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We compare two techniques which enable selective, nucleotide-specific covalent modification of human genomic DNA, as assayed by quantitative ligation- mediated PCR. In the first, a purine motif triplex-forming oligonucleotide with a terminally appended chlorambucil was shown to label a target guanine residue adjacent to its binding site in 80% efficiency at 0.5 microM. Efficiency was higher in the presence of the triplex-stabilizing intercalator coralyne. In the second method, an oligonucleotide targeting a site containing all four bases and bearing chlorambucil on an interior base was shown to efficiently react with a specific nucleotide in the target sequence. The targeted sequence in these cases was in the DQbeta1*0302 allele of the MHC II locus.

Sequence-specificity is the key to effective genetic targeting. With specificity, targeted genes can be manipulated in multiple ways; without it, gene therapy agents become loose canons within cells. Triplex forming oligonucleotides (TFOs) bind in the major groove of duplex DNA with high specificity and affinity. Because of these characteristics, TFOs have been proposed as homing devices for genetic manipulation in vivo. Here we review work demonstrating the ability of TFOs and related molecules to alter gene expression and mediate genome modification in mammalian cells. Recent studies have established that TFOs can mediate targeted gene knock out in mice, laying the foundation for the potential application of these molecules in human gene therapy. [ABSTRACT FROM AUTHOR]

Triplex structures generated by sequence-specific triplex-forming oligonucleotides (TFOs) have proven to be promising tools for gene targeting strategies. In addition, triplex technology has been highly utilized to study the molecular mechanisms of DNa repair, recombination and mutagenesis. however, triplex formation utilizing guanine-rich oligonucleotides as third strands can be inhibited by potassium-induced self-association resulting in G-quadruplex formation. We report here that guanine-rich TFOs partially substituted with 8-aza-7-deaza-guanine (PPG) have improved target site binding in potassium compared with TFOs containing the natural guanine base. We designed PPG-substituted TFOs to bind to a polypurine sequence in the supFG1 reporter gene. The binding efficiency of PPG-substituted TFOs to the target sequence was analyzed using electrophoresis mobility gel shift assays. We have determined that in the presence of potassium, the non-substituted TFO, aG30 did not bind to its target sequence, however binding was observed with the PPG-substituted aG30 under conditions with up to 140 mM Kcl. The PPG-TFOs were able to maintain their ability to induce genomic modifications as measured by an assay for gene-targeted mutagenesis. In addition, these compounds were capable of triplex-induced DNa double strand breaks, which resulted in activation of apoptosis. [ABSTRACT FROM AUTHOR]

Background: Gene targeting depends on the ability of cells to use homologous recombination to integrate exogenous DNA into their own genome. A robust mechanistic model of homologous recombination is necessary to fully exploit gene targeting for therapeutic benefit. Methodology/Principal Findings: In this work, our recently developed numerical simulation model for homology search is employed to develop rules for the design of oligonucleotides used in gene targeting. A Metropolis Monte-Carlo algorithm is used to predict the pairing dynamics of an oligonucleotide with the target double-stranded DNA. The model calculates the base-alignment between a long, target double-stranded DNA and a probe nucleoprotein filament comprised of homologous recombination proteins (Rad51 or RecA) polymerized on a single strand DNA. In this study, we considered different sizes of oligonucleotides containing 1 or 3 base heterologies with the target; different positions on the probe were tested to investigate the effect of the mismatch position on the pairing dynamics and stability. We show that the optimal design is a compromise between the mean time to reach a perfect alignment between the two molecules and the stability of the complex. Conclusion and Significance: A single heterology can be placed anywhere without significantly affecting the stability of the triplex. In the case of three consecutive heterologies, our modeling recommends using long oligonucleotides (at least 35 bases) in which the heterologous sequences are positioned at an intermediate position. Oligonucleotides should not contain more than 10% consecutive heterologies to guarantee a stable pairing with the target dsDNA. Theoretical modeling cannot replace experiments, but we believe that our model can considerably accelerate optimization of oligonucleotides for gene therapy by predicting their pairing dynamics with the target dsDNA. [ABSTRACT FROM AUTHOR]

Gene targeting by single-stranded oligodeoxyribonucleotides (ssODNs) is a promising technique for introducing site-specific sequence alterations without affecting the genomic organization of the target locus. Here, we discuss the significant progress that has been made over the last 5 years in unraveling the mechanisms and reaction parameters underlying ssODN-mediated gene targeting. We will specifically focus on ssODN-mediated gene targeting in murine embryonic stem cells (ESCs) and the impact of the DNA mismatch repair (MMR) system on the targeting process. Implications of novel findings for routine application of ssODN-mediated gene targeting and challenges that need to be overcome for future therapeutic applications are highlighted. [ABSTRACT FROM AUTHOR]

