Taking control of gene expression with light-activated oligonucleotides

The recent development of caged oligonucletides that are efficiently activated by ultraviolet (UV) light creates opportunities for regulating gene expression with very high spatial and temporal resolution. By selectively modulating gene activity, these photochemical tools will facilitate efforts to elucidate gene function and may eventually serve therapeutic aims. We demonstrate how the incorporation of a photocleavable blocking group within a DNA duplex can transiently arrest DNA polymerase activity. Indeed, caged oligonucleotides make it possible to control many different protein-oligonucleotide interactions. In related experiments, hybridization of a reverse complementary (antisense) oligodeoxynucleotide to target mRNA can inhibit translation by recruiting endogenous RNases or sterically blocking the ribosome. Our laboratory recently synthesized caged antisense oligonucleotides composed of phosphorothioated DNA or peptide nucleic acid (PNA). The antisense oligonucleotide, which was attached to a complementary blocking oligonucleotide strand by a photocleavable linker, was blocked from binding target mRNA. This provided a useful method for photomodulating hybridization of the antisense strand to target mRNA. Caged DNA and PNA oligonucleotides have proven effective at photoregulating gene expression in cells and zebrafish embryos.     

A novel potent strategy for gene delivery using a single peptide vector as a carrier.

We have shown previously that a peptide, MPG, derived from the hydrophobic fusion peptide of HIV-1 gp41 and the hydrophilic nuclear localisation sequence of SV40 large T antigen, can be used as a powerful tool for the delivery of oligonucleotides into cultured cells. Now we extend the potential of MPG to the delivery of nucleic acids into cultured cells. In vitro, MPG interacts strongly with nucleic acids, most likely forming a peptide cage around them, which stabilises and protects them from degradation in cell culture media. MPG is non-cytotoxic, insensitive to serum and efficiently delivers plasmids into several different cell lines in only 1 h. Moreover, MPG enables complete expression of the gene products encoded by the plasmids it delivers into cultured cells. Finally, we have investigated the potential of MPG as an efficient delivery agent for gene therapy, by attempting to deliver antisense nucleic acids targeting an essential cell cycle gene. MPG efficiently delivered a plasmid expressing the full-length antisense cDNA of human cdc25C, which consequently successfully reduced cdc25C expression levels and promoted a block to cell cycle progression. Based on our results, we conclude that MPG is a potent delivery agent for the generalised delivery of nucleic acids as well as of oligonucleotides into cultured cells and believe that its contribution to the development of new gene therapy strategies could be of prime interest.     

Regulation of in vitro gene expression using antisense oligonucleotides or antisense expression plasmids transfected using starburst PAMAM dendrimers.

Starburst polyamidoamine (PAMAM) dendrimers are a new type of synthetic polymer characterized by a branched spherical shape and a high density surface charge. We have investigated the ability of these dendrimers to function as an effective delivery system for antisense oligonucleotides and 'antisense expression plasmids' for the targeted modulation of gene expression. Dendrimers bind to various forms of nucleic acids on the basis of electrostatic interactions, and the ability of DNA-dendrimer complexes to transfer oligonucleotides and plasmid DNA to mediate antisense inhibition was assessed in an in vitro cell culture system. Cell lines that permanently express luciferase gene were developed using dendrimer mediated transfection. Transfections of antisense oligonucleotides or antisense cDNA plasmids into these cell lines using dendrimers resulted in a specific and dose dependent inhibition of luciferase expression. This inhibition caused approximately 25-50% reduction of baseline luciferase activity. Binding of the phosphodiester oligonucleotides to dendrimers also extended their intracellular survival. While dendrimers were not cytotoxic at the concentrations effective for DNA transfer, some non-specific suppression of luciferase expression was observed. Our results indicate that Starburst dendrimers can be effective carriers for the introduction of regulatory nucleic acids and facilitate the suppression of the specific gene expression.     

