Dynamic arrest and aging of biomolecular condensates are regulated by low-complexity domains, RNA and biochemical activity

Biomolecular condensates require suitable material properties to properly carry out their function. Yet, modulators that affect the material properties of condensates have remained largely unexplored.Here we apply Differential Dynamic Microscopy (DDM) to probe the material properties of anin vitromodel of processing bodies (P-bodies) consisting of condensates formed by the DEAD-box ATPase Dhh1 in the presence of ATP and RNA. DDM allows us to measure the viscosity of liquid droplets and to distinguish between liquid-like and gel- or glass-like condensates. By applying this single-droplet technique we show that condensates within the same population exhibit a distribution of material properties, which can be drastically affected by several modulators such as the low-complexity domains (LCDs) of the protein, the protein/RNA ratio, the type of RNA as well as the enzymatic activity.In particular, we show that structured RNA leads to a large fraction of dynamically arrested condensates with respect to unstructured polyuridylic acid (polyU), emphasizing the role of RNA structure in condensate dynamics. We further demonstrate that the ageing of the condensates and the formation of gel or glass-like structures is reduced by promoting the enzymatic ATPase activity of Dhh1 and the rate of droplet formation and dissolution.Our study shows that not only the reversible formation and dissolution of condensates but also their material properties are regulated on several levels, and that biochemical activity and material turnover can be important to maintain fluid-like properties over time.

Raman Spectroscopic Study of Nucleobase Pairs and Mispairs - A Potential Method for Analysis of DNA Sequences

To exploit sensitive, accurate and convenient detection techniques for nucleic acid sequences can help to overcome the defects of current fluorescence detection method. Raman spectroscopy represents one family of methods that may offer such potential. In this paper, G•C base pairs and C•U mispairs were studied by Raman spectral characterization of single-strand homopolynucleotides, i.e. polyguanylic acid (PolyG), polycytidylic acid (PolyC) and polyuridylic acid (PolyU), and their equimolar complexes PolyG•PolyC and PolyC•PolyU in solution of 0.14 M Na+, pH7.0. Both of the complexes were found to adopt A type conformation. Helix formation between complementary polynucleotides led to marked and characterisctic changes in frequency and intensity of Raman bands of the biopolymers. Spectral changes were also observed upon the formation of C•U mispairs. According to the Raman data, in C•U mispairs the C2=O of U was engaged in a hydrogen bond to the N4H2group of cytosine, while the uracil C4=O group is free. Some of the rC residues transformed from C3-endo/anti to C2-endo/anti conformation due to base mispairing.The results suggest that Raman spectroscopy provides detailed and highly sensitive information about both the structure of nucleic acids and their base composition, and has great potential for analysis of DNA sequences.

Toll like receptor 3 expression and efficacy of adjuvant treatment with polyadenylic-polyuridylic acid in patients with axillary node positive breast cancer: Results from two randomized trials

10563 Background: Toll like receptor 3 (TLR3) are transmembrane receptors involved in the cellular response to danger signal and specifically activated by double stranded RNA. Based on these data, we hypothesized that the TLR3 expression by tumor cells is associated with the efficacy of a treatment with double stranded RNA in cancer patients. Patients and Methods: TLR3 expression was assessed by immunohistochemistry in 336 patients with axillary node positive breast cancer. These patients were selected to have been included in two randomized trials that compared a post-operative administration of polyadenylic-polyuridylic acid (polyAU) to either no treatment (n = 174, trial I) or an adjuvant chemotherapy by CMF regimen (n = 162, trial II). In the trial II, locoregional and pelvic radiotherapy were given in the polyAU arm. Results: TLR3 was found to be overexpressed in 45 tumors (13%). Median follow-up were 23 and 17 years for living patients included in the trials I and II respectively. When the analysis focused on the first trial, the 20 year overall survival rates were 87% (95% CI: 53–97%) and 32% (95% CI: 12–63%) for patients with TLR3-overexpressing tumour treated (n = 8) or not (n = 11) with polyAU (p = 0.03). In the same trial, the 20 year overall survival rates were 43% (95% CI: 33–54%) and 35% (95% CI: 25–46%) for patients with TLR3-negative tumor treated (n = 81) or not (n = 74) with polyAU (p = 0.32). When the analysis focused on the Trial II, the 15 year overall survival were 62% (95% CI: 45%-91%) and 21% (95% CI: 8%-47%) for patients with TLR3-overexpressing tumors treated (n = 12) or not (n = 14) with polyAU (p = 0.005). In the same trial, 15 year overall survival rates were 31% (95% CI: 21%-42%) and 39% (95% CI: 28%-51%) for patients with TLR3-negative tumors treated (n = 66) or not (n = 70) with polyAU (p = 0.39). Conclusion: This study suggests that the post-operative administration of polyadenylic-polyuridylic acid improves outcome of patient with TLR3-overexpressing, node-positive breast cancer. [Table: see text]