Bashar Mullah

AN EFFICIENT METHOD FOR THE ISOLATION AND PURIFICATION OF OLIGORIBONUCLEOTIDES

Abstract Problems associated with the use of tetrabutylammonium fluoride like incomplete desilylation and removal of the tetrabutylammonium salts during large scale syntheses of oligoribonucleotides (RNA) have been eliminated by the use of triethylamine trihydrofluoride and precipitation of the RNA with 1-butanol. An efficient anion-exchange HPLC method has been developed for the purification of chemically synthesized RNA and the resulting product precipitated directly by the addition of 1-propanol. A new activator, 5-ethylthio-1H-tetrazole significantly enhances the synthesis quality and yield of oligoribonucleotides. RNA synthesized using these improvements has been shown to be biologically active by a comparative ribozyme-substrate assay.

Differential Dependence of the D1 and D5 Dopamine Receptors on the G Protein γ7 Subunit for Activation of Adenylylcyclase

The D1 dopamine receptor, G protein γ7 subunit, and adenylylcyclase are selectively expressed in the striatum, suggesting their potential interaction in a common signaling pathway. To evaluate this possibility, a ribozyme strategy was used to suppress the expression of the G protein γ7 subunit in HEK 293 cells stably expressing the human D1 dopamine receptor. Prior in vitro analysis revealed that the γ7 ribozyme possessed cleavage activity directed exclusively toward the γ7 RNA transcript (Wang, Q., Mullah, B., Hansen, C., Asundi, J., and Robishaw, J. D. (1997)J. Biol. Chem. 272, 26040–26048). In vivoanalysis of cells transfected with the γ7 ribozyme showed a specific reduction in the expression of the γ7 protein. Coincident with the loss of the γ7 protein, there was a noticeable reduction in the expression of the β1 protein, confirming their interaction in these cells. Finally, functional analysis of ribozyme-mediated suppression of the β1 and γ7 proteins revealed a significant attenuation of SKF81297-stimulated adenylylcyclase activity in D1 dopamine receptor-expressing cells. By contrast, ribozyme-mediated suppression of the β1 and γ7 proteins showed no reduction of SKF81297-stimulated adenylylcyclase activity in D5 dopamine receptor-expressing cells. Taken together, these data indicate that the structurally related D1 and D5 dopamine receptor subtypes utilize G proteins composed of distinct βγ subunits to stimulate adenylylcyclase in HEK 293 cells. Underscoring the physiological relevance of these findings, single cell reverse transcriptase-polymerase chain reaction analysis revealed that the D1 dopamine receptor and the G protein γ7 subunit are coordinately expressed in substance P containing neurons in rat striatum, suggesting that the G protein γ7 subunit may be a new target for drugs to selectively alter dopaminergic signaling within the brain.

Automated synthesis of double dye-labeled oligonucleotides using tetramethylrhodamine (TAMRA) solid supports

TAMRA fluorescent dye-labeled non-nucleosidic synthesis supports have been derivatized for automated synthesis of 3′ dye-labeled oligonucleotides.1 Synthesis with 5′ fluorescent dye phosphoramidites leads to 5′ and 3′ double labeled oligonucleotides, useful in Taqman assay and FRET experiments. Cleavage and deprotection of dye-labeled oligonucleotides are carried out with tert-butylamine:methanol:water (1:1:2) without any degradation and/or modification of dyes and nucleobases.

RNA facilitates RecA-mediated DNA pairing and strand transfer between molecules bearing limited regions of homology.

The RecA protein ofEscherichia coli catalyzes homologous pairing and strand exchange between a wide range of molecules showing nucleotide sequence complementarity, including a linear duplex and a single-stranded DNA molecule. We demonstrate that RecA can promote formation of joint molecules when the duplex contains an RNA/DNA hairpin and a single-stranded circle serves as the pairing partner. A chimeric RNA/DNA hairpin can be used to form stable joint molecules with as little as 15 bases of shared homology as long as the RNA stretch contains complementarity to the circle. The joint molecule bears some resemblance to a triple helical structure composed of RNA residues surrounded by two DNA strands which are in a parallel orientation. Evidence is presented that supports the notion that short stretches of RNA can be used in homologous pairing reactions at lengths below that required for DNA-DNA heteroduplex formation.

