Szostak, J. W. | bioRxiv Science

bioRxiv2018

Interpreting turbidity measurements for vesicle studies

Spectrophotometers are routinely used to assess the turbidity of vesicle solutions. Here we provide guidelines for interpreting turbidity measurements of vesicle samples, and highlight potential pitfalls of the approach. We use an exact solution for core-shell scatterers to model and calculate how samples of vesicles scatter light, and provide a comprehensive overview of how the turbidity of vesicle samples can change with vesicle size, contents, and composition. Surprisingly, we find that vesicle lamellarity has a large effect on sample turbidity, while unilamellar vesicles of different sizes have similar turbidity. We use our model in conjunction with experimental data to measure the thickness of oleic acid vesicle membranes and find excellent agreement with values determined by cryo-TEM. We also calculate the effects of potential errors in measurement from forward scattering and multiple scattering.

biophysics↗

bioRxiv2018

Crystallographic observation of nonenzymatic RNA primer extension

The importance of genome replication has inspired detailed crystallographic studies of enzymatic DNA/RNA polymerization. In contrast, the mechanism of nonenzymatic polymerization is less well understood, despite its critical role in the origin of life. Here we report the direct observation of nonenzymatic RNA primer extension through time-resolved crystallography. We soaked crystals of an RNA primer-template-dGMP complex with guanosine-5'-phosphoro-2-aminoimidazolide for increasing times. At early times we see the activated ribonucleotides bound to the template, followed by formation of the imidazolium-bridged dinucleotide intermediate. At later times, we see a new phosphodiester bond forming between the primer and the incoming nucleotide. The intermediate is pre-organized because of the constraints of base-pairing with the template and hydrogen bonding between the imidazole amino group and both flanking phosphates. Our results provide atomic-resolution insight into the mechanism of nonenzymatic primer extension, and set the stage for further structural dissection and optimization of the RNA copying process.

biochemistry↗