Facile functionalization of peptide nucleic acids (PNAs) for antisense and single nucleotide polymorphism detection.

In this report, we show how a convenient on-resin copper-click functionalization of azido-functionalized peptide nucleic acids (PNAs) allows various PNA-based detection strategies. Firstly, a thiazole orange (TO) clicked PNA probe facilitates a binary readout when combined with F/Q labeled DNA, giving increased sensitivity for antisense detection. Secondly, our TO-PNA conjugate also allows single nucleotide polymorphism detection. Since antisense detection is also possible in the absence of the TO label, our sensing platform based on azido-d-ornithine containing PNA even allows for additional and more advanced functionalization and sensing strategies.

Chromatographic methods for the determination of therapeutic oligonucleotides.

Both DNA and RNA are being explored for their therapeutic potential against a wide range of diseases. As these new drugs emerge, new demands arise for the analysis and quantitation of these biomolecules. Pharmacokinetic and pharmacodynamic analysis requirements for drug approval place enormous challenges on the methods for analyzing these therapeutics. This review will focus on bioanalytical methods for DNA antisense and aptamers as well as small-interfering RNA (siRNA) therapeutics. Chromatography methods employing ultraviolet (UV), fluorescence and mass spectrometric (MS) detection along with matrix-assisted laser desorption/ionization (MALDI) will be covered. Sample preparation from biological matrices will be reviewed as well as metabolite analysis and identification. All of these techniques are important contributions toward oligonucleotide therapeutic development. They will also be important in microRNA (miRNA) biomarker discovery and RNomics in general, as more non-coding RNAs are inevitably discovered.

PNA/DNA interstrand cross-links from a modified PNA base upon photolysis or oxidative conditions.

PNA/DNA interstrand cross-links (ICLs) were observed when peptide nucleic acids (PNAs) containing modified thymine derivatives were hybridized with the complementary or one-base mismatched DNA upon photolysis or treatments of oxidative agent. PNA/DNA ICL formation provides a useful method for biological applications such as antisense technologies or PNA chips.

Non-coding antisense transcription detected by conventional and single-stranded cDNA microarray.

BACKGROUND: Recent studies revealed that many mammalian protein-coding genes also transcribe their complementary strands. This phenomenon raises questions regarding the validity of data obtained from double-stranded cDNA microarrays since hybridization to both strands may occur. Here, we wanted to analyze experimentally the incidence of antisense transcription in human cells and to estimate their influence on protein coding expression patterns obtained by double-stranded microarrays. Therefore, we profiled transcription of sense and antisense independently by using strand-specific cDNA microarrays. RESULTS: Up to 88% of expressed protein coding loci displayed concurrent expression from the complementary strand. Antisense transcription is cell specific and showed a strong tendency to be positively correlated to the expression of the sense counterparts. Even if their expression is wide-spread, detected antisense signals seem to have a limited distorting effect on sense profiles obtained with double-stranded probes. CONCLUSION: Antisense transcription in humans can be far more common than previously estimated. However, it has limited influence on expression profiles obtained with conventional cDNA probes. This can be explained by a biological phenomena and a bias of the technique: a) a co-ordinate sense and antisense expression variation and b) a bias for sense-hybridization to occur with more efficiency, presumably due to variable exonic overlap between antisense transcripts.

[Construction and identification of eukaryotic expression vector for sense and antisense human telomerase reverse transcriptase].

OBJECTIVE: To construct eukaryotic expression vectors for sense and antisense human telomerase reverse transcriptase (hTERT), to facilitate further study of the regulation of telomerase activity and gene therapy of malignant tumors. METHODS: According to the published hTERT cDNA sequence in Genbank, a pair of primers containing the sites for given restrictive endonuclease at both ends were designed and synthesized. Reverse transcriptional PCR (RT-PCR) of the total RNA extracted from HeLa cell line was performed, the product of which was cloned into pGEM-T vector by using TA cloning and then subcloned into a highly efficient eukaryotic expression vector pcDNA3.1 (+/-). The recombinants were finally sequenced and identified by restrictive endonuclease digestion. RESULTS: A 210-bp DNA fragment was amplified by PCR as expected. The sense and antisense hTERT eukaryotic expression vector were successfully constructed and identified by double restrictive endonuclease digestion. Sequence analysis of the inserted target fragment revealed the same sequence as that of partial hTERT cDNA published in Genbank. CONCLUSION: The sense and antisense hTERT eukaryotic expression vector has been successfully constructed.

