Fluorescent oligonucleotides can serve as suitable alternatives to radiolabeled oligonucleotides

Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA.
Journal of biomolecular techniques: JBT 10/2009; 20(4):190-4.
Source: PubMed


Prolonged exposure to radiation from radionuclei used in medical research can cause DNA damage and mutation, which lead to several diseases including cancer. Radioactivity-based experiments are expensive and associated with specialized training, dedication of instruments, approvals, and cleanup with potential hazardous waste. The objective of this study was to find an alternative to the use of radioactivity in medical research using nucleic acid chemistry. FITC-labeled oligonucleotides that contain wild-type (wt) and modified base (8-oxo-G) at the same position and their complementary unlabeled strand were synthesized. Purified DNA repair enzyme, OGG1, and nuclear lysates from MCF-7 breast cancer cells were incubated with double-stranded FITC-labeled wt and 8-oxo-G oligonucleotide to demonstrate the OGG1 incision assay. We found that FITC-coupled oligonucleotides do not impose a steric hindrance during duplex formation, and the fluorescence intensity of the oligonucleotide is comparable with the intensity of the radioactive oligonucleotide. Moreover, we have seen that the OGG1 incision assay can be performed using these fluorescence oligonucleotides, replacing conventional use of radiolabeled oligonucleotides in the assay. Although the use of fluorescent-labeled oligonucleotides was described in detail for incision assays, the technique can be applied to replace a broad range of experiments, where radioactive oligonucleotides are used, eliminating the hazardous consequences of radiation.

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    • "The enhanced ability to visualize and track biomolecules has formed the cornerstone of revolutionary growth in molecular biology and biochemistry (Massoud & Gambhir, 2003; Miyakawa, Sawano, & Kogure, 2003; Prescher & Bertozzi, 2005). Over the past couple of decades , advances in fluorescence technology have provided a more accessible route to tagging and imaging of nucleic acids compared with radioactivity (Ballal, Cheema, Ahmad, Rosen, & Saha, 2009). In this regard, the discovery of small molecules that form stable and highly fluorescent complexes with DNA has been a breakthrough in the development of DNA probes (Ahn, Costa, & Emanuel, 1996; Dragan et al., 2010; Glazer, Peck, & Mathies, 1990; Zipper, Brunner, Bernhagen, & Vitzthum, 2004). "
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    ABSTRACT: The oxazole homodimer YOYO-1 has served as a valuable tool for the detection and quantification of nucleic acids. While the base specificity and selectivity of binding of YOYO-1 has been researched to some extent, the effect of unorthodox nucleic acid conformations on dye binding has received relatively less attention. In this work, we attempt to correlate the quadruplex-forming ability of G-rich sequences with binding of YOYO-1. Oligonucleotides differing in the number of tandem G repeats, total length, and length of loop sequence were evaluated for their ability to form quadruplexes in presence of sodium (Na+) or potassium (K+) ions. The fluorescence behavior of YOYO-1 upon binding such G-rich sequences was also ascertained. A distinct correlation was observed between the strength and propensity of quadruplex formation, and the affinity of YOYO-1 to bind such sequences. Specifically, as exemplified by the oligonucleotides 5′-G4T2G4-3′ and 5′-G3TG3TG3-3′, sequences possessing longer G-rich regions and shorter loop sequences formed stronger quadruplexes in presence of K+ which translated to weaker binding of YOYO-1. The dependence of binding of YOYO-1 on sequence and structural features of G-rich DNA has not been explored previously and such studies are expected to aid in more effective interpretation of applications involving the fluorophore.
    Journal of biomolecular Structure & Dynamics 07/2013; DOI:10.1080/07391102.2013.807752 · 2.92 Impact Factor
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    • "The use of oligonucleotides with a fluorescent label has been shown to be a reliable alternative to radiolabelling [17]. For primer extension assays, primer oligonucleotide (P1) with a 5′ 6-FAM label was purchased from the Keck Centre (Yale University). "
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    ABSTRACT: Error-prone DNA synthesis in prokaryotes imparts plasticity to the genome to allow for evolution in unfavorable environmental conditions, and this phenomenon is termed adaptive mutagenesis. At a molecular level, adaptive mutagenesis is mediated by upregulating the expression of specialized error-prone DNA polymerases that generally belong to the Y-family, such as the polypeptide product of the dinB gene in case of E. coli. However, unlike E. coli, it has been seen that expression of the homologs of dinB in Mycobacterium tuberculosis are not upregulated under conditions of stress. These studies suggest that DinB homologs in Mycobacteria might not be able to promote mismatches and participate in adaptive mutagenesis. We show that a representative homolog from Mycobacterium smegmatis (MsDpo4) can carry out template-dependent nucleotide incorporation and therefore is a DNA polymerase. In addition, it is seen that MsDpo4 is also capable of misincorporation with a significant ability to promote G:T and T:G mismatches. The frequency of misincorporation for these two mismatches is similar to that exhibited by archaeal and prokaryotic homologs. Overall, our data show that MsDpo4 has the capacity to facilitate transition mutations and can potentially impart plasticity to the genome.
    Journal of nucleic acids 03/2012; 2012(6):285481. DOI:10.1155/2012/285481
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    ABSTRACT: Damage to mitochondrial DNA (mtDNA) by the production of reactive oxygen species during inflammatory states, such as sepsis, is repaired by poorly understood mechanisms. To test the hypothesis that the DNA repair enzyme, 8-oxoguanine DNA glycosylase (OGG1), contributes to mtDNA repair in sepsis. Using a well-characterized mouse model of Staphylococcus aureus sepsis, we analyzed molecular markers for mitochondrial biogenesis and OGG1 translocation into liver mitochondria as well as OGG1 mRNA expression at 0, 24, 48, and 72 hours after infection. The effects of OGG1 RNA silencing on mtDNA content were determined in control, tumor necrosis factor-α, and peptidoglycan-exposed rat hepatoma cells. Based on in situ analysis of the OGG1 promoter region, chromatin immunoprecipitation assays were performed for nuclear respiratory factor (NRF)-1 and NRF-2α GA-binding protein (GABP) binding to the promoter of OGG1. Mice infected with 10(7) cfu S. aureus intraperitoneally demonstrated hepatic oxidative mtDNA damage and significantly lower hepatic mtDNA content as well as increased mitochondrial OGG1 protein and enzyme activity compared with control mice. The infection also caused increases in hepatic OGG1 transcript levels and NRF-1 and NRF-2α transcript and protein levels. A bioinformatics analysis of the Ogg1 gene locus identified several promoter sites containing NRF-1 and NRF-2α DNA binding motifs, and chromatin immunoprecipitation assays confirmed in situ binding of both transcription factors to the Ogg1 promoter within 24 hours of infection. These studies identify OGG1 as an early mitochondrial response protein during sepsis under regulation by the NRF-1 and NRF-2α transcription factors that regulate mitochondrial biogenesis.
    American Journal of Respiratory and Critical Care Medicine 01/2011; 183(2):226-33. DOI:10.1164/rccm.200911-1709OC · 13.00 Impact Factor
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