Synthesis and properties of oligonucleotides carrying cryptolepine derivatives

A carboxy derivative of the antimalarial cytotoxic drug cryptolepine was introduced into synthetic oligonucleotides by reaction of the carboxy derivative of cryptolepine with oligonucleotides carrying an amino group either at the 3'- or at the 5'-end. Oligonucleotides carrying the cryptolepine derivative bind their complementary sequences with greater affinity than unmodified ones. When cryptolepine is attached to a polypyrimidine oligonucleotide designed to form a parallel triplex, the triplex shows none or weak stabilization.     

8-aza-7-deazaadenine and 7-deazaguanine: synthesis and properties of nucleosides and oligonucleotides with nucleobases linked at position-8

The 8-aza-7-deazaadenine (pyrazolo[3,4-d]pyrimidin-4-amine) N8-(2'-deoxyribonucleoside) (2) and the 7-deazaguanine (pyrrolo[3,4-d]pyrimidine-2-amin-(3H)-4-one) C8-(2'-deoxyribonucleoside) (4) were synthesized and incorporated in oligonucleotides employing phosphoramidite chemistry. Oligonucleotides carrying compound 2 are able to form base pairs with the four canonical DNA constituents without significant structural discrimination. The nucleoside 4 was obtained from the corresponding ribonucleoside by deoxygenation. Oligonucleotides containing compound 4 showed similar base pairing properties as those with 2'-deoxyisoguanosine.     

Reagent for introducing pyrene residues in oligonucleotides.

A novel pyrenyl-containing phosphoramidite reagent, N-[4-(1-pyrenyl)butyryl]-O1-(4,4'-dimethoxytrityl)-O2- [(diisopropylamino)(2-cyanoethoxy)phosphino]-3-amino-1 ,2-propanediol (5), has been synthesized from 4-(1-pyrenyl)butanoic acid in four steps with the 52% overall yield and used to incorporate pyrene residue(s) into oligonucleotides. Oligonucleotides 6 and 7, bearing one or two pyrenes at the 5'-terminus, have been prepared by means of that reagent, characterized with fluorescence spectra, and successfully used as primers in a polymerase chain reaction.     

Affinity modification of E. coli RNA-polymerase in a complex with the promoter by phosphorylating derivatives of primer oligonucleotides

Oligonucleotides 2 to 7 nucleotide residues long, complementary to the codogenic strand of T7 promoter A2, have been synthesized; all of them contained a ribo-unit at the 3'-end. They were converted into 5'-(N-methyl)phosphoimidazolides, and the affinity reagents obtained were allowed to bind covalently to RNA polymerase in the presence of a promoter. Some of the nucleotide residues covalently attached occupied proper positions relative to the active centre of the phosphodiester bond synthesis and on addition of [alpha-32P]UTP were elongated, so that highly selective affinity labelling occurred. With oligonucleotides of various lengths, different distribution of the label between beta, beta' and sigma subunits of RNA polymerase took place. Most efficient was labelling of beta-subunit by the residue--pCpGpCpU, and of sigma-subunit by the residue--pApApApTp-CpGpCpU (p--radioactive phosphorus atom). In both cases, the amino acid residues labelled were histidines.     

Chimeric RNA and 2'-O, 4'-C-ethylene-bridged nucleic acids have stronger activity than phosphorothioate oligodeoxynucleotides in induction of exon 19 skipping in dystrophin mRNA

Antisense phosphorothioate oligodeoxynucleotides against exon 19 of the dystrophin gene have been shown to induce exon 19 skipping and promote the expression of internally deleted dystrophin by correcting the translational reading frame. Because phosphorothioate oligonucleotides are associated with a variety of toxic nonantisense effects, several modifications of nucleic acid have been introduced to alleviate this toxicity. Recently, a 2'-O, 4'-C-ethylene-bridged nucleic acid (ENA trade mark, Sankyo Lifetech Co., Ltd., Tokyo, Japan) was reported to have high affinity to complementary RNA strands and be resistant to nuclease digestion. Here, we examined the ability of this modified nucleic acid to induce exon skipping. Oligonucleotides having the same sequence as the phosphorothioate oligonucleotides but with some stretches of modified backbone (2'-O-methyl RNA with an ENA5-mer at the 5'-end and 3'-end) (RNA/ENA chimera) were transfected into myocytes, and the expressed dystrophin mRNA was analyzed. The RNA/ENA chimera induced exon 19 skipping in a dose-dependent and time-dependent manner. Remarkably, the exon 19-skipping activity of the RNA/ENA chimera was more than 40 times stronger than that of the corresponding conventional phosphorothioate oligodeoxynucleotide. This is the first report of such strong activity of an RNA/ENA chimera in the induction of exon skipping in the dystrophin gene. This new technology will allow the development of less toxic antisense drugs, making long-term therapy possible.     