Sense and antisense transcripts in the histone H1 (HIS-1) locus of Leishmania major.

Histone H1 in the parasitic protozoan Leishmania is a developmentally regulated protein encoded by two genes, HIS-1.1 and HIS-1.2. These genes are separated by approximately 20 kb of sequence and are located on the same DNA strand of chromosome 27. When Northern blots of parasite RNA were probed with HIS-1 strand-specific riboprobes, we detected sense and antisense transcripts that were polyadenylated and developmentally regulated. When the HIS-1.2 coding region was replaced with the coding region of the neomycin phosphotransferase gene, antisense transcription of this gene was unaffected, indicating that the regulatory elements controlling antisense transcription were located outside of the HIS-1.2 gene, and that transcription in Leishmania can occur from both DNA strands even in the presence of transcription of a selectable marker in the complementary strand. A search for other antisense transcripts within the HIS-1 locus identified an additional transcript (SC-1) within the intervening HIS-1 sequence, downstream of adenine and thymine-rich sequences. These results show that gene expression in Leishmania is not only regulated polycistronically from the sense strand of genomic DNA, but that the complementary strand of DNA also contains sequences that could drive expression of open reading frames from the antisense strand of DNA. These findings suggest that the parasite has evolved in such a way as to maximise the transcription of its genome, a mechanism that might be important for it to maintain virulence.

[Construction and identification of a tumor-specific expression vector driven by human telomerase reserve transcriptase gene promotor].

OBJECTIVE: To construct the tumor-specific expression vector driven by human telomerase reserve transcriptase gene promotor. METHODS: The fragment of the enhanced green fluorescent protein (EGFP) gene was PCR amplified from the pEGFP-N1 plasmid and cloned into the multiple cloning site of pLNCX vector, and the recombinant was named as pLNCX-EGFP. The fragment of human telomerase reserve transcriptase gene promoter was amplified from the human genome by using the human telomerase reserve transcriptase gene specific primers, and cloned into the pLNCX-EGFP vector, where the cytomegalovirus promoter was previously removed using restriction enzymes, in sense orientation relative to the green fluorescent protein coding sequence. Then the expression vector pLNT-EGFP under the control of the human telomerase reserve transcriptase gene promoter, containing green fluorescent protein reporter gene, was successfully constructed. To detect the transcriptional activity of the human telomerase reserve transcriptase gene promoter, transient transfection of this specific expression vector into HLF cell lines with high telomerase activity and WI38 cell lines without telomerase activity was performed. RESULTS: The expression vector proven by restriction enzymes digestion and sequencing was in correspondence with the design. The results of transient transfection showed that the pLNT-EGFP vector could highly expressed green fluorescent protein reporter gene in telomerase-positive cells, but not in telomerase-negative cells. CONCLUSION: A tumor-specific expression vector driven by human telomerase reserve transcriptase gene promotor has been successfully constructed.

Substrate shape specificity of E coli RNase P ribozyme is dependent on the concentration of magnesium ion.

