Bulge insertions of (R)-1-O-[4-(1-pyrenylethynyl)phenylmethyl]glycerol (5) into the middle of homopyrimidine oligodeoxynucleotides (twisted intercalating nucleic acids, TINA) obtained via postsynthetic Sonogashira coupling reaction led to extraordinary high thermal stability of Hoogsteen-type triplexes and duplexes, whereas Watson-Crick-type duplexes of the same nucleotide content were destabilized. Modified oligonucleotides were synthesized using the phosphoramidite of (S)-1-(4,4'-dimethoxytriphenylmethyloxy)-3-(4-iodo-benzyloxy)-propan-2-ol followed by treatment of the oligonucleotide on a CPG-support with the Sonogashira-coupling reaction mixture containing different ethynylaryls. Bulged insertion of the pyrene derivative 5 into oligonucleotides was found to be the best among the tested modifications for binding to the Hoogsteen-type triplexes and duplexes. Thus, at pH 7.2 an oligonucleotide with cytidine content of 36% possessing two bulged insertions of 5 separated by three bases formed a stable triplex (T(m) = 43.0 degrees C), whereas the native oligonucleotide was unable to bind to the target duplex. The corresponding Watson-Crick-type duplex with the same oligonucleotide had T(m) of 38.0 degrees C at pH 7.2, while the T(m) of unmodified dsDNA was 47.0 degrees C. Experiments with mismatched oligonucleotides, luminescent properties, and potential applications of TINA technology is discussed.
APOBEC3
enzymes form part of the innate immune system by deaminating
cytosine to uracil in single-stranded DNA (ssDNA) and thereby preventing
the spread of pathogenic genetic information. However, APOBEC mutagenesis
is also exploited by viruses and cancer cells to increase rates of
evolution, escape adaptive immune responses, and resist drugs. This
raises the possibility of APOBEC3 inhibition as a strategy for augmenting
existing antiviral and anticancer therapies. Here we show that, upon
incorporation into short ssDNAs, the cytidine nucleoside analogue
2′-deoxyzebularine (dZ) becomes capable of inhibiting the catalytic
activity of selected APOBEC variants derived from APOBEC3A, APOBEC3B,
and APOBEC3G, supporting a mechanism in which ssDNA delivers dZ to
the active site. Multiple experimental approaches, including isothermal
titration calorimetry, fluorescence polarization, protein thermal
shift, and nuclear magnetic resonance spectroscopy assays, demonstrate
nanomolar dissociation constants and low micromolar inhibition constants.
These dZ-containing ssDNAs constitute the first substrate-like APOBEC3
inhibitors and, together, comprise a platform for developing nucleic
acid-based inhibitors with cellular activity.
A new quadruplex motif located in the promoter of the human KRAS gene, within a nuclease hypersensitive element (NHE), has been characterized. Oligonucleotides mimicking this quadruplex are found to compete with a DNA-protein complex between NHE and a nuclear extract from pancreatic cancer cells. When modified with (R)-1-O-[4-1-(1-pyrenylethynyl) phenylmethyl]glycerol insertions (TINA), the quadruplex oligonucleotides showed a dramatic increase of the T(m) (deltaT(m) from 22 to 32 degrees C) and a strong antiproliferative effects in Panc-1 cells.
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