LNA-modified oligonucleotides effectively drive intramolecular-stable hairpin to intermolecular-duplex state

Sequence-specific hybridization of antisense and antigene agent to the target nucleic acid is an important therapeutic strategy to modulate gene expression. However, efficiency of such agents falls due to inherent intramolecular-secondary-structures present in the target that pose competition to intermolecular hybridization by complementary antisense/antigene agent. Performance of these agents can be improved by employing structurally modified complementary oligonucleotides that efficiently hybridize to the target and force it to transit from an intramolecular-structured-state to an intermolecular-duplex state. In this study, the potential of variably substituted locked nucleic acid-modified oligonucleotides (8mer) to hybridize and disrupt highly stable, secondary structure of nucleic acid has been biophysically characterized and compared with the conventionally used unmodified DNA oligonucleotides. The target here is a stem-loop hairpin oligonucleotide-a structure commonly present in most structured-nucleic acids and known to exhibit an array of biological functions. Using fluorescence-based studies and EMSA we prove that LNA-modified oligonucleotides hybridize to the target hairpin with higher binding affinity even at lower concentration and subsequently, force it to assume a duplex conformation. LNA-modified oligonucleotides may thus, prove as potential therapeutic candidates to manipulate gene expression by disruption of biologically relevant nucleic acid secondary structure.     

Exogenous nucleic acids aggregate in non-P-body cytoplasmic granules when transfected into cultured cells

To modulate gene expression in research studies or in potential clinical therapies, transfection of exogenous nucleic acids including plasmid DNA and small interference RNA (siRNA) are generally performed. However, the cellular processing and the fate of these nucleic acids remain elusive. By investigating the cellular behavior of transfected nucleic acids using confocal imaging, here we show that when siRNA was co-transfected into cultured cells with other nucleic acids, including single-stranded RNA oligonucleotides, single and double-stranded DNA oligonucleotides, as well as long double-stranded plasmid DNA, they all aggregate in the same cytoplasmic granules. Interestingly, the amount of siRNA aggregating in granules was found not to correlate with the gene silencing activity, suggesting that assembly of cytoplasmic granules triggered by siRNA transfection may be separable from the siRNA silencing event. Our results argue against the claim that the siRNA-aggregating granules are the functional site of RNA interference (RNAi). Taken together, our studies suggest that, independent of their types or forms, extraneously transfected nucleic acids are processed through a common cytoplasmic pathway and trigger the formation of a new type of cytoplasmic granules “transfection granules”.     

Design of nuclease resistant protein kinase calpha DNA enzymes with potential therapeutic application

For the therapeutic application of catalytic nucleic acids it is desirable to have small, stable and inexpensive compounds that are active at physiological Mg(2+) concentrations. We have explored the possibility of using the versatile 10-23 DNA catalytic core to suppress the expression of the protein kinase Calpha (PKCalpha) isoform in malignant cells. By introducing either a 3'-3'-inverted thymidine nucleotide or site-specific phosphorothioate modification into a PKCalpha DNA enzyme, we have designed stable catalysts that retained a significant in vitro cleavage activity. In particular, a DNA enzyme containing phosphorothioate analogues in the antisense arms and in the pyrimidine residues of the catalytic core was found to be remarkably stable in 50 % human serum (t(1/2)>90 hours) and inhibited in vitro cell growth by up to 90 % at nanomolar concentrations. The inhibition of PKCalpha gene expression is sequence-specific, as a DNA enzyme with reversed antisense arms was found to be ineffective. Epifluorescence microscopic analysis of cells transfected with a 5' fluorescein isothiocyanate-conjugated DNA enzyme showed that the DNA enzyme molecules are mainly localised in the nuclei. Most of the DNA enzyme-treated cells were killed by apoptosis. The ability of the described PKCalpha DNA enzymes to trigger apoptosis (apoptozymes) in malignant cells illustrates their therapeutic potential. Furthermore, such agents can be a valuable tool for probing gene function.     