The bacterial RNase P ribozyme can accept a hairpin RNA with CCA-3' tag sequence as well as a cloverleaf pre-tRNA as substrate in vitro, but the details are not known. By switching tRNA structure using an antisense guide DNA technique, we examined the Escherichia coli RNase P ribozyme specificity for substrate RNA of a given shape. Analysis of the RNase P reaction with various concentrations of magnesium ion revealed that the ribozyme cleaved only the cloverleaf RNA at below 10 mM magnesium ion. At 10 mM magnesium ion or more, the ribozyme also cleaved a hairpin RNA with a CCA-3' tag sequence. At above 20 mM magnesium ion, cleavage site wobbling by the enzyme in tRNA-derived hairpin occurred, and the substrate specificity of the enzyme became broader. Additional studies using another hairpin substrate demonstrated the same tendency. Our data strongly suggest that raising the concentration of metal ion induces a conformational change in the RNA enzyme.

Monitoring liposome-mediated delivery and fate of an antisense drug in cell extracts and in single cells by capillary electrophoresis with laser-induced fluorescence.

Capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection was used for evaluation of the effectiveness of delivery and fate of a model 25-mer DNA-based phosphorothiate antisense drug in cells. The antisense molecule was delivered to the cells through a simple incubation and by using a cationic liposome (Cytofectin GS 3815). The cationic lipid interacted with the negatively charged antisense to form a more positively charged complex. It was observed that uptake of the liposome-antisense complex by the cell was dependent on concentration of lipid, duration of transfection, and the cell type. The antisense drug interacted with intracellular components such as proteins and additional steps were needed to quantify the free antisense. Proteinase-K was able to release antisense from proteins. However, the addition of sodium dodecyl sulfate (SDS) to the sample or running buffer was more effective than Proteinase-K to release both naked and liposome-bound antisense from the cellular materials. Analysis of single HeLa cells for uptake of the unbound and liposome-complexed antisense revealed averages of 8.9x10(-19) moles and 4.9x10(-18) moles, respectively. The amount of uptake, however, varied greatly among individual cells and depended on the delivery method. With liposome-mediated delivery, the relative standard deviation (RSD) for the amount of antisense in individual cells was 130%, while the variation was much smaller (RSD = 45%) when the cells were incubated with the unbound antisense. These uptake variations agreed with those obtained from flow cytometry analysis.

Quantitative nuclear and cytoplasmic localization of antisense oligonucleotides by capillary electrophoresis with laser-induced fluorescence detection.

We demonstrate the use of simple extraction procedures to separate nuclear and cytoplasmic material from cell extracts, which have been scrape-loaded with a 2-O-methyl phosphorothioate antisense oligonucleotide. Separation and quantitation of the fluorescein-labeled antisense and the flourescein isothiocyanate (FITC)-dextran (molecular weight 40000) as an internal standard is done using capillary electrophoresis coupled with laser-induced fluorescence detection (CE-LIF). The bulky FITC-dextran is unable to penetrate the nuclear membrane thereby making it a quantitative indicator of any overlap between the nuclear and cytoplasmic materials during separation of the two phases. Using this procedure, the fluorescein-labeled phosphorothioate oligomer was quantitated at 4.1 x 10(-13) and 3.4x 10(-14) mol antisense/microg-total cellular protein in the nuclear and cytoplasmic extracts respectively following scrape-load delivery of the phosphorothioate to a batch of confluent HeLa cells at a concentration of 0.5 microM (5 x 10(-10) total moles of oligomer). Additionally, gene expression was monitored by measurement of the luciferase reporter protein activity. Scrape-load, spontaneous and liposomal delivery were investigated and compared for subcellular distribution of the oligomer and subsequent gene expression.

Capillary electrophoresis of drugs: current status in the analysis of pharmaceuticals.

The current status of capillary electrophoresis (CE) in pharmaceutical analyses is reviewed with about 300 references, mainly from 1996 until 1999. This article covers the use of CE for assay and purity determination of the main component, analysis of natural medicines, antisense DNA, peptides, and proteins. Analysis of hydrophobic and/or electrically neutral drugs by electrokinetic chromatography, capillary electrochromatography and nonaqueous CE is critically evaluated. Detailed techniques for the separation of enantiomers are given in the text with some actual applications. Furthermore, this review includes sensitivity and regulatory aspects for the actual use of CE in new drug applications (NDA). The analytical validation required for CE in NDA is also treated.