Altered gene activities are underlying causes of many neurological disorders. The ability to detect, image, and report endogenous gene transcription using magnetic resonance (MR) holds great potential for providing significant clinical benefits. In this review, we present the development of conjugates consisting of gene-targeting short nucleic acids (oligodeoxynucleotides, or sODN) and superparamagnetic iron oxide nanoparticles (SPION, an MR susceptibility T2 agent) for reporting gene activity using transcription MRI (tMRI). We will discuss 1) the target specificity of sODN, 2) selection of contrast agents for tMRI, 3) the distribution and uptake, 4) sequence specificity, 5) histology of SPION and sODN, 6) data acquisition and quantitative analysis for tMRI, and 7) application of gene transcript—targeting nanoparticles in biology and medicine. We will also discuss methods of validating the correlation between results from conventional assays (in situ hybridization, PCR, histology Prussian blue stain and immunohistochemistry) in postmortem samples and retention of SPION-sODN using tMRI. The application of our novel contrast probe to report and target gene transcripts in the mesolimbic pathways of living mouse brains after amphetamine exposure will be discussed. Because of the targeting ability in the nucleic acid sequence, the concept of tMRI probes with complementary nucleic acid (antisense DNA or short interfering RNA) allows not only tracking, targeting, binding to intracellular mRNA, and manipulating gene action but also tracing cells with specific gene action in living brains. Transcription MRI will lend itself to myriad applications in living organs. [ABSTRACT FROM AUTHOR]

Single-stranded oligodeoxynucleotide (ssODN) gene targeting may facilitate animal model creation and gene repair therapy. Lipofection of ssODN can introduce point mutations into target genes. However, typical efficiencies in mouse embryonic stem cells (ESC) are <10−4, leaving corrections too rare to effectively identify. We developed ESC lines with an integrated mutant neomycin resistance gene (Tyr22Ter). After targeting with ssODN, repaired cells survive selection in G418. Correction efficiencies varied with different lipofection procedures, clonal lines, and ssODN designs, ranging from 1 to 100 corrections per million cells plated. Uptake studies using cell sorting of Cy5-labelled ssODN showed 40% of the corrections concentrated in the best transfected 22% of cells. Four different basepair mismatches were tested and results show that the base-specificity of the mismatch is critical. Dual mismatch ssODN also showed mismatch preferences. These ESC lines may facilitate development of improved ssODN targeting technologies for either animal production or ex vivo gene therapy.Gene Therapy (2007) 14, 304–315. doi:10.1038/sj.gt.3302866; published online 5 October 2006 [ABSTRACT FROM AUTHOR]

Gene repair, the precise modification of the genome, offers a number of advantages over replacement gene therapy. In practice, gene targeting strategies are limited by the inefficiency of homologous recombination in mammalian cells. A number of strategies, including RNA-DNA oligonucleotides (RDOs) and short DNA fragments (SDFs), show promise in improving the efficiency of gene correction. We are using GFP as a reporter for gene repair in living cells. A single base substitution was introduced into GFP to create a nonsense mutation (STOP codon, W399X). RDOs and SDFs are used to repair this mutation episomally in transient transfections and restore green fluorescence. The correction efficiency is determined by FACS analysis. SDFs appear to correct GFP W399X in a number of different cell lines (COS7, A549, HT1080, HUH-7), although all at a similar low frequency (∼0.6% of transfected cells). RDOs correct only one of our cell lines significantly (HT1080-RAD51), these cells overexpress the human RAD51 gene; the bacterial RecA homologue. The GFP W399X reporter is a fusion gene with hygromycin (at the 5' end), this has allowed us to make stable cell lines (A549, HT1080) to study genomic correction. Initial studies using our correction molecules show only low efficiencies of genomic repair (∼ 10[sup -4]). Polyethylenimine (PEI) is used to deliver RDOs and SDFs into mammalian cells in culture for our study. We have used fluorescently labelled RDOs and SDFs to study the effectiveness of this process. FACS analysis of transfected nuclei implied efficient delivery (>90%) both with SDFs and RDOs. However, confocal fluorescence microscopy suggests that a large proportion of the complexed RDO/SDF appears to remain outside the nucleus (or attached to the nuclear membrane). On the basis of these data we are assessing new delivery methods and factors that may alter recombination status to optimise gene repair. [ABSTRACT FROM AUTHOR]