Disulfide-crosslinked oligonucleotides

Ferentz, Ann E.; Verdine, Gregory L.

doi:10.1021/ja00010a057

Cited by 101 publications

(60 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Oligonucleotides containing convertible nucleosides were synthesized on an Applied Biosystems 392 DNA synthesizer. The two carbon tether (cystamine) was added post-synthetically using the convertible nucleoside approach (15), reduced with excess dithiothreitol, and purified using a NAP-5 column. Incubation of the DNA with a 10-fold molar excess of 5,5Ј-dithiobis(2-nitrobenzoic acid) (Sigma) using pH 8.5 phosphate buffer and subsequent ethanol precipitation provided the activated DNA (16).…”

Section: Methodsmentioning

confidence: 99%

Integration Requires a Specific Interaction of the Donor DNA Terminal 5′-Cytosine with Glutamine 148 of the HIV-1 Integrase Flexible Loop

Johnson¹,

Santos

Pais

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

Integration is essential for retroviral replication and gene therapy using retroviral vectors. Human immunodeficiency virus, type 1 (HIV-1), integrase specifically recognizes the terminal sequences of each long terminal repeat (LTR) and cleaves the 3-end terminal dinucleotide 5-GT. The exposed 3-hydroxyl is then positioned for nucleophilic attack and subsequent strand transfer into another DNA duplex (target or chromosomal DNA). We report that both the terminal cytosine at the protruding 5-end of the long terminal repeats (5-C) and the integrase residue Gln-148 are critical for strand transfer. Proximity of the 5-C and Gln-148 was demonstrated by disulfide cross-linking. Cross-linking is inhibited by the inhibitor 5CITEP 1-(5-chloroindol-3-yl)-3-hydroxy-3-(2H-tetrazol-5-yl)-propenone. We propose that strand transfer requires a conformational change of the integrase-viral (donor) DNA complex with formation of an H-bond between the N-3 of the 5-C and the amine group of Gln-148. These findings have implications for the molecular mechanisms coupling 3-processing and strand transfer as well as for the molecular pharmacology of integrase inhibitors.

show abstract

Section: Methodsmentioning

confidence: 99%

Integration Requires a Specific Interaction of the Donor DNA Terminal 5′-Cytosine with Glutamine 148 of the HIV-1 Integrase Flexible Loop

Johnson¹,

Santos

Pais

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…B, representative gel showing inhibition of disulfide cross-link formation by the bis-DKA inhibitor (see chemical structure in Fig. 4 (Ferentz and Verdine, 1991), and the resulting cystaminemodified oligonucleotide was gel-purified. The cystamine was reduced with 100-fold molar excess of DTT for 20 min at 37°C and purified using a mini Quickspin Oligo column (Roche, Indianapolis, IN).…”

Section: Methodsmentioning

confidence: 99%

Probing HIV-1 Integrase Inhibitor Binding Sites with Position-Specific Integrase-DNA Cross-Linking Assays

Johnson¹,

Marchand²,

Patil³

et al. 2006

Mol Pharmacol

View full text Add to dashboard Cite

HIV-1 integrase binds site-specifically to the ends of the viral cDNA. We used two HIV-1 integrase-DNA cross-linking assays to probe the binding sites of integrase inhibitors from different chemical families and with different strand transfer selectivities. The disulfide assay probes cross-linking between the integrase residue 148 and the 5Ј-terminal cytosine of the viral cDNA, and the Schiff base assay probes cross-linking between an integrase lysine residue and an abasic site placed at selected positions in the viral cDNA. Cross-linking interference by eight integrase inhibitors shows that the most potent cross-linking inhibitors are 3Ј-processing inhibitors, indicating that crosslinking assays probe the donor viral cDNA (donor binding site). In contrast, strand transfer-selective inhibitors provide weak cross-linking interference, consistent with their binding to a specific acceptor (cellular DNA) site. Docking and crystal structure studies illustrate specific integrase-inhibitor contacts that prevent cross-linking formation. Four inhibitors that prevented Schiff base cross-linking to the conserved 3Ј-terminal adenine position were examined for inhibition at various positions within the terminal 21 bases of the viral cDNA. Two of them selectively inhibited upper strand cross-linking, whereas the other two had a more global effect on integrase-DNA binding. These findings have implications for elucidating inhibitor binding sites and mechanisms of action. The cross-linking assays also provide clues to the molecular interactions between integrase and the viral cDNA.

show abstract

“…It was not until early in 1991, several months prior to our first paper in this area, that Ferenz and Verdine reported synthesizing the first disulfide cross-linked oligonucleotide. 87,88 Using convertible nucleoside chemistry,…”

Section: Other Disulfide Cross-linked Nucleic Acidsmentioning

confidence: 99%

Design, synthesis, and analysis of conformationally constrained nucleic acids

Glick

1998

Biopolymers

View full text Add to dashboard Cite

In this review I discuss straightforward and general methods to modify nucleic acid structure with disulfide cross-links. A motivating factor in developing this chemistry was the notion that disulfide bonds would be excellent tools to probe the structure, dynamics, thermodynamics, folding, and function of DNA and RNA, much in the way that cystine cross-links have been used to study proteins. The chemistry described has been used to synthesize disulfide cross-linked hairpins and duplexes, higher order structures like triplexes, nonground-state conformations, and tRNAs. Since the cross-links form quantitatively by mild air oxidation and do not perturb either secondary or tertiary structure, this modification should prove quite useful for the study of nucleic acids.

show abstract

Disulfide-crosslinked oligonucleotides

Cited by 101 publications

References 0 publications

Integration Requires a Specific Interaction of the Donor DNA Terminal 5′-Cytosine with Glutamine 148 of the HIV-1 Integrase Flexible Loop

Integration Requires a Specific Interaction of the Donor DNA Terminal 5′-Cytosine with Glutamine 148 of the HIV-1 Integrase Flexible Loop

Probing HIV-1 Integrase Inhibitor Binding Sites with Position-Specific Integrase-DNA Cross-Linking Assays

Design, synthesis, and analysis of conformationally constrained nucleic acids

Contact Info

Product

Resources

About