Cellular uptake, distribution, and stability of 10-23 deoxyribozymes.

Johnson and Johnson Research Laboratories, Eveleigh, Australia.
Antisense and Nucleic Acid Drug Development 11/2002; 12(5):289-99. DOI: 10.1089/108729002761381276
Source: PubMed

ABSTRACT The cellular uptake, intracellular distribution, and stability of 33-mer deoxyribozyme oligonucleotides (DNAzymes) were examined in several cell lines. PAGE analysis revealed that there was a weak association between the DNAzyme and DOTAP or Superfect transfection reagents at charge ratios that were minimally toxic to cultured cells. Cellular uptake was analyzed by cell fractionation of radiolabeled DNAzyme, by FACS, and by fluorescent microscopic analysis of FITC-labeled and TAMRA-labeled DNAzyme. Altering DNAzyme size and chemistry did not significantly affect uptake into cells. Inspection of paraformaldehyde-fixed cells by fluorescence microscopy revealed that DNAzyme was distributed primarily in punctate structures surrounding the nucleus and that substantial delivery to the nucleus was not observed up to 24 hours after initiation of transfection. Incubation in human serum or plasma demonstrated that a 3'-inversion modification greatly increased DNAzyme stability (t(1/2) approximately 22 hours) in comparison to the unmodified form (t(1/2) approximately 70 minute). The 3'-inversion-modified DNAzymes remained stable during cellular uptake, and catalytically active oligonucleotide could be extracted from the cells 24 hours posttransfection. In smooth muscle cell proliferation assay, the modified DNAzyme targeting the c-myc gene showed a much stronger inhibitory effect than did the unmodified version. The present study demonstrates that DNAzymes with a 3'-inversion are readily delivered into cultured cells and are functionally stable for several hours in serum and within cells.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 350 words maximum: (PLEASE TYPE) Today, malaria remains the biggest killer of the third world, killing over a million people every year, despite intensive research efforts. Carbamoyl phosphate synthetase II (CPSII) is the first and rate-limiting enzyme in pyrimidine biosynthesis of Plasmodium falciparum, the causative agent of malaria. PfCPSII is a unique target for DNAzyme therapy due to the presence of two unique insertion sequences of 700bp and 1800bp that exist within the mature mRNA transcript. Previous studies have demonstrated that exogenous delivery of nucleic acids such as ribozymes and DNAzymes targeting PfCPSII insertion II effectively inhibited the growth of P. falciparum cultures at sub-micromolar levels. The objective of this study was to investigate the insertion sequences within CPSII from rodent malaria species P. berghei, P. chabaudi and P. yoelii in order to further validate the insertions as DNAzyme targets in vivo. In addition, the insertions were isolated from another human malaria parasite, P. vivax. All Plasmodium CPSII genes investigated encoded two highly hydrophilic insertion sequences of similar size and nature, in the precise position seen in PfCPSII. Although these insertions are poorly conserved, border and internal regions of high homology are present.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The development of large-scale molecular computational networks is a promising approach to implementing logical decision making at the nanoscale, analogous to cellular signaling and regulatory cascades. DNA strands with catalytic activity (DNAzymes) are one means of systematically constructing molecular computation networks with inherent signal amplification. Linking multiple DNAzymes into a computational circuit requires the design of substrate molecules that allow a signal to be passed from one DNAzyme to another through programmed biochemical interactions. In this paper, we chronicle an iterative design process guided by biophysical and kinetic constraints on the desired reaction pathways and use the resulting substrate design to implement heterogeneous DNAzyme signaling cascades. A key aspect of our design process is the use of secondary structure in the substrate molecule to sequester a downstream effector sequence prior to cleavage by an upstream DNAzyme. Our goal was to develop a concrete substrate molecule design to achieve efficient signal propagation with maximal activation and minimal leakage. We have previously employed the resulting design to develop high-performance DNAzyme-based signaling systems with applications in pathogen detection and autonomous theranostics.
    PLoS ONE 10/2014; 9(10):e110986. DOI:10.1371/journal.pone.0110986 · 3.53 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Objective Doxorubicin (Dox) is used clinically against various neoplasias, but suffers from serious side effects, and for the past three decades, this shortcoming has spurred research towards finding better drug delivery systems (DDSs) for this frontline drug. Methods A non-targeted nucleotropic Dox-loaded nanoparticle (DNP) DDS is described, which has a simple chemical design, is easy to formulate and administer, is inexpensive, non-biohazardous and may prove to be useful clinically. Key findings The DNP formulated via vortex-assisted complex coarcevation enhanced (300-fold) cell-inhibitory activity of the drug in a panel of human cancer cells (osteosarcoma, breast, prostate and colorectal cancer) and enhanced (10-fold) efficacy against osteosarcoma (OS) in vivo. The slow-release DNPs localised to the endoplasmic reticulum disrupted the mitochondria and entered the nucleus. Prominent cytosolic vacuolisation, budding off of portions of the cytoplasm, both suggestive of autophagy, were observed. Mice that were administered with DNPs intratumorally had the smallest tumours at the end of the study, with more necrotic hotspots. Conclusion This promising nucleotropic DDS enhances the cell delivery and activity of Dox against a variety of human cancer cell lines and in OS tumours in mice.
    09/2014; 67(1). DOI:10.1111/jphp.12322