Facile preparation of nuclease resistant 3′ modified oligodeoxynucleotides

Gamper, Howard; Reed, Michael W.; Cox, Thomas C.; Virosco, J S; Adams, Alan D.; Gall, A. A.; Scholler, John; Meyer, Rich B.

doi:10.1093/nar/21.1.145

Cited by 127 publications

(123 citation statements)

References 26 publications

(63 reference statements)

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“…It has been shown that oligonucleotides containing cholesterol adducts at the 3Ј-terminus are cytotoxic (41,42). Conjugation of the cholesterol group increases the cellular uptake of the antisense oligonucleotide and improves its nuclease resistance (43). Finally it has been demonstrated that the addition of a cholesterol group at the 5Ј-end of self-complementary oligonucleotides increases the stability of the duplex, probably via better stacking interactions with the terminal base pairs (44).…”

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confidence: 99%

Effect of Bulky Lesions on DNA

Gómez‐Pinto

Cubero

Kalko

et al. 2004

Journal of Biological Chemistry

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The three-dimensional solution structure of two DNA decamers of sequence d(CCACXGGAAC)-(GTTCCG-GTGG) with a modified nucleotide containing a cholesterol derivative (X) in its C1(chol)␣ or C1(chol)␤ diastereoisomer form has been determined by using NMR and restrained molecular dynamics. This DNA derivative is recognized with high efficiency by the UvrB protein, which is part of the bacterial nucleotide excision repair, and the ␣ anomer is repaired more efficiently than the ␤ one. The structures of the two decamers have been determined from accurate distance constraints obtained from a complete relaxation matrix analysis of the NOE intensities and torsion angle constraints derived from J-coupling constants. The structures have been refined with molecular dynamics methods, including explicit solvent and applying the particle mesh Ewald method to properly evaluate the long range electrostatic interactions. These calculations converge to well defined structures whose conformation is intermediate between the A-and B-DNA families as judged by the root mean square deviation but with sugar puckerings and groove shapes corresponding to a distorted B-conformation. Both duplex adducts exhibit intercalation of the cholesterol group from the major groove of the helix and displacement of the guanine base opposite the modified nucleotide. Based on these structures and molecular dynamics calculations, we propose a tentative model for the recognition of damaged DNA substrates by the UvrB protein.DNA is susceptible to a number of lesions, such as those caused by chemicals, ionizing radiation, and ultraviolet light.Failure to repair DNA damage is thought to play a significant role in cancer and aging. Nucleotide excision repair (NER) 1 is a universal DNA repair mechanism that recognizes and removes many structurally diverse DNA lesions. This system repairs the damage in the DNA by a three step mechanism: (i) recognition of the lesion, (ii) dual nicking (3Ј and 5Ј) of the damaged strand around the altered site, and (iii) DNA synthesis and ligation to fill the 12-13-base gap. The NER system in eucaryotes is very complex and involves the action of at least 16 different polypeptides (1). On the contrary, in procaryotic organisms the NER mechanism involves only four specific proteins, UvrA, UvrB, UvrC, and UvrD (in addition to DNA polymerase and ligase). Despite the fact that the general mechanism of the UvrABCD system is known, many key issues remain unclear. The most important one is the determination of the structural characteristics of DNA lesions that trigger the repair system. The wide range of lesions that are recognized by the UvrABCD system suggests that the feature that induces repair is an alteration of the structural characteristics of the damaged DNA rather than a particular chemical structure of the damaging agent (2-4). After the analysis of a large number of lesions that are substrates of the UvrABCD system, van Houten (3) classified them into six categories: (i) covalent damage, (ii) bulky substituents, (iii) localize...

