine, cytosine, thymine, and adenine, or complementary nucleotide strands that are tagged with a different fluorochrome which radiates light at a predetermined wavelength. The treated substrate is then washed and the light patterns radiating therefrom are compared with predetermined light patterns of various diseases that were prepared on identical substrates

Hybridization properties of long nucleic acid probes for detection of variable target sequences, and development of a hybridization prediction algorithm

One of the main problems in nucleic acid-based techniques for detection of infectious agents, such as influenza viruses, is that of nucleic acid sequence variation. DNA probes, 70-nt long, some including the nucleotide analog deoxyribose-Inosine (dInosine), were analyzed for hybridization tolerance to different amounts and distributions of mismatching bases, e.g. synonymous mutations, in target DNA. Microsphere-linked 70-mer probes were hybridized in 3M TMAC buffer to biotinylated single-stranded (ss) DNA for subsequent analysis in a Luminex® system. When mismatches interrupted contiguous matching stretches of 6 nt or longer, it had a strong impact on hybridization. Contiguous matching stretches are more important than the same number of matching nucleotides separated by mismatches into several regions. dInosine, but not 5-nitroindole, substitutions at mismatching positions stabilized hybridization remarkably well, comparable to N (4-fold) wobbles in the same positions. In contrast to shorter probes, 70-nt probes with judiciously placed dInosine substitutions and/or wobble positions were remarkably mismatch tolerant, with preserved specificity. An algorithm, NucZip, was constructed to model the nucleation and zipping phases of hybridization, integrating both local and distant binding contributions. It predicted hybridization more exactly than previous algorithms, and has the potential to guide the design of variation-tolerant yet specific probes

The ligase detection reaction: the evolution of a mutation detection strategy

Early detection of genetic mutations is important for control of diseases such as cancer and Alzheimer\u27s. Early detection requires methods that detect small amounts of mutated DNA in very large amounts of normal or wild type DNA. One method to detect mutated DNA is the ligase detection reaction (LDR). Since its inception LDR has evolved greatly from a simple detection reaction after PCR amplification to PCR/RE/LDR, a scheme which uses nucleoside base analogs in PCR to convert wild type sequences to sequences containing restriction endonuclease (RE) sites which can then be cleaved leaving only mutant sequences for detection by LDR. Analysis of LDR has also evolved from slab gel electrophoresis to microarray analysis. Understanding the structure and DNA polymerase recognition of nucleoside base analogs used in PCR/RE/LDR is key to improving this detection scheme. The use of higher fidelity DNA polymerase containing 3\u27→5\u27 exonuclease domains for error correction is also important in early detection of genetic diseases. Pyrazole-based nucleoside analogs have been studied computationally and enzymatically. The stability a DNA containing these analogs depends largely on the dipole moment of the analogs, rather than polarizability or surface area. Reduced DNA polymerase recognition is due in part to altered base pair geometry, either inherent or created by DNA polymerase. Thiazole and thiazole N-oxide analogs to be used in the PCR/RE/LDR assay have been synthesized and characterized computationally, thermodynamically, and enzymatically. The N-oxide, a pyrimidine O2 mimic, enhances DNA stability and DNA polymerase recognition. The N-oxide increases electrostatic properties and solvation by the formation of a hydrogen bond when base paired with guanine. Enzymatic analysis indicated a preference for the base pairing of thiazole N-oxide with guanine and thiazole with adenine. An N3\u27→P5\u27 phosphoramidate backbone analog has shown to inhibit the exonuclease activity of higher fidelity DNA polymerases for use in PCR/RE/LDR. The evolution of the analysis of LDR continues with the adaptation to capillary and microdevice electrophoresis. These formats were used to analyze model samples and LDR reactions mimicking low abundant mutations. These improved techniques greatly improve the resolution of LDR analysis

Synthesis, Thermodynamic Behavior, and Enzymatic Properties of Oligonucleotides Containing Nucleoside Analogs.