EFFICIENT AUTOMATED SYNTHESIS OF MOLECULAR BEACONS

Abstract Automated synthesis of molecular beacons using 4-(4-dimethylamino phenylazo) benzoic acid (dabcyl) support and phosphoramidite is described.

Purification of 5′-O-Trityl-on Oligoribonucleotides. Investigation of Phosphate Migration During Purification and Detritylation.

Abstract Synthetic oligoribonucleotides (RNA) are efficiently prepared with 2′-O-tert-butyldimethylsilyl nucleoside 3′-O-phosphoramidites with labile base-protection; Admf or APac, Gdmf, Cibu, U. After cleavage from the polystyrene support, the exocyclic amine protecting groups are removed with conc. NH4OH: ethanol/3:1 by heating at 55°C for 3–5 h. The 2′-O- silyl protecting groups are removed with tetra-n-butylammonium fluoride in THF or more conveniently with neat triethylamine trihydrofluoride. To gain the advantages of increased capacity on reverse phase HPLC and the convenience of cartridge based purification (OPC, Oligonucleotide Purification Cartridge), the 5′ trityl was left on the RNA as the final protecting group to be removed. The mild conditions which are effective for trityl removal are shown to preserve 3′-5′ phosphate linkage integrity in RNA. The absence of phosphate migration is demonstrated by model studies, utilizing N4 -isobutyryl-5′-O-DMT-3′-O-TBDMS-2′-O-(2-cyanoethyl-N,N-diisopropylp...

Oxidative conversion of N-dimethylformamidine nucleosides to N-cyano nucleosides

Abstract Reaction of the N -dimethylformamidine (dmf) derivatives of 2′-deoxyguanosine, guanosine, and 2′-deoxyadenosine with iodine and aqueous ammonia gives the corresponding N -cyano nucleosides. This reaction occurs in oligonucleotides under conditions where iodine is reatined on the solid support, or in the synthesis column, prior to cleavage with aqueous ammonia. This base modification can be eliminated with lower iodine concentration in the oxidation reagent.

Oxidation of biotin during oligonucleotide synthesis.

A new “polystyrene biotin support” has been synthesized for the solid support synthesis of the 3′-biotinylated oligonucleotides. Several oligos were synthesized and were analyzed by the HPLC and Mass Spec. Oligo analysis revealed that the biotin gets oxidized to “biotin sulfoxide” during the synthesis.

Quantitative PCR Technology

Higuchi et al. (1992, 1993) pioneered the analysis of PCR kinetics by constructing a system that detects PCR products as they accumulate. This “real-time” system includes the intercalator ethidium bromide in each amplification reaction, an adapted thermal cycler to irradiate the samples with ultraviolet light, and detection of the resulting fluorescence with a computer-controlled cooled CCD camera. Amplification produces increasing amounts of double-stranded DNA, which binds ethidium bromide, resulting in an increase in fluorescence. By plotting the increase in fluorescence versus cycle number, the system produces amplification plots that provide a more complete picture of the PCR process than assaying product accumulation after a fixed number of cycles.

Targeted Gene Correction: Synthesis and Characterization of Double-Hairpin 2′-O-Methyl RNA/DNA Chimera Oligonucleotides

Abstract A series of 2′-O-methyl RNA/DNA chimera oligonucleotides were synthesized with a double-hairpin structural motif. Liposome formulated delivery of the chimeras effected targeted, high conversion of mutant alleles in mammalian cell culture. The chimera oligonucleotides were prepared with DNA and 2′-OMe RNA phosphoramidite nucleoside monomers on the ABI 394 synthesizer.