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confidence: 99%

Effect of Bulky Lesions on DNA

Gómez‐Pinto

Cubero

Kalko

et al. 2004

Journal of Biological Chemistry

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“…It was next investigated whether fluorescently labeled alginate strands could home in vivo to a target gel through sequence-mediated targeting. ODNs were conjugated to alginate through the 3′ end to increase serum exonuclease stability (33)(34)(35). ODN-conjugated alginate strands were still competent to bind in a sequencespecific manner after 6 h, 12 h, and 24 h of circulation in the blood (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…One challenge to this type of application is ODN stability. In this work alginate was conjugated to the 3′ end of ODNs to overcome 3′-5′ exonucleases (the major serum-based nuclease) (33)(34)(35) and the backbone was modified with phosphorothioate groups for increased endonuclease and chemical stability (46). These changes were sufficient to protect alginate strand-conjugated ODNs for up to 12 h in circulation (Fig.…”

Section: Discussionmentioning

confidence: 99%

Refilling drug delivery depots through the blood

Brudno

Silva

Kearney

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Local drug delivery depots have significant clinical utility, but there is currently no noninvasive technique to refill these systems once their payload is exhausted. Inspired by the ability of nanotherapeutics to target specific tissues, we hypothesized that bloodborne drug payloads could be modified to home to and refill hydrogel drug delivery systems. To address this possibility, hydrogels were modified with oligodeoxynucleotides (ODNs) that provide a target for drug payloads in the form of free alginate strands carrying complementary ODNs. Coupling ODNs to alginate strands led to specific binding to complementary-ODN-carrying alginate gels in vitro and to injected gels in vivo. When coupled to a drug payload, sequencetargeted refilling of a delivery depot consisting of intratumor hydrogels completely abrogated tumor growth. These results suggest a new paradigm for nanotherapeutic drug delivery, and this concept is expected to have applications in refilling drug depots in cancer therapy, wound healing, and drug-eluting vascular grafts and stents.nanoparticle | targeting | DNA nanotechnology | controlled release | biomaterials

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“…The phosphodiester oligonucleotides facing the medium were sensitive to degradation by DNase I at high concentrations (Fig. 1), but as they are coupled to cholesterol at their 3' extremity they should be protected to some extent against nucleases present in serum (Boutorine et al, 1992;Gamper et al, 1993) which are principally Y-exonucleases (Shaw et al, 1991).…”

Section: Discussionmentioning

confidence: 99%

Synthesis and anti-HIV activity of thiocholesteryl-coupled phosphodiester antisense oligonucleotides incorporated into immunoliposomes

1994

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Encapsulation of oligonucleotides in antibody-targeted liposomes (immunoliposomes) which bind to target cells permits intracellular delivery of the oligonucleotides. This approach circumvents problems of extracellular degradation by nucleases and poor membrane permeability which free phosphodiester oligonucleotides are subject to, but leaves unresolved the inefficiency of encapsulation of oligonucleotides in liposomes. We have coupled oligonucleotides to cholesterol via a reversible disulfide bond. This modification of oligonucleotides improved their association with immunoliposomes by a factor of about 10 in comparison to unmodified oligonucleotides. The presence of cholesteryl-modified oligonucleotides incorporated in the bilayer of liposomes did not interfere with the coupling of the targeting protein to the liposome surface. Free or cholesterol coupled oligonucleotides associated with liposomes and directed against the tat gene of HIV-1 were tested for inhibition of HIV-1 proliferation in acutely infected cells. We demonstrate that the cholesteryl-modified as well as unmodified oligonucleotides acquire the target specificity of the antibody on the liposome. Their antiviral activity when delivered into cells is sequence-specific. The activity of these modified or unmodified oligonucleotides to inhibit the replication of HIV was the same on an equimolar basis (ECs0 around 0.1 /~M). Cholesterol coupled oligonucleotides thus offer increased liposome association without loss of antiviral activity.

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Facile preparation of nuclease resistant 3′ modified oligodeoxynucleotides

Cited by 127 publications

References 26 publications

Effect of Bulky Lesions on DNA

Effect of Bulky Lesions on DNA

Refilling drug delivery depots through the blood

Synthesis and anti-HIV activity of thiocholesteryl-coupled phosphodiester antisense oligonucleotides incorporated into immunoliposomes

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