Enhanced delivery of antisense oligonucleotides with fluorophore-conjugated PAMAM dendrimers

PAMAM dendrimers are cationic polymers that have been used for the delivery of genes and oligonucleotides to cells. However, little is known about the behavior of dendrimer–nucleic acid complexes once they reach the cell interior. To pursue this issue, we prepared dendrimers conjugated with the fluorescent dye Oregon green 488. These were used in conjunction with oligonucleotides labeled with a red (TAMRA) fluorophore in order to visualize the sub-cellular distribution of the dendrimer–oligonucleotide complex and of its components by two-color digital fluorescence microscopy. The 2′-O-methyl antisense oligonucleotide sequence used in these studies was designed to correct splicing at an aberrant intron inserted into a luciferase reporter gene; thus effective delivery of the antisense agent results in the expression of the reporter gene product. The dendrimer–oligonucleotide complex remained associated during the process of uptake into vesicular compartments and eventual entry into the nucleus. Since the pharmacological activity of the antisense compound was manifest under these conditions, it suggests that the dendrimer–oligonucleotide complex is functionally active. A surprising result of these studies was that the Oregon green 488-conjugated dendrimer was a much better delivery agent for antisense compounds than unmodified dendrimer. This suggests that coupling of relatively hydrophobic small molecules to PAMAM dendrimers may provide a useful means of enhancing their capabilities as delivery agents for nucleic acids.     

Photocleavable linkage between genotype and phenotype for rapid and efficient recovery of nucleic acids encoding affinity-selected proteins

In vitro display technologies, such as mRNA display and DNA display are powerful tools to screen peptides and proteins with desired functions from combinatorial libraries in the fields of directed protein evolution and proteomics. When screening combinatorial libraries of polypeptides (phenotype), each of which is displayed on its gene (genotype), the problem remains, how best to recover the genotype moiety whose phenotype moiety has bound to the desired target. Here, we describe the use of a photocleavable 2-nitrobenzyl linker between genotype (DNA or mRNA) and phenotype (protein) in our DNA and mRNA display systems. This technique allows rapid and efficient recovery of selected nucleic acids by simple UV irradiation at 4 degrees C for 15 min. Further, we confirmed that the photocleavable DNA display and mRNA display systems are useful for in vitro selection of epitope peptides, recombinant antibodies, and drug-receptor interactions. Thus, these improved methods should be useful in therapeutics and diagnostics, e.g., for screening high-affinity binders, such as enzyme inhibitors and recombinant antibodies from random peptide and antibody libraries, as well as for screening drug-protein interactions from cDNA libraries.     

Covalent coupling of a PIM-1 oncogene targeted PNA with an antennapedia derived peptide

Peptide nucleic acids (PNA) are promising antisense molecule for blocking gene expression in cell culture or in vivo. Nevertheless because they are poor efficient to pass the cellular membrane, it is necessary to use a vectorisation agent to observe an inhibitory effect. We describe the coupling of the rhodamine labeled 17-mer antisense PNA to a fusogenic peptide from antenapedia via S-S linkage, the studies of the penetration of this complex into fibroblast cells and its inhibitory effect on pim1 targeted protononcogene.     

Inhibition of gene expression in Entamoeba histolytica with antisense peptide nucleic acid oligomers

Peptide nucleic acids (PNAs) may be a potent tool for gene function studies in medically important parasitic organisms, especially those that have not before been accessible to molecular genetic knockout approaches. One such organism is Entamoeba histolytica, the causative agent of amebiasis, which infects about 500 million people and is the cause of clinical disease in over 40 million each year, mainly in the tropical and subtropical world. We used PNA antisense oligomers to inhibit expression of an episomally expressed gene (neomycin phosphorotransferase, NPT) and a chromosomal gene (EhErd2, a homolog of Erd2, a marker of the Golgi system in eukaryotic cells) in axenically cultured trophozoites of E. histolytica. Measurement of NPT enzyme activity and EhErd2 protein levels, as well as measurement of cellular proliferation, revealed specific decreases in expression of the target genes, and concomitant inhibition of cell growth, in trophozoites treated with micromolar concentrations of unmodified antisense PNA oligomers.     

