Design and applications of single and double stranded DNA analogs.

Chemical ligation method for assembling modified DNA-duplexes as well as solid support method and postsynthetic modification in aqueous media was used to synthesize a variety of single and double stranded oligonucleotide derivatives. Novel oligodeoxynucleotides with cluster or alternating sugar modifications and probes resistant to cell nuclease degradation were obtained. The use of such probes for effective and regiospecific hybridase RNA-hydrolysis is provided. Effective synthesis of new DNA duplexes containing modified and chemical cleavable internucleotide bonds was developed. A novel type of affinity reagent was suggested for covalent attachment of substrate to active site nucleophiles in the restriction/modification systems.     

Site specific functionalization of oligonucleotides for attaching two different reporter groups.

The synthesis of an oligonucleotide functionalized to attach two different reporter groups at specific internucleotide linkages is described. To incorporate the amine specific reporter group the internucleotide linkage is modified to phosphoramidate (N-1-aminoalkyl) and for a thiol specific reporter group the internucleotide linkage is modified to a phosphorothioate diester. The synthetic cycle for introducing the modified internucleotide linkages at specific sites can be carried out using an automated DNA synthesizer. Combination of reporter groups have been attached successfully.     

Use of a temporary protective group during the synthesis of oligonucleotides with an unnatural bond

Methoxygroup has been used for transient P-protection on H-phosphonate oligonucleotide synthesis. Whereas other H-phosphonate linkages can be irreversibly transformed into phosphoramidates by treatment with alkylamines, the protected phosphoralkoxyl groups generate natural phosphodiester linkages after the final deprotection. The method allows for synthesis of oligonucleotides with addressed position of internucleotide phosphoralkylamidate groups.     

Chemical reactions in double helical nucleic acids. XIII. Directed introduction of acylphosphate internucleotide bonds into the DNA-duplex structure]

DNA duplex, containing an acylphosphate internucleotide bond in a predetermined position of the sugar-phosphate backbone, was synthesized. The synthesis was carried out by condensing on the complementary matrix two heptanucleotides, one of which possessed at the 3'-end a glycine residue, connected with the oligonucleotide by the phosphoramide bond, whereas the 5'-end phosphate group of the other was activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC). The yield of the oligonucleotide with an acylphosphate bond was 24%. The stability and chemical properties of the synthesized compound were studied in comparison with analogous oligonucleotide containing a substituted pyrophosphate internucleotide bond. The former was shown to be an effective acylating agent in the aqueous medium in contrast to the latter which is a phosphorylating agent.     

Use of Modified Flap Structures for Study of Base Excision Repair Proteins

To investigate interactions between proteins participating in the long-patch pathway of base excision repair (BER), DNA duplexes with flap strand containing modifications in sugar phosphate backbone within the flap-forming oligonucleotides were designed. When the flap-forming oligonucleotide consisted of two sequences bridged by a decanediol linker located in the flap strand near the branch point, the efficiency and position of cleavage by flap endonuclease 1 (FEN1) differed from those for natural flap. The cleavage rate of chimeric structure by FEN1 was lower than that of a normal substrate. When we introduced the second modification in the flap-forming oligonucleotide, the cleavage rate decreased significantly. To estimate efficiency of recognition and processing of the chimeric structures by BER proteins, we studied the rate of DNA synthesis by DNA polymerase beta (Pol beta) and the rate of nucleotide excision at the 3'-end of the initiating primer by apurinic/apyrimidinic endonuclease 1 (APE1) compared with those for the natural DNA duplexes. Efficiency of strand-displacement DNA synthesis catalyzed by Pol beta was shown to be higher for flap structures containing non-nucleotide linkers. The chimeric structures were processed by the 3'-exonuclease activity of APE1 with efficiency lower than that for a normal flap structure. Thus, DNA duplexes with modifications in sugar phosphate backbone can be used to mimic intermediates of the long-patch pathway of BER in reconstituted systems containing FEN1. Based on chimeric and natural oligonucleotides, photoreactive DNA structures were designed. The photoreactive dCMP moiety was introduced into the 3'-end of DNA primer via the activity of Pol beta. The photoreactive DNA duplexes--3'-recessed DNA, nicked DNA, and flap structures containing natural and chimeric oligonucleotides--were used for photoaffinity labeling of BER proteins.     

