Rapid, inexpensive, sequence-independent fluorescent labeling of phosphorothioate DNA.

Fluorescently labeled oligonucleotides are powerful tools for characterizing DNA processes; however, their use is limited by the cost and sequence requirements of current labeling technologies. Here, we develop an easy, inexpensive, and sequence-independent method for site-specifically labeling DNA oligonucleotides. We utilize commercially synthesized oligonucleotides containing phosphorothioate diester(s) in which a nonbridging oxygen is replaced with a sulfur (PS-DNA). The increased nucleophilicity of the thiophosphoryl sulfur relative to the phosphoryl oxygen permits selective reactivity with iodoacetamide compounds. As such, we leverage a long-existing bifunctional linker, N,N'-bis(α-iodoacetyl)-2-2'-dithiobis(ethylamine) (BIDBE), that reacts with PS-DNAs to leave a free thiol, allowing conjugation of the wide variety of commercial maleimide-functionalized compounds. We optimized BIDBE synthesis and its attachment to PS-DNA and then fluorescently labeled the BIDBE-PS-DNA using standard protocols for labeling cysteines. We purified the individual epimers, and using single-molecule Förster resonance energy transfer (FRET), we show that the FRET efficiency is independent of the epimeric attachment. Subsequently, we demonstrate that an epimeric mixture of double-labeled Holliday junctions (HJs) can be used to characterize their conformational properties in the absence and presence of the structure-specific endonuclease Drosophila melanogaster Gen. Finally, we use a biochemical activity assay to show that this double-labeled HJ is functional for cleavage by Gen and that the double-labeled HJ allows multiple DNA species to be identified in a single experiment. In conclusion, our results indicate that dye-labeled BIDBE-PS-DNAs are comparable to commercially labeled DNAs at a significantly reduced cost. Notably, this technology could be applied to other maleimide-functionalized compounds, such as spin labels, biotin, and proteins. The sequence independence of labeling, coupled with its ease and low cost, enables unrestricted exploration of dye placement and choice, providing the potential for creation of differentially labeled DNA libraries and opening previously inaccessible experimental avenues.

Comparison of angiopoietin-like protein 3 and 4 reveals structural and mechanistic similarities.

Elevated plasma triglycerides are a risk factor for coronary artery disease, which is the leading cause of death worldwide. Lipoprotein lipase (LPL) reduces triglycerides in the blood by hydrolyzing them from triglyceride-rich lipoproteins to release free fatty acids. LPL activity is regulated in a nutritionally responsive manner by macromolecular inhibitors including angiopoietin-like proteins 3 and 4 (ANGPTL3 and ANGPTL4). However, the mechanism by which ANGPTL3 inhibits LPL is unclear, in part due to challenges in obtaining pure protein for study. We used a new purification protocol for the N-terminal domain of ANGPTL3, removing a DNA contaminant, and found DNA-free ANGPTL3 showed enhanced inhibition of LPL. Structural analysis showed that ANGPTL3 formed elongated, flexible trimers and hexamers that did not interconvert. ANGPTL4 formed only elongated flexible trimers. We compared the inhibition of ANGPTL3 and ANGPTL4 using human very-low-density lipoproteins as a substrate and found both were noncompetitive inhibitors. The inhibition constants for the trimeric ANGPTL3 (7.5 ± 0.7 nM) and ANGPTL4 (3.6 ± 1.0 nM) were only 2-fold different. Heparin has previously been reported to interfere with ANGPTL3 binding to LPL, so we questioned if the negatively charged heparin was acting in a similar fashion to the DNA contaminant. We found that ANGPTL3 inhibition is abolished by binding to low-molecular-weight heparin, whereas ANGPTL4 inhibition is not. Our data show new similarities and differences in how ANGPTL3 and ANGPTL4 regulate LPL and opens new avenues of investigating the effect of heparin on LPL inhibition by ANGPTL3.