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Author: search.filters.author.Filichev VVSubject: search.filters.subject.DNA

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    Understanding intercalative modulation of G-rich sequence folding: solution structure of a TINA-conjugated antiparallel DNA triplex.
    (Oxford University Press, 2024-01-28) Garavís M; Edwards PJB; Serrano-Chacón I; Doluca O; Filichev VV; González C
    We present here the high-resolution structure of an antiparallel DNA triplex in which a monomer of para-twisted intercalating nucleic acid (para-TINA: (R)-1-O-[4-(1-pyrenylethynyl)phenylmethyl]glycerol) is covalently inserted as a bulge in the third strand of the triplex. TINA is a potent modulator of the hybridization properties of DNA sequences with extremely useful properties when conjugated in G-rich oligonucleotides. The insertion of para-TINA between two guanines of the triplex imparts a high thermal stabilization (ΔTM = 9ºC) to the structure and enhances the quality of NMR spectra by increasing the chemical shift dispersion of proton signals near the TINA location. The structural determination reveals that TINA intercalates between two consecutive triads, causing only local distortions in the structure. The two aromatic moieties of TINA are nearly coplanar, with the phenyl ring intercalating between the flanking guanine bases in the sequence, and the pyrene moiety situated between the Watson-Crick base pair of the two first strands. The precise position of TINA within the triplex structure reveals key TINA-DNA interactions, which explains the high stabilization observed and will aid in the design of new and more efficient binders to DNA.
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    Structure-guided inhibition of the cancer DNA-mutating enzyme APOBEC3A.
    (Springer Nature Limited, 2023-10-11) Harjes S; Kurup HM; Rieffer AE; Bayarjargal M; Filitcheva J; Su Y; Hale TK; Filichev VV; Harjes E; Harris RS; Jameson GB
    The normally antiviral enzyme APOBEC3A is an endogenous mutagen in human cancer. Its single-stranded DNA C-to-U editing activity results in multiple mutagenic outcomes including signature single-base substitution mutations (isolated and clustered), DNA breakage, and larger-scale chromosomal aberrations. APOBEC3A inhibitors may therefore comprise a unique class of anti-cancer agents that work by blocking mutagenesis, slowing tumor evolvability, and preventing detrimental outcomes such as drug resistance and metastasis. Here we reveal the structural basis of competitive inhibition of wildtype APOBEC3A by hairpin DNA bearing 2'-deoxy-5-fluorozebularine in place of the cytidine in the TC substrate motif that is part of a 3-nucleotide loop. In addition, the structural basis of APOBEC3A's preference for YTCD motifs (Y = T, C; D = A, G, T) is explained. The nuclease-resistant phosphorothioated derivatives of these inhibitors have nanomolar potency in vitro and block APOBEC3A activity in human cells. These inhibitors may be useful probes for studying APOBEC3A activity in cellular systems and leading toward, potentially as conjuvants, next-generation, combinatorial anti-mutator and anti-cancer therapies.
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    DNA with zwitterionic and negatively charged phosphate modifications: Formation of DNA triplexes, duplexes and cell uptake studies
    (Beilstein-Institut, 2021-03-29) Su Y; Bayarjargal M; Hale TK; Filichev VV; Kumar P; Brown T
    Two phosphate modifications were introduced into the DNA backbone using the Staudinger reaction between the 3',5'-dinucleoside β-cyanoethyl phosphite triester formed during DNA synthesis and sulfonyl azides, 4-(azidosulfonyl)-N,N,N-trimethylbutan-1-aminium iodide (N+ azide) or p-toluenesulfonyl (tosyl or Ts) azide, to provide either a zwitterionic phosphoramidate with N+ modification or a negatively charged phosphoramidate for Ts modification in the DNA sequence. The incorporation of these N+ and Ts modifications led to the formation of thermally stable parallel DNA triplexes, regardless of the number of modifications incorporated into the oligodeoxynucleotides (ONs). For both N+ and Ts-modified ONs, the antiparallel duplexes formed with complementary RNA were more stable than those formed with complementary DNA (except for ONs with modification in the middle of the sequence). Additionally, the incorporation of N+ modifications led to the formation of duplexes with a thermal stability that was less dependent on the ionic strength than native DNA duplexes. The thermodynamic analysis of the melting curves revealed that it is the reduction in unfavourable entropy, despite the decrease in favourable enthalpy, which is responsible for the stabilisation of duplexes with N+ modification. N+ONs also demonstrated greater resistance to nuclease digestion by snake venom phosphodiesterase I than the corresponding Ts-ONs. Cell uptake studies showed that Ts-ONs can enter the nucleus of mouse fibroblast NIH3T3 cells without any transfection reagent, whereas, N+ONs remain concentrated in vesicles within the cytoplasm. These results indicate that both N+ and Ts-modified ONs are promising for various in vivo applications.