Synthesis and reaction of DNA oligomers containing modified cytosines related to bisulfite sequencing.

Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan.
Organic Letters (Impact Factor: 6.14). 04/2009; 11(6):1377-9. DOI: 10.1021/ol900195z
Source: PubMed

ABSTRACT The synthesis of DNA oligomers containing N(4)-hydroxy-5,6-dihydrocytosine-6-sulfonate by using ligand-induced base flipping of cytosine followed by the simultaneous addition of bisulfite and hydroxylamine is reported. In contrast to C, the flipped-out 5-methylcytosine was selectively oxidized over thymines and cytosines in the duplex by potassium permanganate. Ligand-induced base flipping is a convenient and powerful strategy for the synthesis of modified cytosines and 5-methylcytosines related to bisulfite sequencing at the predetermined site of DNA.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Mismatched (non-Watson-Crick) base pairs represent the most common type of DNA damage, as they are permanently formed in living cells due to erroneous insertion, deletion and misincorporation of bases. In vivo, they are readily recognised and repaired by the proteins of the DNA mismatch repair system, which identify the mismatch sites with high efficiency and fidelity. Notably, the last decades have witnessed the development of several chemically diverse families of small organic molecules and metal complexes that selectively bind to mismatched base pairs (and not to fully paired double-stranded DNA), much like the proteins of the mismatch repair system. This review focuses on these DNA mismatch-binding ligands, with an emphasis on their (often unusual) binding modes, as well as on the similarities and differences between the different classes of mismatch binders. Also discussed are the potential bioanalytical and therapeutic applications of mismatch-binding ligands.
    Chemical Society Reviews 03/2014; · 24.89 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: DNA methylation is an epigenetic mechanism for transcriptional regulation. The methylation process controls cellular differentiation and is defective in many diseases including cancer. Therefore, the development of a simple method for analysing cytosine methylation in a target gene is required. Here we report a conceptually new method for sequence-selective chemical modification of a single cytosine in single-stranded DNA (ssDNA) using two DNA probes to form a DNA three-way junction with the ssDNA. The method was successfully used in a simple quantitative polymerase-chain-reaction-based assay for discrimination of a single methylated cytosine.
    The Analyst 03/2014; · 4.23 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The presence of the methylated nucleobase (5Me)dC in CpG islands is a key factor that determines gene silencing. False methylation patterns are responsible for deteriorated cellular development and are a hallmark of many cancers. Today genes can be sequenced for the content of (5Me)dC only with the help of the bisulfite reagent, which is based exclusively on chemical reactivity differences established by the additional methyl group. Despite intensive optimization of the bisulfite protocol, the method still has specificity problems. Most importantly ∼95% of the DNA analyte is degraded during the analysis procedure. We discovered that the reagent O-allylhydroxylamine is able to discriminate between dC and (5Me)dC. The reagent, in contrast to bisulfite, does not exploit reactivity differences but gives directly different reaction products. The reagent forms a stable mutagenic adduct with dC, which can exist in two states (E versus Z). In case of dC the allylhydroxylamine adduct switches into the E-isomeric form, which generates dC to dT transition mutations that can easily be detected by established methods. Significantly, the (5Me)dC-adduct adopts exclusively the Z-isomeric form, which causes the polymerase to stop. O-allylhydroxylamine does allow differentiation between dC and (5Me)dC with high accuracy, leading towards a novel and mild chemistry for methylation analysis.
    Nucleic Acids Research 11/2010; 38(21):e192. · 8.81 Impact Factor