Carboranyl oligonucleotides: 4. synthesis and physicochemical studies of oligonucleotides containing 2'-O-(o-carboran-1-yl)methyl group

Boronated oligonucleotides are potential candidates for antisense oligonucleotide technology (AOT), boron neutron capture therapy (BNCT), and as tools in molecular biology. A method was developed for the solid phase synthesis of oligonucleotides containing 2'-O-(o-carboran-1-yl-methyl) (2'-CBM) group. Synthesis was performed using a standard beta-cyanoethyl cycle and automated DNA synthesizer. Manual steps were performed for the insertion of a modified monomer bearing the 2'-CBM group. Several tetradecanucleotides complementary to DNA-HCMV, and bearing 2'-CBM modification near the 3'-end or 5'-end or in the middle of the oligonucleotide chain were synthesized. The resulting oligomers were characterized by polyacrylamide gel electrophoresis (PAGE), reverse phase high-performance liquid chromatography (RP-HPLC), matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and ultraviolet spectroscopy (UV), circular dichroism (CD), and melting temperature (Tm) measurements. Tm of duplexes formed between 2'-CBM-modified tetradecanucleotides and complementary DNA and RNA template were compared with those formed by the unmodified oligonucleotide and complementary sequence. The stability of 2'-CBM oligonucleotides in the presence of phosphodiesterase I from Crotalus atrox venom and in human serum was studied. Oligonucleotides bearing the 2'-CBM group are characterized by increased resistance to enzymatic digestion, increased lipophilicity, and the ability to form stable duplexes with complementary templates.     

8-AZA-7-DEAZAADENINE AND 7-DEAZAGUANINE: SYNTHESIS AND PROPERTIES OF NUCLEOSIDES AND OLIGONUCLEOTIDES WITH NUCLEOBASES LINKED AT POSITION-8

The 8-aza-7-deazaadenine (pyrazolo[3,4-d]pyrimidin-4-amine) N⁸-(2′-deoxy-ribonucleoside) (2) and the 7-deazaguanine (pyrrolo[3,4-d]pyrimidine-2-amin-(3H)-4-one) C⁸-(2′-deoxyribonucleoside) (4) were synthesized and incorporated in oligonucleotides employing phosphoramidite chemistry. Oligonucleotides carrying compound 2 are able to form base pairs with the four canonical DNA constituents without significant structural discrimination. The nucleoside 4 was obtained from the corresponding ribonucleoside by deoxygenation. Oligonucleotides containing compound 4 showed similar base pairing properties as those with 2′-deoxyisoguanosine.     

DNA primase-DNA polymerase alpha from simian cells. Modulation of RNA primer synthesis by ribonucleoside triphosphates

DNA primase-DNA polymerase alpha, purified 53,000-fold from CV-1 cells, synthesized predominantly (p)ppA(pA)6-primed DNA on a poly(dT) template. About 80% of the RNA primers synthesized on an M13 DNA template were (p)ppA/G(pN)5-7, and 20% were (p)ppA/G(pN)0-4. RNA primer size was determined by gel electrophoresis after removing nascent DNA with phage T4 DNA polymerase 3'-5' exonuclease, leaving a single dNMP at the 3'-end of the RNA primer, and the terminal 5'-(p)ppN residue was determined by "capping" with [alpha-32P]GTP using vaccinia guanylyl-transferase. The processivity of DNA synthesis initiated by de novo synthesis of RNA primers was the same as that initiated on pre-existing RNA primers (10-15 dNMPs), although initiation on pre-existing primers was strongly preferred. Primers always began with A or G, even at high levels of CTP or UTP, although the ratio of A to G varied from 4:1 to 1:1 depending on the relative concentrations of ATP and GTP in the assay. ATP and GTP had no effect on primer length, but the fraction of shorter RNA primers increased 2-fold with higher concentrations of CTP or UTP. Nearest-neighbor analysis revealed a preference for purine ribonucleotides at RNA covalently linked to the 5'-end of DNA (RNA-p-DNA) junctions, and increasing the concentration of a single rNTP increased slightly its presence at RNA-p-DNA junctions. Thus, the base composition and size of RNA primers synthesized by DNA primase-DNA polymerase alpha is modulated by the relative concentrations of ribonucleoside triphosphates.     

