Search

Advances in peroxidase- and biotin ligase-mediated signal amplification have enabled high-resolution subcellular mapping of endogenous RNA localization and protein-protein interactions. Application of these technologies has been limited to RNA and proteins because of the reactive groups required for biotinylation in each context. Here we report several novel methods for proximity biotinylation of exogenous oligodeoxyribonucleotides by application of well-established and convenient enzymatic tools. We describe approaches using simple and efficient conjugation chemistries to modify deoxyribonucleotides with “antennae” sensitive to phenoxy radical or biotinoyl-5’-adenylate. In addition, we report chemical details of a previously undescribed adduct between tryptophan and a phenoxy radical group. These developments have potential application in the selection of exogenous nucleic acids capable of unaided entry into living cells.

rHIgM22 is a recombinant human monoclonal IgM designed to promote remyelination, and it is currently in Phase I clinical trials in patients with multiple sclerosis (MS). In animal models of demyelination, a single low dose of rHIgM22 stimulates oligodendrocyte maturation, induces remyelination, preserves axons, and slows the decline of locomotor deficits. Natural autoantibodies like rHIgM22 typically bind to multiple antigens with weak affinity. rHIgM22 binds to oligodendrocytes and myelin. Because the antigens for rHIgM22 is prevalent within and exclusive to central nervous system (CNS) myelin, we used CNS myelin particles in combination with surface plasmon resonance to determine the kinetic and affinity constants for the interaction of rHIgM22 to myelin. We found that both the serum and recombinant forms of the antibody bind to myelin with very small dissociation constants in the 100 pM range, which is highly unusual for natural autoantibodies. The extraordinary affinity between rHIgM22 and myelin may explain why such a low effective dose can stimulate CNS repair in animal models of demyelination and underlie the accumulation of rHIgM22 in the CSF in treated MS patients by targeting myelin.

The intrinsic curvature of seven 98-base pair DNA molecules containing up to four centrally located A6-tracts has been measured by gel and capillary electrophoresis as a function of the number and arrangement of the A-tracts. At low cation concentrations, the electrophoretic mobility observed in polyacrylamide gels and in free solution decreases progressively with the increasing number of phased A-tracts, as expected for DNA molecules with increasingly curved backbone structures. Anomalously slow electrophoretic mobilities are also observed for DNA molecules containing two pairs of phased A-tracts that are out of phase with each other, suggesting that out-of-phase distortions of the helix backbone do not cancel each other out. The mobility decreases observed for the A-tract samples are due to curvature, not cation binding in the A-tract minor groove, because identical free solution mobilities are observed for a molecule with four out-of-phase A-tracts and one with no A-tracts. Surprisingly, the curvature of DNA A-tracts is gradually lost when the monovalent cation concentration is increased to ~200 mM, regardless of whether the cation is a hydrophilic ion like Na+, NH4+ or Tris+ or a hydrophobic ion like tetrabutylammonium (TBA+). The decrease of A-tract curvature with increasing ionic strength, along with the known decrease of A-tract curvature with increasing temperature, suggests that DNA A-tracts are not significantly curved under physiological conditions.

HMGB proteins are abundant non-histone components of eukaryotic chromatin. The biological function of DNA sequence-nonspecific HMGB proteins is obscure. These proteins are composed of one or two conserved HMG box domains, each forming three alpha-helices that fold into a sequence-nonspecific DNA-binding module recognizing the DNA minor groove. Box A and box B homology domains have subtle sequence differences such that box B domains bend DNA strongly while DNA bending by isolated box A domains is weaker. Both box A and box B domains preferentially bind to distorted DNA structures. Here we show using DNA cyclization kinetics assays in vitro and Escherichia coli DNA looping assays in vivo that an isolated HMG box A domain derived from human HMGB2 folds poorly and does not enhance apparent DNA flexibility. Surprisingly, substitution of a small number of cationic residues from the N-terminal leader of a functional yeast box B protein, Nhp6Ap, confers the ability to enhance DNA flexibility. These results demonstrate important roles for cationic leader amino acids in HMGB folding, DNA interaction, and DNA bending.

A procedure is presented for preparing an oligonucleotide-biotin conjugate that is chemically cleavable through the reduction of a disulfide bond within the linker. Conjugation involves reaction of a primary amine with an N-hydroxysulfosuccinimide ester linked to biotin. The oligonucleotide can be liberated from streptavidin agarose containing immobilized conjugate under mild conditions (neutral pH, 50 mM dithiothreitol). This cleavable conjugate is useful for affinity purification applications.

An experimental approach is presented for the creation of an artificial and functional repressor/operator interaction that does not involve polypeptides. This in vitro approach confers oligonucleotide regulation upon a bacteriophage T7 RNA polymerase promoter by introducing an overlapping homopurine operator that can be recognized by oligonucleotide-directed DNA triple-helix formation. Recognition of optimized operator sequences in either of two triple-helix motifs is shown to efficiently inhibit T7 RNA polymerase transcription initiation in both a promoter- and oligonucleotide-specific manner. Inhibition due to triple helices of the pyrimidine motif is pH-dependent, as expected. Inhibition by purine motif triple helices is not pH-dependent and occurs efficiently under optimum T7 RNA polymerase transcription conditions. Repression by triple-helix formation can be observed rapidly after addition of purine motif repressor oligonucleotides, even when polymerase has been given prior access to the promoter. The mechanism of repression is shown to be occlusion of polymerase from the promoter rather than trapping of the polymerase in unproductive preinitiation or initiation complexes. In contrast to their inhibition of T7 RNA polymerase initiation, the triple-helical complexes studied here do not detectably inhibit transcription elongation.

A site-specific triple-helical DNA complex has previously been shown to inhibit DNA binding by eukaryotic transcription factor Sp1. To examine the functional consequences of such inhibition, homopurine target sequences for oligonucleotide-directed triple-helix formation were inserted in various configurations relative to Sp1 transcription activator binding sites, upstream of the TATA element of recombinant eukaryotic promoters. The resulting promoters were tested for activity in the presence or absence of recombinant human Sp1 in a Drosophila in vitro transcription system lacking endogenous Sp1. When triple-helical complexes were assembled on the promoters by incubation with specific oligodeoxyribonucleotides, promoter-specific repression of basal transcription was observed in the absence of Sp1. Transcriptional repression required the preassembly of triple-helical complexes before addition of nuclear extract. The degree of basal repression was a function of the number and proximity of triple-helical complexes relative to the basal promoter complex. Repression did not result from triple-helix-induced template degradation. Addition of recombinant Sp1 did not cause derepression. These results suggest that triple-helical complexes can repress transcription primarily by blocking promoter DNA assembly into initiation complexes rather than by occluding Sp1 binding. One of several plausible mechanisms for triple-helix-induced repression involves changes in DNA flexibility. Evidence in favor of this model is provided by a permutation-dependent gel mobility assay in which formation of site-specific triple-helical complexes is shown to stiffen double-helical DNA.