Analysis of non-canonical three- and four-way DNA junctions.

The development of compounds that can selectively bind with non-canonical DNA structures has expanded in recent years. Junction DNA, including three-way junctions (3WJs) and four-way Holliday junctions (HJs), offer an intriguing target for developmental therapeutics as both 3WJs and HJs are involved in DNA replication and repair processes. However, there are a limited number of assays available for the analysis of junction DNA binding. Here, we describe the design and execution of multiplex fluorescent polyacrylamide gel electrophoresis (PAGE) and microscale thermophoresis (MST) assays that enable evaluation of junction-binding compounds. Two well characterised junction-binding compounds-a C6 linked bis-acridine ligand and an iron(II)-bound peptide helicate, which recognise HJs and 3WJs, respectively-were employed as probes for both MST and PAGE experiments. The multiplex PAGE assay expands beyond previously reported fluorescent PAGE as it uses four individual fluorophores that can be combined to visualise single-strands, pseudo-duplexes, and junction DNA present during 3WJ and HJ formation. The use of MST to identify the binding affinity of junction binding agents is, to our knowledge, first reported example of this technique. The combined use of PAGE and MST provides complementary results for the visualisation of 3WJ and HJ formation and the direct binding affinity (Kd and EC50) of these agents. These assays can be used to aid the discovery and design of new therapeutics targeting non-canonical nucleic acid structures.

DNA Three-Way Junction for Differentiation of Single-Nucleotide Polymorphisms with Fluorescent Copper Nanoparticles.

A label- and enzyme-free fluorescent sensor for the detection of single-nucleotide polymorphisms (SNPs) at room temperature is proposed, using new copper nanoparticles (CuNPs) as fluorescent reporters. The CuNPs were constructed by using a DNA three-way junction (3WJ) template. In this assay, two complementary adenine/thymine-rich probes can hybridize with the wild-type target simultaneously to construct a 3WJ structure, serving as an efficient scaffold for the generation of CuNPs. However, the CuNPs produce weak fluorescence when the probes bind with a mutant-type target. SNPs can be identified by the difference in fluorescence intensity of the CuNPs. This SNPs detection strategy is straightforward, cost-effective, and avoids the complicated procedures of labeling or enzymatic reactions. The fluorescent sensor is versatile and can be applied to all types of mutation because the probes are programmable. Moreover, the sensor exhibits good detection performance in biological samples.

Crystal structure of 3WJ core revealing divalent ion-promoted thermostability and assembly of the Phi29 hexameric motor pRNA.

The bacteriophage phi29 DNA packaging motor, one of the strongest biological motors characterized to date, is geared by a packaging RNA (pRNA) ring. When assembled from three RNA fragments, its three-way junction (3WJ) motif is highly thermostable, is resistant to 8 M urea, and remains associated at extremely low concentrations in vitro and in vivo. To elucidate the structural basis for its unusual stability, we solved the crystal structure of this pRNA 3WJ motif at 3.05 Å. The structure revealed two divalent metal ions that coordinate 4 nt of the RNA fragments. Single-molecule fluorescence resonance energy transfer (smFRET) analysis confirmed a structural change of 3WJ upon addition of Mg²âº. The reported pRNA 3WJ conformation is different from a previously published construct that lacks the metal coordination sites. The phi29 DNA packaging motor contains a dodecameric connector at the vertex of the procapsid, with a central pore for DNA translocation. This portal connector serves as the foothold for pRNA binding to procapsid. Subsequent modeling of a connector/pRNA complex suggests that the pRNA of the phi29 DNA packaging motor exists as a hexameric complex serving as a sheath over the connector. The model of hexameric pRNA on the connector agrees with AFM images of the phi29 pRNA hexamer acquired in air and matches all distance parameters obtained from cross-linking, complementary modification, and chemical modification interference.