Rapid purification of synthetic oligonucleotides: a convenient alternative to high-performance liquid chromatography and polyacrylamide gel electrophoresis

A new method has been developed to purify and detritylate milligram amounts of synthetic oligonucleotides. Dimethoxytrityl oligonucleotides from 15 to 100 nucleotides in length are applied in triethylammonium acetate or concentrated ammonium hydroxide to a disposable chromatographic cartridge, the NENSORB PREP Nucleic Acid Purification Cartridge. Salts, failure sequences and synthetic by-products are washed away while the desired, full-length, dimethoxytrityl oligonucleotide remains bound to the cartridge. The trityl group is hydrolyzed from the 5'-end of the oligonucleotide with an acid wash and then the purified oligonucleotide is eluted with 35% methanol. Oligonucleotides are recovered salt-free with purities greater than 95%. NENSORB PREP-purified primers provide superior sequence data compared to similar primers used without purification and equivalent data to primers purified by polyacrylamide gel electrophoresis when used in manual radiometric Sanger sequencing.     

Localization of a segment of 16S RNA on the surface of the small ribosomal subunit by immune electron microscopy of complementary oligodeoxynucleotides.

Oligonucleotides that complement Escherichia coli 16S ribosomal RNA residues 685-696 and 694-705 have been synthesized so as to incorporate antibody-recognizable markers: a 3'-terminal residue of N6-delta 2-isopentenyladenosine, a 5'-dinitrophenyl group, or both. Each oligonucleotide is able to bind RNA within the small ribosomal subunit, whether free or in 70S ribosomes. Immune electron microscopy places probes at nucleotides 685, 694 and 705 within a single area, at the tip of the subunit platform, very near the position of the 3'-end of the 16S RNA.     

Determination of the reopening temperature of a DNA hairpin structure in vitro.

A novel method, based upon primer extension, has been developed for measuring the reopening temperature of a single type of DNA hairpin structure. Two DNA oligonucleotides have been utilized and designated as primers 1 and 2. Primer 1, with its 5- and 3'-termini fully complementary to the hairpin flanking sequences, was used to evaluate primer extension conditions, and primer 2, with its 3'-end competing with the DNA hairpin stem, was used to detect the DNA hairpin reopening temperature. A single DNA hairpin structure was formed on the DNA template by thermal denaturation and renaturation, and this hairpin structure was predicted to prevent the annealing of the 3'-end of primer 2 with the template DNA, which leads to no primer extension. By incubating at different temperatures, the DNA hairpin structure can be reopened at a particular temperature where the primer extension can be carried out. This resulted in the appearance of double-stranded DNA that was detected on an agarose gel. This temperature is defined here as the hairpin reopening temperature.     

Elimination of introns from the interleukin 1 beta genome by DNA amplification and expression of interleukin 1 beta in Escherichia coli]

The use of the polymerase chain reaction was proposed for intron excision from genomic genes with known nucleotide sequences. Three exons (5, 6 and 7) of genomic interleukin 1 beta gene were amplified by means of thermostable DNA polymerase TthI from Thermus thermophilus on the base of cloned in M13 phage human genomic interleukin 1 beta gene. Synthetic oligonucleotides complementary to sequences flanking exons were used as primers. The fragments obtained by exon DNA amplification were joined in the correct order due to reciprocal complementation of end sequences, that was foreseen during synthesis of oligonucleotide primers followed by amplification of the enlarged fragments. As a result the structural interleukin-1 beta gene consisting of three exons was assembled. DNA sequences carrying the ATG initiation codon and XbaI recognition site at the 5'-end, and PstI recognition site at the 3'-end (essential for insertion into the expression vector) were formed by the additional end sequences of primers. The nucleotide sequence analysis of the obtained structural gene revealed its complete identity with natural interleukin 1 beta human gene. We created the expression vector pPR114 with phage lambda promoter PR thermo-inducible in case of the cIts857 repressor presence in cells. It was used for expression of the present gene. The interleukin 1 beta synthesized in E. coli had biological activity.     

PCR fragmentation of DNA

A method has been developed to prepare random DNA fragments using PCR. First, two cycles are carried out at 16 degrees C with the Klenow's fragment and oligonucleotides (random primers) with random 3'-sequences and the 5'-constant part containing the site for cloning with the site-specific endonuclease. The random primers can link to any DNA site, and random DNA fragments are formed during DNA synthesis. During the second cycle, after denaturation of the DNA and addition of the Klenow's fragment, the random primers can link to newly synthesized DNA strands, and after DNA synthesis single-stranded DNA fragments are produced which have a constant primer sequence at the 5'-end and a complementary to it sequence at the 3'-end. During the third cycle, the constant primer is added and double-stranded fragments with the constant primer sequences at both ends are formed during DNA synthesis. Incubation for 1 h at 37 degrees C degrades the oligonucleotides used at the first stage due to endonuclease activity of the Klenow's fragment. Then routine PCR amplification is carried out using the constant primer. This method is more advantageous than hydrodynamic methods of DNA fragmentation widely used for "shotgun" cloning.     