Characterization of synthetic oligonucleotides containing biologically important modified bases by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Oligonucleotides containing modified bases are commonly used for biochemical and biophysical studies to assess the impact of specific types of chemical damage on DNA structure and function. In contrast to the synthesis of oligonucleotides with normal DNA bases, oligonucleotide synthesis with modified bases often requires modified synthetic or deprotection conditions. Furthermore, several modified bases of biological interest are prone to further damage during synthesis and oligonucleotide isolation. In this article, we describe the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to the characterization of a series of modified synthetic oligonucleotides. The potential for and limits in obtaining high mass accuracy for confirming oligonucleotide composition are discussed. Examination of the isotope cluster is also proposed as a method for confirming oligonucleotide elemental composition. MALDI-TOF-MS analysis of the unpurified reaction mixture can be used to confirm synthetic sequence and to reveal potential problems during synthesis. Analysis during and after purification can yield important information on depurination and base oxidation. It can also reveal unexpected problems that can occur with nonstandard synthesis, deprotection, or purification strategies. Proper characterization of modified oligonucleotides is essential for the correct interpretation of experiments performed with these substrates, and MALDI-TOF-MS analysis provides a simple yet extensive method of characterization that can be used at multiple stages of oligonucleotide production and use.     

Properties of circular dumbbell RNA/DNA chimeric oligonucleotides containing antisense phosphodiester oligonucleotides.

We have designed a new class of oligonucleotides, "dumbbell RNA/DNA chimeric phosphodiesters", containing two alkyl loop structures with RNA/DNA base pairs (sense (RNA) and antisense (DNA) in the double helical stem. The reaction of nicked (NDRDON) and circular (CDRDON) dumbbell RNA/DNA chimeric oligonucleotides with RNaseH gave the corresponding antisense phosphodiester oligonucleotide together with the sense RNA cleavage products. The liberated antisense phosphodiester oligodeoxynucleotide was bound to the target 35mer RNA, which gave 35mer RNA cleavage products by treatment with RNaseH. The circular dumbbell RNA/DNA chimeric oligonucleotide showed more nuclease resistance than the linear antisense phosphodiester oligodeoxynucleotide(anti-ODN) and the nicked dumbbell RNA/DNA chimeric oligonucleotide.     

Hydrazide oligonucleotides: new chemical modification for chip array attachment and conjugation

We report the synthesis of new phosphoramidite building blocks and their use for the modification of oligonucleotides with hydrazides. The reaction of these hydrazide oligonucleotides with active esters and aldehydes is demonstrated for solution conjugation and immobilization. Compared with the established amino modified oligonucleotides, hydrazides show enhanced reactivity at neutral and acidic buffer conditions. One method to introduce hydrazides is using amidites with preformed, protected hydrazides. A completely novel approach is the generation of the hydrazide functionality during the oligonucleotide cleavage and deprotection with hydrazine. Therefore, building blocks for the introduction of esters as hydrazide precursors are described. For the enhanced attachment on surfaces branched modifier amidites, which introduce up to four reactive groups to the oligonucleotide, are applied. The efficiency of branched hydrazide oligonucleotides compared with standard amino modified oligonucleotides for the immobilization of DNA on active electronic Nanogen chips is demonstrated.     

Interaction of the MvaI restriction enzyme with synthetic DNA fragments

The cleavage of synthetic DNA duplexes by the restriction endonuclease MvaI has been studied. The main result of the cleavage experiments is that MvaI cleaves unmodified duplexes in two single strand scissions in separate events and that the two strands are cleaved at significantly different rates. One strand nicks within the recognition site do not affect the cleavage. Furthermore, neither a pyrophosphate internucleotide bond modification in one strand nor the absence of one phosphate group at the central dA-residue of the recognition site do inhibit the cleavage of the second strand.     

Localized in vivo genotypic and phenotypic correction of the albino mutation in skin by RNA-DNA oligonucleotide

We recently demonstrated that an RNA-DNA oligonucleotide corrected a point mutation in the mouse tyrosinase gene, resulting in permanent and inheritable restoration of tyrosinase enzymatic activity, melanin synthesis, and pigmentation changes in cultured melanocytes. In this study, we extended gene correction of melanocytes from tissue culture to live animals, using a chimeric oligonucleotide designed to correct a point mutation in the tyrosinase gene. Both topical application and intradermal injection of this oligonucleotide to albino BALB/c mouse skin resulted in dark pigmentation of several hairs in a localized area. The restored tyrosinase enzymatic activity was detected by dihydroxyphenylacetic acid (DOPA) staining of hair follicles in the treated skin. Tyrosinase gene correction was also confirmed by restriction fragment length polymorphism analysis and DNA sequencing from skin that was positive for DOPA staining and melanin synthesis. Localized gene correction was maintained three months after the last application of the chimeric oligonucleotides. These results demonstrated correction of the tyrosinase gene point mutation by chimeric oligonucleotides in vivo.     

