Can DyeCycling break the photobleaching limit in single-molecule FRET?

Biomolecular systems, such as proteins, crucially rely on dynamic processes at the nanoscale. Detecting biomolecular nanodynamics is therefore key to obtaining a mechanistic understanding of the energies and molecular driving forces that control biomolecular systems. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to observe in real-time how a single biomolecule proceeds through its functional cycle involving a sequence of distinct structural states. Currently, this technique is fundamentally limited by irreversible photobleaching, causing the untimely end of the experiment and thus, a narrow temporal bandwidth of ≤ 3 orders of magnitude. Here, we introduce "DyeCycling", a measurement scheme with which we aim to break the photobleaching limit in smFRET. We introduce the concept of spontaneous dye replacement by simulations, and as an experimental proof-of-concept, we demonstrate the intermittent observation of a single biomolecule for one hour with a time resolution of milliseconds. Theoretically, DyeCycling can provide > 100-fold more information per single molecule than conventional smFRET. We discuss the experimental implementation of DyeCycling, its current and fundamental limitations, and specific biological use cases. Given its general simplicity and versatility, DyeCycling has the potential to revolutionize the field of time-resolved smFRET, where it may serve to unravel a wealth of biomolecular dynamics by bridging from milliseconds to the hour range. Electronic Supplementary Material: Supplementary material is available for this article at 10.1007/s12274-022-4420-5 and is accessible for authorized users.

Control over the fibrillization yield by varying the oligomeric nucleation propensities of self-assembling peptides.

Self-assembling peptides are an exemplary class of supramolecular biomaterials of broad biomedical utility. Mechanistic studies on the peptide self-assembly demonstrated the importance of the oligomeric intermediates towards the properties of the supramolecular biomaterials being formed. In this study, we demonstrate how the overall yield of the supramolecular assemblies are moderated through subtle molecular changes in the peptide monomers. This strategy is exemplified with a set of surfactant-like peptides (SLPs) with different ß-sheet propensities and charged residues flanking the aggregation domains. By integrating different techniques, we show that these molecular changes can alter both the nucleation propensity of the oligomeric intermediates and the thermodynamic stability of the fibril structures. We demonstrate that the amount of assembled nanofibers are critically defined by the oligomeric nucleation propensities. Our findings offer guidance on designing self-assembling peptides for different biomedical applications, as well as insights into the role of protein gatekeeper sequences in preventing amyloidosis.

Identification of a novel murine pancreatic tumour antigen, which elicits antibody responses in patients with pancreatic carcinoma.

Pancreatic cancer is the fourth leading cause of cancer related death in the United States. Despite numerous efforts in developing new therapies, the prognosis for patients with pancreatic cancer remains poor. Mouse models for spontaneous pancreatic cancer represent an ideal system to develop immunotherapeutic approaches. The aim of this study was to identify new tumour antigens in a murine model that mimics human disease closely, and to verify the results in patients with pancreatic cancer. We analysed a murine pancreatic complementary DNA expression library with serum from tumour-bearing mice, which led to the identification and isolation of several antigens. One of the antigens repeatedly identified in this screening was Tankyrase-2. Here, we show Tankyrase-2 as an antigen eliciting humoral responses not only in mice with established tumours, but also in mice with pre-malignant lesions. Finally, antibody responses to Tankyrase-2 were found in the serum of patients with pancreatic cancer. Reverse transcriptase-polymerase chain reaction analysis showed Tankyrase-2 expression in human pancreatic tumour. These findings show the relevance of spontaneous murine tumour models for the identification of human tumour antigens.

Genetically induced pancreatic adenocarcinoma is highly immunogenic and causes spontaneous tumor-specific immune responses.

Treatment options for pancreatic cancer are limited and often ineffective. Immunotherapeutic approaches are one possible option that needs to be evaluated in appropriate animal models. The aim of the present study was to analyze tumor-specific immune responses in a mouse model of pancreatic cancer, which mimics the human disease closely. C57BL/6 EL-TGF-alpha x Trp53-/- mice, which develop spontaneous ductal pancreatic carcinoma, were generated. EL-TGF-alpha x Trp53-/- mice developed spontaneous pancreatic tumors with pathomorphologic features close to the human disease. Tumor-specific CD8+ T-cell responses and IgG responses were analyzed in EL-TGF-alpha x Trp53-/- mice during tumor development and compared with mice with s.c. growing pancreatic tumors. In contrast to spontaneous pancreatic tumors, cell lines generated from these tumors were rejected after s.c. injection into wild-type mice but not in nude or RAG knockout mice. Direct comparison of spontaneous and s.c. injected tumors revealed an impaired infiltration of CD8+ T cells in spontaneous pancreatic tumors, which was also evident after adoptive transfer of tumor-specific T cells. Intratumoral cytokine secretion of tumor necrosis factor-alpha, IFN-gamma, IL-6, and MCP-1 was lower in spontaneous tumors as well as the number of adoptively transferred tumor-specific T cells. Our data provide clear evidence for tumor-specific immune responses in a genetic mouse model for pancreatic carcinoma. Comparative analysis of s.c. injected tumors and spontaneous tumors showed significant differences in tumor-specific immune responses, which will help in improving current immune-based cancer therapies against adenocarcinoma of the pancreas.