The objective of this study is to evaluate the structure and protein recognition properties of hybrid four-way junctions (4WJs) composed of DNA and peptide nucleic acid (PNA) strands. We compare a classic immobile DNA junction, J1, vs. six PNA-DNA junctions, including a number with blunt DNA ends and multiple PNA strands. Circular dichroism (CD) analysis reveals that hybrid 4WJs are composed of helices that possess structures intermediate between A- and B-form DNA, the apparent level of A-form structure correlates with the PNA content. The structure of hybrids that contain one PNA strand is sensitive to Mg+2. For these constructs, the apparent B-form structure and conformational stability (Tm) increase in high Mg+2. The blunt-ended junction, b4WJ-PNA3, possesses the highest B-form CD signals and Tm (40.1°C) values vs. all hybrids and J1. Protein recognition studies are carried out using the recombinant DNA-binding protein, HMGB1b. HMGB1b binds the blunt ended single-PNA hybrids, b4WJ-PNA1 and b4WJ-PNA3, with high affinity. HMGB1b binds the multi-PNA hybrids, 4WJ-PNA1,3 and b4WJ-PNA1,3, but does not form stable protein-nucleic acid complexes. Protein interactions with hybrid 4WJs are influenced by the ratio of A- to B-form helices: hybrids with helices composed of higher levels of B-form structure preferentially associate with HMGB1b.

Holliday or DNA four-way junctions (4WJs) are cruciform/bent structures composed of four DNA duplexes. 4WJs are key intermediates in homologous genetic recombination and double-strand break repair. To investigate 4WJs in vitro, junctions are assembled using four asymmetric DNA strands. The presence of four asymmetric strands about the junction branch point eliminates branch migration, and effectively immobilizes the resulting 4WJ. The purpose of these experiments is to show that immobile 4WJs composed of DNA and peptide nucleic acids (PNAs) can be distinguished from contaminating labile nucleic acid structures. These data compare the electrophoretic mobility of hybrid PNA–DNA junctions vs. i) a classic immobile DNA 4WJ, J1 and ii) contaminating nucleic acid structures.

Aminoacyl‐tRNA synthetases (aaRSs) are a well‐known class of enzymes that act as sentinels of genome. During protein translation, aaRSs ensure that L‐amino acids are charged or acylated onto their cognate tRNA isoacceptor(s). Hence, aaRSs help ensure that ribosomally translated proteins contain the proper sequence of amino acids. aaRS catalyze tRNA charging in a two‐step process. In step one, aaRSs selectively form a high energy amino acid adenylate. This step, refereed to amino acid activation, is the first level of selectivity in protein translation. Here, aaRSs effectively discriminate non‐cognate L‐amino acids. In step two, aaRSs transfer adenylated amino acids onto the cognate tRNA isoacceptor. This step is referred to as aminoacylation or tRNA charging. A great deal is known about aaRS reaction mechanisms but a number of confounding idiosyncrasies remain. The focus of this work aims to elucidate idiosyncrasies associated with amino acid activation. Here, a 32P‐based assay is used to measure the level to which E. coliaaRSs activate non‐cognate L‐amino acids. The purpose of these assays is to measure the plasticity of aaRS active sites to define additional underlying binding interactions that modulate amino acid recognition. This information could also be used to develop small molecule inhibitors of aaRS. Moreover, these data could reveal underlying clues to the potential origins of aaRSs. Here, three aaRSs are investigated: ArgRS, IleRS and LysRS. The activation data shows that each aaRS perhaps somewhat surprisingly activates more than five non‐cognate AAs to varying levels. Next, computer docking experiments are used to visualize the binding interaction of synthetase active site residues to each non‐cognate amino acid. These data are currently being used to construct protein‐ligand interaction maps of each aaRS active site. The resulting interactions maps can be used to more clearly elucidate idiosyncrasies associated with amino activation.