A new affinity reagent for the site-specific, covalent attachment of DNA to active-site nucleophiles: application to the EcoRI and RsrI restriction and modification enzymes.

A modified oligodeoxyribonucleotide duplex containing an unnatural internucleotide trisubstituted 3' to 5' pyrophosphate bond in one strand [5'(oligo1)3'-P(OCH3)P-5'(oligo2) 3'] reacts with nucleophiles in aqueous media by acting as a phosphorylating affinity reagent. When interacted with a protein, a portion of the oligonucleotide [--P-5'(oligo2)3'] becomes attached to an amino acid nucleophilic group through a phosphate of the O-methyl-modified pyrophosphate linkage. We demonstrate the affinity labeling of nucleophilic groups at the active sites of the EcoRI and RsrI restriction and modification enzymes with an oligodeoxyribonucleotide duplex containing a modified scissile bond in the EcoRI recognition site. With the EcoRI and RsrI endonucleases in molar excess approximately 1% of the oligonucleotide becomes attached to the protein, and with the companion methyltransferases the yield approaches 40% for the EcoRI enzyme and 30% for the RsrI methyltransferase. Crosslinking proceeds only upon formation of a sequence-specific enzyme-DNA complex, and generates a covalent bond between the 3'-phosphate of the modified pyrophosphate in the substrate and a nucleophilic group at the active site of the enzyme. The reaction results in the elimination of an oligodeoxyribonucleotide remnant that contains the 3'-O-methylphosphate [5'(oligo1)3'-P(OCH3)] derived from the modified phosphate of the pyrophosphate linkage. Hydrolysis properties of the covalent protein-DNA adducts indicate that phosphoamide (P-N) bonds are formed with the EcoRI endonuclease and methyltransferase.     

Detection of Nucleic Acids by Cycling Probe Technology on Magnetic Particles: High Sensitivity and Ease of Separation

Abstract

Cycling Probe Technology (CPT) is a signal amplification system that allows detection of nucleic acid target sequences without target amplification. CPT employs a sequence specific chimeric probe, typically DNA-RNA-DNA, which hybridizes to a complementary target DNA sequence and becomes a substrate for RNase H. Cleavage occurs at the RNA internucleotide linkages and results in dissociation of the probe from the target, thereby making it available for the next probe molecule. This communication describes the use of oligonucleotides attached to solid supports for target capture and release followed by solution and solid phase cycling. Through the attachment of chimeric probes to Sera-Mag^TM magnetic particles (SMP) a simple and effective method of separating the cleaved probe from non-cycled probe has been developed. By capturing the target DNA on particles and separating it from the extraneous non-specific DNA we are able to dramatically reduce background and thus discriminate between samples of Methicillin Resistant (MRSA) and Methicillin Sensitive (MSSA) Staphylococcus Aureus. We conjugated oligonucleotide probes to SMPs (～1 um) and Nylon beads (NB) which were coated with ID Biomedical's proprietary coating materials (R, patent pending). The general structure of the constructs is shown below:     

Study of the mutagenic properties of oligonucleotides containing and internucleotide pyrophosphate bond using a mutation system based on phage M13]

Mutagenic properties of oligonucleotides with pyrophosphate internucleotide bond was studied. It was shown that the pyrophosphate bond in the oligo structure does not induce mutations but promotes a more efficient induction of marker deletions predetermined by the nucleotide sequence as compared to the native oligonucleotide. Marker deletion induction proceeds according to the repair mechanism as homozygotes dominate in the mutant generation.     

Ligation-based synthesis of oligonucleotides with block structure

We describe here a method for the synthesis of oligonucleotides with block structure (padlock probes, primers for multiplex polymerase chain reaction (PCR), and ligation-independent cloning), based on the ligation of presynthesized parts by T4 DNA ligase. The advantages of this approach are: (i) suitability of the technology for any producer-from synthesis company to laboratory, (ii) high quality and adjustable scale of synthesis, and (iii) possibility of including any modified bases inexpensively in the common part of the oligonucleotide. Clear difference of sizes of products and substrates makes the synthesis amenable to automation. For large series of padlock probes, the price per one primer approaches the price of the locus-specific parts. We demonstrate the application of this method to two different tasks: preparative-scale production of padlock probes and small-scale synthesis of PCR primers.     

