Intracellular production of DNA enzyme by a novel single-stranded DNA expression vector

Abstract

A set of single-stranded DNA (ssDNA) expression vectors, which can generate intracellularly any ssDNA or oligodeoxynucleotide (ODN) molecules, have been developed in our laboratory. Studies from our laboratory as well as our collaborators demonstrated that these ssDNA expression vectors are capable of producing: (1) 10-23 DNA enzyme for downregulating c-raf kinase gene expression and (2) triplexforming oligodeoxynucleotide (TFO) for inducing genomic recombination. We report here the construction of a new version of ssDNA expression vector. A b-galactosidase (bgal) reporter gene was used as a test target so that the alteration of gene expression can be easily measured using b-gal activity assay. We designed a 10-23 DNA enzyme molecule that specifically cleaves b-gal mRNA at protein translation starting site (ATG). Using a cell-free RNA cleavage assay, we confirmed that this DNA enzyme molecule could effectively cleave b-gal RNA. However, a single substitution from T to G in the catalytic domain of this DNA enzyme molecule abolished its RNA cleavage activity. We also constructed an expression vector that can generate DNA enzyme molecules in cells. A549 lung carcinoma cells were cotransfected with both DNA enzyme expression vector and the b-gal reporter gene. Compared to the cells that were transfected with the mutated DNA enzyme expression vector, significant reduction of b-gal gene expression (up to 76%) was observed in the cells transfected with DNA enzyme expression vector as indicated by the protein expression level as well as its enzyme activity. These results further suggest that the ssDNA expression vector has potential applications in the study of gene function and target validation.

Bcl-2 Protein in 518A2 Melanoma Cells In vivo and In vitro

“…The ssDNA expression system employed in this work, or its prototypes, has been successfully used to down-regulate the intracellular expression of C-raf via production of a DNAzyme (19,20), to generate a 34-mer G-rich oligomer capable of triplex formation (28), and to produce a quadruplex-forming 29-mer G-rich species (29) that has been shown to have significant antiproliferative activity (30,31). In this work, stable transfection of 518A2 melanoma cells with the pssXF-BCL2-AS plasmid produced two clones (as32 and as44) in which Bcl-2 protein and mRNA expression was significantly reduced (95-97%).…”

“…Newly synthesized reverse transcriptase, after binding of endogenous tRNA pro to the 3 ¶ terminus of the transcript, reverse transcribes the ssDNA target sequence (i.e., Bcl-2), until transcription is terminated by the stem-loop structure. Subsequently, the ssDNA is released after template degradation either by endogenous RNase H or by the RNase H activity of the reverse transcriptase (19,20).…”

“…This vector was constructed following the method of Chen and McMicken (19). To create a multiple cloning site, two oligodeoxyribonucleotides, 5 ¶-E/S/P/P-linker (5 ¶-TCGAGCGGCCAGGGGTCTC-CCGATCCCGGACGAGCCCCCAAAG-AATTCCGCGGCTGCAGTT-AAT-3 ¶) and 3 ¶-E/S/P/P-linker (5 ¶-TAAC-TGCAGCCGCGGAATTCTTTG-GGGGCTCGTCCGGGATCGGGAGACCCCTGGCCGC-3 ¶), were annealed and inserted into the PacI and XhoI sites of a ssDNA expression vector, pssXD (19). This new construct was designated as pssXF.…”

Targeting Essential Genes in Salmonella enterica Serovar Typhimurium with Antisense Peptide Nucleic Acid

“…The rising incidence of multidrug-resistant (MDR) strains provides a strong impetus to develop novel methods to combat salmonellosis. Antisense oligonucleotides (ODN) have successfully been employed in downregulating specific target genes, and they represent an innovative advancement in gene-silencing technology (5,6). Peptide nucleic acid (PNA) is an ODN analog built off a pseudopeptide backbone (19,20).…”

Peptide Nucleic Acid Antisense Oligomer as a Therapeutic Strategy against Bacterial Infection: Proof of Principle Using Mouse Intraperitoneal Infection

“…The use of oligodeoxynucleotides (ODNs) has been successful in a variety of applications to silence gene expression in eukaryotic systems for drug target validation and therapeutic purposes (3)(4)(5)11). With the development of ODN analogs such as phosphorothioate, peptide nucleic acid (PNA), and phosphodiamidate morpholino oligomer (PMO) (for reviews, see references 6 and 19), the ODN gene silencing technology has been increasingly utilized to down-regulate target genes that are critical for bacterial viability, thus inhibiting bacterial growth (14,15,31).…”