Demonstration and characterization of a transport system capable of lysine and leucine absorption that is encoded for in porcine jejunal epithelium by expression of mRNA in Xenopus laevis oocytes.

Defolliculated Xenopus laevis oocytes were injected with size-fractionated poly(A)+ RNA (RNA) isolated from the jejunal epithelium of growing pigs (average BW 33.8 kg) to identify proteins capable of Na+ -independent amino acid transport. The ability of oocytes to absorb L-lysine (lysine) or L-leucine (leucine) from Na+- free media was quantified in oocytes after injection of RNA fractions or water. Specific RNA fractions were identified that induced saturable uptake of lysine (Kt = 52 microM) and leucine (Kt = 97 microM), whereas endogenous oocyte uptake was not saturable. Induced uptake of .05 mM lysine by oocytes was inhibited (P < .05) 68.1% by 5 mM leucine and 38.9% by .2 mM L-cystine (cystine). Induced uptake of .05 mM leucine was inhibited (P < .05) 83.1% by 5 mM lysine and 23.2% by .2 mM cystine. Although not significant (P > .05), 5 mM L-glutamate (glutamate) quantitatively stimulated the induced uptake of .05 mM lysine by 18.8% and the induced uptake of .05 mM leucine by 60%. To identify mRNA species responsible for this bo,+ transporter-like activity, oocytes were co-injected with the RNA fractions and degenerate DNA oligomers complementary (antisense) to the cloned human kidney bo,+ amino acid transporter, or (as a negative control) with a DNA oligomer complementary to the rabbit intestinal Na+/glucose cotransporter, or with water. Only those oocytes injected with two specific RNA fractions and the antisense DNA oligomer complementary to the bo,+ transporter displayed reduced (P < .05) uptake of lysine (45.7, 55.4%) and leucine (44.1, 65.9%). These results indicate that messenger RNA encoding for a protein capable of stimulating the competitive absorption of lysine and leucine is expressed by the jejunal epithelia of growing pigs.

A targeted glucocorticoid receptor antisense transgene increases thymocyte apoptosis and alters thymocyte development.

The exquisite sensitivity of thymocytes to steroid-induced apoptosis, the steroidogenic potential of thymic epithelial cells, and the ability of steroid synthesis inhibitors to enhance antigen-specific deletion of thymocytes in fetal thymic organ cultures suggest a role for glucocorticoids in thymocyte development. To address this further, transgenic mice that express antisense transcripts to the glucocorticoid receptor (GR) specifically in immature thymocytes were generated. The consequent hyporesponsiveness of thymocytes to glucocorticoids was accompanied by a reduction in thymic size, primarily owing to a decrease in the number of CD4+CD8+ cells. While an enhanced susceptibility to T cell receptor (TCR)-mediated apoptosis appeared to be partially responsible for this reduction, thymocyte loss could also be detected before thymocytes progressed to the CD4+CD8+ TCR alpha beta-expressing stage. These results suggest that glucocorticoids are necessary for survival and maturation of thymocytes, and are consistent with a role for steroids in both the transition from CD4-CD8- to CD4+CD8+ cells and the survival of CD4+CD8+ cells stimulated via the TCR.

Expression of adrenomedullin in normal human lung and in pulmonary tumors.

Adrenomedullin (AM) is a potent hypotensive peptide recently discovered in extracts of human pheochromocytoma. In this report we present evidence, using reverse transcriptase-polymerase chain reaction, immunocytochemistry, and in situ reverse transcriptase-polymerase chain reaction, that AM is synthesized by several cell populations of the normal lung, tumor cell lines of pulmonary origin, and tumor specimens. Among the normal cell populations of the lung, we found AM expression in the columnar epithelium, some glands, neurons of the pulmonary parasympathetic nervous system, endothelial cells, chondrocytes, alveolar macrophages, and smooth muscle cells. In tumors, AM expression was located in most of the nonsmall cell lung carcinomas and in half of the small cell lung carcinomas studied. These findings suggest that AM may play a broad role in respiratory homeostasis and lung carcinogenesis.