Method for one-step preparation of double-stranded DNA template applicable for use with Pyrosequencing technology

A new one-step method for fast and efficient preparation of double-stranded DNA template, suitable for use with Pyrosequencing technology, has been developed. In the new method, two different types of oligonucleotides were used to prevent reannealing of remaining PCR primers to the template: oligonucleotides complementary to the PCR primers and 3'-end modified oligonucleotides with the same sequence as the PCR primers. Advantages with the new strategy are: (i) faster and simpler template preparation procedure (one-step); (ii) no need for exonuclease I treatment; and (iii) less problem with unspecific priming from loop structures and dimers. By careful oligonucleotide design, and/or by addition of single-stranded DNA-binding protein, problems with unspecific sequence signals due to mispriming can be reduced. The new method was used for analysis of genotype variations within the renin-angiotensin-aldosterone system.     

Microinjected oligonucleotides complementary to the alpha-sarcin loop of 28 S RNA abolish protein synthesis in Xenopus oocytes

The integrity of the alpha-sarcin loop in 28 S ribosomal RNA is critical during protein synthesis. The toxins alpha-sarcin, ricin, Shiga toxin, and Shiga-like toxin inhibit protein synthesis in oocytes by attacking specific nucleotides within this loop (Ackerman, E.J., Saxena, S. K., and Ulbrich, N. (1988) J. Biol. Chem. 263, 17076-17083; Saxena, S.K., O'Brien, A.D., and Ackerman, E.J. (1989) J. Biol. Chem. 264, 596-601). We injected Xenopus oocytes with deoxyoligonucleotides complementary to the 17-nucleotide alpha-sarcin loop of Xenopus 28 S rRNA. Only injected oligonucleotides fully covering the alpha-sarcin loop or slightly beyond inhibited oocyte protein synthesis. Shorter alpha-sarcin domain deoxyoligonucleotides complementary to the alpha-sarcin and ricin sites but not spanning the entire loop were less effective inhibitors of protein synthesis. The alpha-sarcin domain oligonucleotides covering the entire loop were more effective inhibitors of protein synthesis than injected cycloheximide at equivalent concentrations. Control oligonucleotides complementary to nine other regions of Xenopus 28 S rRNA as well as universal M13 DNA sequencing primers had no effect on oocyte protein synthesis. Oligonucleotides complementary to the highly conserved alpha-sarcin domain therefore represent an alternative to catalytic toxins for causing cell death and may prove effective in immunotherapy.     

Highly selective labeling of the E. coli RNA-polymerase promoter complex with reactive derivatives of oligonucleotide primers of various specificity

A technique of highly selective affinity labelling, which includes covalent modification of the enzyme-T7A2 promoter complex with reactive oligonucleotide derivatives and subsequent elongation of the attached oligonucleotide residue with a radioactive substrate was used to study the product-binding site of E. coli RNA polymerase. Different oligonucleotides complementary to the T7A2 promoter (with lengths ranging from 2 to 8 residues) containing 5'-terminal phosphorylating, alkylating or aldehyde groups were used for the labelling. The procedure resulted in labelling DNA and beta-, beta'- or sigma-subunits of the enzyme, which are therefore believed to contact with growing RNA in the course of initiation. Consideration of the labelling patterns as a functions of the oligonucleotide's length as well as of the structure and chemical specificity of the reactive groups led to a tentative topographic scheme of the RNA polymerase product-binding region.     

Isolation of partial complementary DNA encoding human thromboxane synthase

Thromboxane synthase catalyzes the biosynthesis of thromboxane A2 which plays a key role in the proaggregatory and vasoconstrictive processes. In this communication, we reported the successful cloning of thromboxane synthase cDNA from a human lung cDNA library. Oligonucleotides were synthesized according to the direct amino acid sequence of 2 peptides derived from purified human thromboxane synthase. Polymerase chain reaction was carried out using these oligonucleotides as primers to isolate a complementary DNA from human lung cDNA library. The longest cDNA thus obtained was 687 base pairs in length. Amino acid sequences deduced from the cDNA contained all three peptide sequences reported, confirming the authenticity of the cDNA clone.