Bo-Ra Choi

Oligodeoxyribozymes That Cleave β-Catenin Messenger RNA Inhibit Growth of Colon Cancer Cells via Reduction of β-Catenin Response Transcription

Abnormal regulation of Wnt/β-catenin signaling followed by increased levels of the β-catenin protein have been identified in enhanced cellular proliferation and development of colon polyps and cancers. To inhibit β-catenin gene expression in colon cancer cells, RNA-cleaving oligodeoxyribozyme (DNAzyme) was employed to destroy the β-catenin mRNA. We designed a strategy to identify the cleavage sites in β-catenin RNA with a pool of random sequences from a DNAzyme library and identified four potential DNAzyme-working sites. DNAzymes were constructed for the selected target sites and were tested for the ability to cleave β-catenin RNA. When introduced into the cells, the selected DNAzymes decreased the expression of β-catenin significantly as well as its downstream gene, cyclin D1. Additionally, we designed short hairpin RNA that targets the same cleavage site for the selected DNAzyme. The designed short hairpin RNA also inhibited β-catenin gene expression in colon cancer cells. Our studies show that RNA-cleaving DNAzymes and RNA interference targeted to β-catenin significantly reduced β-catenin–dependent gene expression, resulting in inhibition of colon cancer cell growth. These results indicate that the functional antisense oligonucleotides directed against β-catenin might have potential as a therapeutic intervention to treat colon cancer. Mol Cancer Ther; 9(6); 1894–902. ©2010 AACR.

Apoptotic Cell Imaging Using Phosphatidylserine‐Specific Receptor‐Conjugated Ru(bpy)32+‐Doped Silica Nanoparticles

Apoptosis, or programmed cell death, is a normal physiological process that occurs during embryonic development; the process plays an important role in maintaining tissue homeostasis. Defective apoptosis can lead to several pathological conditions, such as neurodegenerative disorders, cardiovascular diseases, and cancers. Diagnosis of apoptosis is therefore of great importance for the early determination of therapy efficiency and the evaluation of disease progression. Traditional intracellular methods for detecting apoptosis are based on changes in caspase activity and cytoplasmic compartments. A popular extracellular method for the detection of apoptosis involves monitoring of the distribution of phospholipids on the cell surface. In particular, phosphatidylserine (PS),which usually constitutes less than 10% of the total phospholipids in cell membranes, plays an important role in phospholipid scrambling that occurs during the early stages of apoptosis. The appearance of PS on the outer leaflet of cell membranes is a universal indicator of the initial stage of apoptosis. The most popular method for detecting PS on the cell surface involves the use of annexin V (AnxV), which is a calcium-dependent 35 kDa PSbinding protein. Theuse of dye-labeledAnxVsimplifies the method and makes it effective for the detection of apoptotic cells. However, this method is expensive and has some technical limitations. For example, it has been reported that AnxV can associate with membrane surfaces containing the

Detection of single-base mutation in RNA using T4 RNA ligase-based nick-joining or DNAzyme-based nick-generation

A single nucleotide polymorphism (SNP) is a common genetic variation when a single nucleotide differs between members of a species or paired chromosome. Due to its association with disease susceptibility and drug resistance, SNP detection is of great value in studying the variation in drug responses. Here we present two quantitative SNP detection methods for a single-base mismatch in RNA, based on nick-joining and nick-generating activities of T4 RNA ligase and DNAzyme, respectively. T4 RNA ligase successfully discriminated a one-base mismatch in the ligation junction, and the designed DNAzyme cleaved RNA by discerning a single-base mismatch in the cleaving site.

Design and kinetic analysis of hammerhead ribozyme and DNAzyme that specifically cleave TEL–AML1 chimeric mRNA

In order to develop the oligonucleotides to abolish an expression of TEL-AML1 chimeric RNA, which is a genetic aberration that causes the acute lymphoblastic leukemia (ALL), hammerhead ribozymes and deoxyoligoribozymes that can specifically cleave TEL-AML1 fusion RNA were designed. Constructs of the deoxyribozyme with an asymmetric substrate binding arm (Dz26) and the hammerhead ribozyme with a 4nt-bulged substrate binding arm in the stem III (buRz28) were able to cleave TEL-AML1 chimeric RNA specifically at sites close to the junction in vitro, without cleaving the normal TEL and AML1 RNA. Single-turnover kinetic analysis under enzyme-excess condition revealed that the buRz28 is superior to the Dz26 in terms of substrate binding and RNA-cleavage. In conjunction with current progress in a gene-delivery technology, the designed oligonucleotides that specifically cleave the TEL-AML1 chimeric mRNA are hoped to be applicable for the treatment of ALL in vivo.

RETRACTED: Replication of an RNA ligase ribozyme under alternating temperature condition

Felix Wieland, Managing Editor