New chemically reactive dsDNAs containing single internucleotide monophosphoryldithio links: reactivity of 5'-mercapto-oligodeoxyribonucleotides

Novel modified DNA duplexes with single bridging 5′-SS-monophosphoryldithio links [-OP(=O)-O^–-SS-CH₂-] were synthesized by autoligation of an oligonucleotide 3′-phosphorothioate and a 5′-mercapto-oligonucleotide previously converted to a 2-pyridyldisulfide adduct. Monophosphoryldisulfide link formation is not a stringent template-dependent process under the conditions used and does not require strong binding of the reactive oligomers to the complementary strand. The modified internucleotide linkage, resembling the natural phosphodiester bond in size and charge density, is stable in water, easily undergoes thiol–disulfide exchange and can be specifically cleaved by the action of reducing reagents. DNA molecules containing an internal -OP(=O)-O^–-SS-CH₂- bridge are stable to spontaneous exchange of disulfide-linked fragments (recombination) even in the single-stranded state and are promising reagents for autocrosslinking with cysteine-containing proteins. The chemical and supramolecular properties of oligonucleotides with 5′-sulfhydryl groups were further characterized. We have shown that under the conditions of chemical ligation the 5′-SH group of the oligonucleotide has a higher reactivity towards N-hydroxybenzotriazole-activated phosphate in an adjacent oligonucleotide than does the OH group. This autoligation, unlike disulfide bond formation, proceeds only in the presence of template oligonucleotide, necessary to provide the activated phosphate in close proximity to the SH-, OH- or phosphate function.     

Preparation of DNA-duplexes, containing internucleotide thiophosphate groups in various positions of the recognition site for the EcoRII restriction endonuclease

Twenty-four 12-mer DNA duplexes, each containing a chiral phosphorothioate group successively replacing one of the internucleotide phosphate groups either in the EcoRII recognition site (5'CCA/TGG) or near to it, were obtained for studying the interaction of the restriction endonuclease EcoRII with internucleotide DNA phosphates. Twelve of the 12-mer oligonucleotides were synthesized as Rp and Sp diastereomeric mixtures. Six of them were separated by reversed-phase HPLC using various buffers. Homogeneous diastereomers of the other oligonucleotides were obtained by enzymatic ligation of the Rp and Sp diastereomers of 5- to 7-mer oligonucleotides preliminarily separated by HPLC with the corresponding short oligonucleotides on a complementary DNA template. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.     

Hybridization specificity, enzymatic activity and biological (Ha-ras) activity of oligonucleotides containing 2,4-dideoxy-beta-D-erythro-hexopyranosyl nucleosides.

Antisense oligonucleotides with a 2,4-dideoxyhexopyranosyl nucleoside incorporated at the 3'-end and at a mutation site of the Ha-ras oncogene mRNA were synthesized. Melting temperature studies revealed that an A*-G mismatch is more stable than an A*-T mismatch with these hexopyranosyl nucleosides incorporated at the mutation site. The oligonucleotides are stable against enzymatic degradation. RNase H mediated cleavage studies revealed selective cleavage of mutated Ha-ras mRNA. The oligonucleotide containing two pyranose nucleosides at the penultimate position activates RNase H more strongly than natural oligonucleotides. No correlation, however, was found between DNA - DNA or RNA - DNA melting temperatures and RNase H mediated cleavage capacity. Although the A*-G mismatch gives more stable hybridization than the A*-T base pairing, only the oligonucleotides containing an A*-T base pair are recognized by RNase H. This modification is situated 3 base pairs upstream to the cleavage site. Finally, the double pyranose modified oligonucleotide was able to reduce the growth of T24 cells (bladder carcinoma) while the unmodified antisense oligonucleotide was not.     

Anti-Influenza Virus Activities of Nicked and Circular Dumbbell RNA/DNA Chimeric Oligonucleotides

Abstract

We have designed a new type of antisense oligonucleotide, containing two hairpin loop structures with RNA/DNA base pairs (sense (RNA) and antisense (DNA)) in the double helical stem (nicked and circular dumbbell DNA/RNA chimeric oligonucleotides). The reaction of the nicked and circular dumbbell DNA/RNA chimeric oligonucleotides with RNase H gave the corresponding anti-DNA together with the sense RNA cleavage products. These oligonucleotides were more resistant to exonuclease attack. We also describe the anti-Fluv activities of nicked and circular dumbbell DNMA chimeric oligonucleotides.     

Chemical and Photochemical Ligation of Oligonucleotide Blocks

Abstract

Several efficient means for joining oligonucleotides in dilute solution by non-natural internucleotide bridges are discussed. It is also shown that an oligonucleotide containing a -OP(O)(O^?)S- link can hnction as an effective template in PCR amplification and that oligonucleotide probes containing stilbenedicarboxamide groups can serve in monitoring the presence of mismatched bases in an oligonucleotide target.