Gene expression analysis by a competitive and differential PCR with antisense competitors.

We report a sensitive method for the reproducible and accurate measurement of gene expression from small samples of RNA. This method is based on a combination of two PCR techniques: First, an endogenous reporter gene and the gene of interest are simultaneously amplified in one tube after random-primed reverse transcription (RT) of RNA (differential RT-PCR). Second, exogenous homologous fragments of both genes with artificially introduced mutations are added and coamplified in the same reaction (competitive PCR). The first-strand cDNA, and the mutated antisense homologues of the reporter as well as the target gene compete for their respective primers and are therefore amplified with equal efficiencies. After PCR, restriction enzyme digestion allows visualization of the quantitative differences between the four resulting reaction products. The ratios of products that competed during PCR provide the quantitative information. The initial amount of a specific cDNA can be calculated from any competitor/cDNA ratio of reliably measurable PCR product amounts. Extensive competitor titration to experimentally approach the equilibrium is therefore unnecessary. The differential counterpart of competitive and differential RT-PCR (CD-RT-PCR) allows expression of the levels in reference to a reporter gene. MDR1 expression was determined in tumor cells by CD-RT-PCR.

Downregulation of endothelial cell thrombospondin 1 enhances in vitro angiogenesis.

Vascular endothelial cells are an established source of thrombospondin 1 (TSP1), a multifunctional extracellular matrix molecule. TSP1 appears to play an important role in modulating endothelial cell functions such as proliferation, migration, and capillary morphogenesis. In addition, TSP1 has recently been reported to potently inhibit angiogenesis both in vitro and in vivo. To better understand the mechanism underlying the antiangiogenic property of TSP1, endogenous TSP1 production was disrupted in bovine aortic endothelial cells (BAEC) by stable transfection with a vector expressing a TSP1 antisense RNA. Stable transfectants in which the antisense vector caused a decrease in TSP1 production were assayed for their ability to form capillary-like cords on gelled basement membrane matrix and for their responsiveness to the angiogenic/chemotactic mediator basic fibroblast growth factor. BAEC in which TSP1 production was disrupted exhibited a ten-fold increase over control BAEC in chemotactic activity to basic fibroblast growth factor and a twofold increase over control cells in the number of capillary-like cords that formed on gelled basement membrane matrix. Thus, the down-regulation of endogenous TSP1 appears to facilitate endothelial cell chemotaxis and capillary morphogenesis. These studies suggest that the modulation of TSP1 production is an important component of the angiogenic response, and support the idea that soluble TSP1 inhibits angiogenesis by interfering with endothelial cell chemotaxis and capillary formation.

Expression of antisense E1A in 293 cells results in altered cell morphologies and cessation of proliferation.

To determine whether continued E1A expression is required to maintain immortalization, 293 cells, a cell line that has been immortalized and transformed by adenovirus E1A and E1B, respectively, were transfected by an antisense E1A expression vector. In the presence of low serum, 293 cells underwent a transient, viable, nonproliferative phase wherein cells with altered morphologies were detected. However, only those clones which did not integrate the antisense sequences and exhibited a morphology similar to the original 293 cells were able to survive. High serum concentrations resulted in fewer clones, which rapidly discarded the antisense DNA. Similar experiments with an antisense CAT vector did not give rise to cells with either altered morphologies or growth rates. Furthermore, CAT antisense DNA was integrated into the 293 cell DNA. Hela cells, which were not immortalized or transformed by E1, were unaffected by the expression of antisense E1A and integrated the antisense DNA into genomic DNA. Consistently, the addition of synthetic antisense, but not sense oligonucleotides resulted in a transient inhibition of 293 cell DNA synthesis. These data demonstrate that even after extended periods of time in culture, cells immortalized by E1A still require E1A expression to activate the cell cycle and prevent them from senescing.