Peptide nucleic acids (PNAs) antisense effect to bacterial growth and their application potentiality in biotechnology

Peptide nucleic acids (PNAs) are nucleic acid analogs having attractive properties such as quiet stability against nucleases and proteases, and they form strong complexes with complementary strands of DNA or RNA. Because of this attractive nature, PNA is often used in antisense technology to inhibit gene expression and microbial cell growth with high specificity. Many bacterial antisense or antiribosomal studies using PNA oligomers have been reported so far, and parameters to design effective antisense PNAs and to improve PNA cell entry for efficient inhibition of bacterial growth have been presented. However, there are still several obstacles such as low cellular uptake of PNA while applying antisense PNAs to a complex microbial community. On overcoming these problems, the PNA antisense technique might become a very attractive tool not only for controlling the microbial growth but also for further elucidating microbial ecology in complex microbial consortia. Here, we summarize and present recent studies on the development of antimicrobial PNAs targeting mRNAs and rRNAs. In addition, the application potentiality of antisense techniques in nonclinical biotechnology fields is discussed.     

Zif268的锌指DNA结合区在大肠杆菌中的功能性表达

锌指蛋白是最大的蛋白家族,是识别核酸最常见的、最有效的结构元件。通过选择合适的表达载体及诱导表达条件,实现了小鼠转录因子Zif268的锌指DNA结合区在大肠杆菌中的部分可溶性表达。凝胶迁移率移动试验证实纯化的可溶部分锌指DNA结合区可以特异性识别、结合其天然靶序列,提示锌指DNA结合区在大肠杆菌中得到了功能性表达。锌指DNA结合区在大肠杆菌中的功能性表达成功为锌指蛋白DNA相互作用的胞内遗传筛选模型的建立奠定了基础。     

Covalent Coupling of a PIM-1 Oncogene Targeted PNA with an Antennapedia Derived Peptide

Abstract

Peptide nucleic acids (PNA) are promising antisense molecule for blocking gene expression in cell culture or in vivo. Nevertheless because they are poor efficient to pass the cellular membrane, it is necessary to use a vectorisation agent to observe an inhibitory effect. We describe the coupling of the rhodamine labeled 17-mer antisense PNA to a fusogenic peptide from antenapedia via S-S linkage, the studies of the penetration of this complex into fibroblast cells and its inhibitory effect on piml targeted protononcogene.     

Comparative evaluation of target-specific GFP gene silencing efficiencies for antisense ODN, synthetic siRNA, and siRNA plasmid complexed with PEI-PEG-FOL conjugate

Cell specific gene silencing effects of antisense oligodeoxynucleotide (AS-ODN), synthetic small interfering RNA (siRNA-S), and siRNA expressing plasmid (siRNA-P) were comparatively evaluated. Poly(ethylenimine) (PEI) and PEI-graft-poly(ethylene glycol)-folate (PEI-PEG-FOL) conjugate were used to form nanosized polyelectrolyte complexes with the above three nucleic acids coding for inhibition of green fluorescent protein (GFP) expression. The three nucleic acid complexes formulated with either PEI or PEI-PEG-FOL had comparable sizes and surface zeta potential values. Among the three inhibitory nucleic acids, siRNA-S, when complexed with PEI-PEG-FOL, exhibited the most dose-effective and fastest gene silencing effect for FOL receptor overexpressing KB cells, because the siRNA-S could be directly delivered, via FOL receptor-mediated endocytosis, into the cytoplasm compartment where the degradation processing of target GFP mRNA occurred in a sequence-specific manner.