Specific conjugation of DNA binding proteins to DNA templates through thiol–disulfide exchange

Metelev, Valeri; Кубарева, Е. А.; Воробьева, О. В.; Romanenkov, A. S.; Oretskaya, Tatiana S.

doi:10.1016/s0014-5793(03)00122-4

Cited by 24 publications

(9 citation statements)

References 18 publications

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“…Oligonucleotides containing phosphoryldisulfide group (PDG) in a specified position of the sugar phos phate backbone were prepared by chemical ligation as pre viously described [21,22]. We synthesized DNA duplexes XII and XIII containing PDG(pSS) in the Ig κ κB site ( Table 2).…”

Section: ′ G a G C C T T T C A G G G G A G A 5′mentioning

confidence: 99%

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Analysis of DNA-Protein Interactions in Complexes of Transcription Factor NF-κB with DNA

Romanenkov

Ustyugov

Zatsepin

et al. 2005

Biochemistry (Moscow)

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We have applied bioinformatic analysis of X-ray 3D structures of complexes of transcription factor NF-kappaB with DNAs. We determined the number of possible Van der Waals contacts and hydrogen bonds between amino acid residues and nucleotides. Conservative contacts in the NF-kappaB dimer-DNA complex composed of p50 and/or p65 NF-kappaB subunit and DNA sequences like 5 -GGGAMWTTCC-3 were revealed. Based on these results, we propose a novel scheme for interactions between NF-kappaB p50 homodimer and the kappaB region of the immunoglobulin light chain gene enhancer (Ig-kappaB). We applied a chemical cross-linking technique to study the proximity of some Lys and Cys residues of NF-kappaB p50 subunit with certain reactive nucleotides into its recognition site. In all cases, the experimentally determined protein-DNA contacts were in good agreement with the predicted ones.

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Section: ′ G a G C C T T T C A G G G G A G A 5′mentioning

confidence: 99%

“…3b) [21,22]. Cross linking of 3′ 32 P labeled duplexes XII XIV with NF κB p50 homodimer was performed in a buffer, pH 7.5, for 30 min in the absence of reducing agents.…”

Section: ′ G a G C C T T T C A G G G G A G A 5′mentioning

confidence: 99%

Analysis of DNA-Protein Interactions in Complexes of Transcription Factor NF-κB with DNA

Romanenkov

Ustyugov

Zatsepin

et al. 2005

Biochemistry (Moscow)

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“…[4] Other proteins that have been used in DNAo rigami assemblies are DNA-antibody conjugates [5] and engineered proteins containing His-, [6] Snap-, and Halo-tags. [7] Other covalent conjugation techniques such as coupling by transglutaminase [8] or methyltransferases, [9] expressed protein ligation, and enzymatic ligation [10] also provide site-specific conjugation methods with controlled stoichiometry.A lternatively,c hemical crosslinkers containing maleimide or Nhydroxysuccinimide groups are extensively used for nonspecific covalent coupling (for amore complete overview see Ref. [11]).…”

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confidence: 99%

Orthogonal Protein Assembly on DNA Nanostructures Using Relaxases

et al. 2016

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DNA‐binding proteins are promising reagents for the sequence‐specific modification of DNA‐based nanostructures. Here, we investigate the utility of a series of relaxase proteins—TrwC, TraI, and MobA—for nanofunctionalization. Relaxases are involved in the conjugative transfer of plasmids between bacteria, and bind to their DNA target sites via a covalent phosphotyrosine linkage. We study the binding of the relaxases to two standard DNA origami structures—rodlike six‐helix bundles and flat rectangular origami sheets. We find highly orthogonal binding of the proteins with binding yields of 40–50 % per binding site, which is comparable to other functionalization methods. The yields differ for the two origami structures and also depend on the position of the binding sites. Due to their specificity for a single‐stranded DNA target, their orthogonality, and their binding properties, relaxases are a uniquely useful addition to the toolbox available for the modification of DNA nanostructures with proteins.

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“…It is well-known that disulfide bonds can form between Cys-bearing proteins and 5Ј-thiol groups of synthetic DNA (47)(48)(49). The formation of disulfide bonds between Cys residues and internal phosphorothioate groups would be expected to be strongly affected by steric hindrance and nucleophilic properties.…”

Section: Discussionmentioning

confidence: 99%

Cysteine-Tailed Class I-Binding Peptides Bind to CpG Adjuvant and Enhance Primary CTL Responses

Wettstein¹,

Borson²,

Park

et al. 2005

The Journal of Immunology

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Immunostimulatory CpG motifs in synthetic oligonucleotides can be effective adjuvants for the priming of CTLs. We first observed that a single male-specific peptide (KCSRNRQYL) (HY2) was more efficient than another male-specific peptide (WMHHNMDLI) (HY1) at priming IFN-γ-secreting CTLs in vivo when combined with lipid A and CpG and that it also visibly precipitated CpG. The addition of the six N-terminal residues (KCSRNR) from HY2 to HY1 yielded a peptide, KCSRNR-HY1, that both precipitated CpG and primed increased numbers of HY1-specific CTLs. We refer to this type of peptide as a primotope that includes a class I binding peptide tailed with amino acids that increase priming. Ala residues were substituted for the Arg/Lys residues (ACSANA-HY1), and these substitutions did not reduce in vivo priming potential. However, the substitution of Ala for Cys (KASRNR-HY1) resulted in the complete loss of priming, demonstrating the importance of Cys for in vivo priming when mixed with CpG. This result suggested that increased priming was based in disulfide bonding between Cys residues and internal phosphorothioate groups of synthetic CpG. The addition of Cys-bearing primotopes to radiolabeled CpG with a single thioate group resulted in the appearance of a new band that was inhibited by 1) Cys > Ala substitution and 2) reduction and alkylation of CpG. These results reveal a novel mechanism for complexing class I binding peptides and CpG adjuvant for development of new peptide-adjuvant combinations for vaccines for cancer and infectious diseases.

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Specific conjugation of DNA binding proteins to DNA templates through thiol–disulfide exchange

Cited by 24 publications

References 18 publications

Analysis of DNA-Protein Interactions in Complexes of Transcription Factor NF-κB with DNA

Analysis of DNA-Protein Interactions in Complexes of Transcription Factor NF-κB with DNA

Orthogonal Protein Assembly on DNA Nanostructures Using Relaxases

Cysteine-Tailed Class I-Binding Peptides Bind to CpG Adjuvant and Enhance Primary CTL Responses

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