The focus of this research is on base modified nucleosides. By generating these nucleosides we hope to find information about the role of base-stacking in the stability of the DNA duplex and generate nucleoside analogues that could be used as conversion agents or in ambiguous positions in probes and primers. The target nucleosides included one C-nucleoside 5-(2\u27-deoxy-beta-D-ribofuranosyl)-2-hydroxypyrimidine and four 4-substituted pyrazole nucleosides; 1-(2\u27-deoxy-beta- D-ribofuranosyl)-4-iodopyrazole, 1-(2\u27-deoxy-beta- D-ribofuranosyl)-4-, 1-(2\u27-deoxy-beta- D-ribofuranosyl)-4-propynylpyrazole, and 1-(2\u27-deoxy-beta- D-ribofuranosyl)-4-(2-thiazolyl)pyrazole. The C-nucleoside was of interest because of its potential anti-viral and anti cancer activity and its potential to function as a convertide . There are several potential routes for the synthesis of C-nucleosides. The two routes we attempted were based on a palladium catalyzed Heck type coupling and a Grignard reaction. Neither route was successful. The palladium coupling was hindered mostly by the poorly defined reaction conditions and the complex mix of product produced. It was initially hampered by the difficulty in obtaining the requisite glycals for the reaction. This problem; however, was addressed by generating protected glycals by the treatment of appropriately protected thymidines with hexamethyldisilazane. The Grignard coupling route produced mostly addition of the base to the toluoyl protecting group. In addition both methods may have been hindered by acid lability of the base. The pyrazole nucleosides were easily synthesized by addition of the sodium salt of the base to a p-toluoyl protected 1-chlororibose. The pyrazole nucleosides were then converted to the phosphoramidites and incorporated into oligonucleotides using standard solid phase coupling conditions. Once incorporated into oligonucleotides the relative stabilities of the nucleosides were measured by thermodynamic melting. Overall the thiazolylpyrazole nucleoside was the most stable and the iodopyrazole was the least stable. The propynylpyrazole and the nitropyrazole were similar. All four nucleosides showed a preference for the purines and were lease stable versus dC. The nucleosides were also found to function in PCR reactions. The initial preference for the natural nucleoside incorporated versus each modified nucleoside was a dC; however, this is most likely a result of impure starting materials

Solid-phase synthesis of modified oligonucleotides

Synthetic oligonucleotides are ubiquitously found in most laboratories since solid-phase synthesis protocols have become highly optimized. These protocols make it possible to synthesize a large variety of modified oligonucleotides. As one example, we will review some of the developments regarding oligonucleotide synthesis from our own group. In particular, we will describe the synthesis of oligonucleotides carrying non-natural bases, of oligonucleotide-peptide conjugates, and of modified oligonucleotides used in the assembly of nanomaterials.The work summarized in this review has been conducted by a large number of colleagues and collaborators whom I thank for their contributions and friendship. We also thank E.E.C.C., E.M.B.L., the Spanish Ministry of Education and the Fundació La Caixa for funding this work.Peer reviewe

Use of continuous/contiguous stacking hybridization as a diagnostic tool

A method for detecting disease-associated alleles in patient genetic material is provided whereby a first group of oligonucleotide molecules, synthesized to compliment base sequences of the disease associated alleles is immobilized on a predetermined position on a substrate, and then contacted with patient genetic material to form duplexes. The duplexes are then contacted with a second group of oligonucleotide molecules which are synthesized to extend the predetermined length of the oligonucleotide molecules of the first group, and where each of the oligonucleotide molecules of the second group are tagged and either incorporate universal bases or a mixture of guanine, cytosine, thymine, and adenine, or complementary nucleotide strands that are tagged with a different fluorochrome which radiates light at a predetermined wavelength. The treated substrate is then washed and the light patterns radiating therefrom are compared with predetermined light patterns of various diseases that were prepared on identical substrates. A method is also provided for determining the length of a repeat sequence in DNA or RNA, and also for determining the base sequence of unknown DNA or RNA

Chemical synthesis, DNA incorporation and biological study of a new photocleavable 2′-deoxyadenosine mimic

The phototriggered cleavage of chemical bonds has found numerous applications in biology, particularly in the field of gene sequencing through photoinduced DNA strand scission. However, only a small number of modified nucleosides that are able to cleave DNA at selected positions have been reported in the literature. Herein, we show that a new photoactivable deoxyadenosine analogue, 3-nitro-3-deaza-2′-deoxyadenosine (d(3-NiA)), was able to induce DNA backbone breakage upon irradiation (λ > 320 nm). The d(3-NiA) nucleoside was chemically incorporated at desired positions into 40-mer oligonucleotides as a phosphoramidite monomer and subsequent hybridization studies confirmed that the resulting modified duplexes display a behaviour that is close to that of the related natural sequence. Enzymatic action of the Klenow fragment exonuclease free revealed the preferential incorporation of dAMP opposite the 3-NiA base. On the other hand, incorporation of the analogous 3-NiA triphosphate to a primer revealed high enzyme efficiency and selectivity for insertion opposite thymine. Furthermore, only the enzymatically synthesized base pair 3-NiA:T was a substrate for further extension by the enzyme. All the hybridization and enzymatic data indicate that this new photoactivable 3-NiA triphosphate can be considered as a photochemically cleavable dATP analogue