Deprotection of methylphosphonate oligonucleotides using a novel one-pot procedure

Hogrefe, Richard I.; Vaghefi, Morteza M.; Reynolds, Mark A.; Young, Kevin M.; Arnold, Lyle J.

doi:10.1093/nar/21.9.2031

Cited by 81 publications

(58 citation statements)

References 17 publications

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“…DNA oligonucleotides were obtained from Gibco-BRL or GlaxoWellcome, Inc. Methylphosphonate oligonucleotides were synthesized and purified as previously described (21). All oligonucleotides were purified on 20% denaturing polyacrylamide gels before use.…”

Section: Methodsmentioning

confidence: 99%

Processing of Nontelomeric 3′ Ends by Telomerase: Default Template Alignment and Endonucleolytic Cleavage

Melek

Greene

Shippen

1996

Molecular and Cellular Biology

117

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Telomerase is a specialized reverse transcriptase that maintains telomeres at chromosome ends by extending preexisting tracts of telomeric DNA and forming telomeres de novo on broken chromosomes. Whereas the interaction of telomerase with telomeric DNA has been studied in some detail, relatively little is known about how this enzyme processes nontelomeric DNA. In this study we recruited the Euplotes telomerase to nontelomeric 3 termini in vitro using chimeric DNA primers that carried one repeat of a telomeric sequence at various positions upstream of a nontelomeric 3 end. Such primers were processed in two distinct pathways. First, nontelomeric 3 ends could be elongated directly by positioning a primer terminus at a specific site on the RNA template. Delivery to this default site was precise, always resulting in the addition of 4 dG residues to the nontelomeric 3 ends. These same residues initiate new telomeres formed in vivo. Alternatively, 3 nontelomeric nucleotides were removed from primers prior to initiating the first elongation cycle. As with default positioning of nontelomeric 3 ends, the cleavage event was extremely precise and was followed by the addition of dG residues to the primer 3 ends. The specificity of the cleavage reaction was mediated by primer interaction with the RNA template and, remarkably, proceeded by an endonucleolytic mechanism. These observations suggest a mechanism for the precision of developmentally regulated de novo telomere formation and expand our understanding of the enzymatic properties of telomerase.

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Section: Methodsmentioning

confidence: 99%

Processing of Nontelomeric 3′ Ends by Telomerase: Default Template Alignment and Endonucleolytic Cleavage

Melek

Greene

Shippen

1996

Molecular and Cellular Biology

117

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“…Five sets of partially complementary synthetic oligonucleotides containing a single methylphosphonate linkage were prepared using a commercially available methylphosphonamidite monomer (Glen Research, Sterling, VA). Oligonucleotides containing methylphosphonate linkages were deprotected and purified according to the procedure described by Hogrefe et al (1993). Each set of oligonucleotides was complementary over a 26 base pair region which included the methylphosphonate linkages.…”

Section: Methodsmentioning

confidence: 99%

Electrostatic Mechanism for DNA Bending by bZIP Proteins

et al. 1997

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Biology is replete with examples of protein-induced DNA bending, yet the forces responsible for bending have been neither established nor quantified. Mirzabekov and Rich proposed in 1979 that asymmetric neutralization of the anionic phosphodiester backbone by basic histone proteins could provide a thermodynamic driving force for DNA bending in the nucleosome core particle [Mirzabekov, A. D., & Rich, A. (1979) A. 93, 9515-9520]. Here it is shown that bZIP proteins bend DNA via a mechanism involving direct contacts between one or two basic side chains and a symmetry-related pair of unique, nonbridging phosphate oxygens. The locations of these phosphates provide direct experimental support for a protein-induced bending mechanism based on asymmetric charge neutralization. This straightforward mechanism is compatible with many DNA-recognition motifs and may represent a general strategy for the assembly of protein-DNA complexes of defined stereochemistries.

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“…Oligonucleotides containing site-specific racemic methylphosphonate substitutions were synthesized at 1-mol scale using methylphosphonamidite monomers obtained from Glen Research (Sterling, VA). Isobutyryl derivatives of cytosine were used to facilitate cleavage from the solid support and deprotection as described previously (11). All oligomers were purified by denaturing polyacrylamide gel electrophoresis, eluted from the gel, and desalted using C 18 reverse phase cartridges.…”

Section: Methodsmentioning

confidence: 99%