Multi-pass, single-molecule nanopore reading of long protein strands with single-amino acid sensitivity.

The ability to sequence single protein molecules in their native, full-length form would enable a more comprehensive understanding of proteomic diversity. Current technologies, however, are limited in achieving this goal. Here, we establish a method for long-range, single-molecule reading of intact protein strands on a commercial nanopore sensor array. By using the ClpX unfoldase to ratchet proteins through a CsgG nanopore, we achieve single-amino acid level sensitivity, enabling sequencing of combinations of amino acid substitutions across long protein strands. For greater sequencing accuracy, we demonstrate the ability to reread individual protein molecules, spanning hundreds of amino acids in length, multiple times, and explore the potential for high accuracy protein barcode sequencing. Further, we develop a biophysical model that can simulate raw nanopore signals a priori, based on amino acid volume and charge, enhancing the interpretation of raw signal data. Finally, we apply these methods to examine intact, folded protein domains for complete end-to-end analysis. These results provide proof-of-concept for a platform that has the potential to identify and characterize full-length proteoforms at single-molecule resolution.

Deep generative models for peptide design.

Computers can already be programmed for superhuman pattern recognition of images and text. For machines to discover novel molecules, they must first be trained to sort through the many characteristics of molecules and determine which properties should be retained, suppressed, or enhanced to optimize functions of interest. Machines need to be able to understand, read, write, and eventually create new molecules. Today, this creative process relies on deep generative models, which have gained popularity since powerful deep neural networks were introduced to generative model frameworks. In recent years, they have demonstrated excellent ability to model complex distribution of real-word data (e.g., images, audio, text, molecules, and biological sequences). Deep generative models can generate data beyond those provided in training samples, thus yielding an efficient and rapid tool for exploring the massive search space of high-dimensional data such as DNA/protein sequences and facilitating the design of biomolecules with desired functions. Here, we review the emerging field of deep generative models applied to peptide science. In particular, we discuss several popular deep generative model frameworks as well as their applications to generate peptides with various kinds of properties (e.g., antimicrobial, anticancer, cell penetration, etc). We conclude our review with a discussion of current limitations and future perspectives in this emerging field.

A quiescent state following mild sensory arousal in Caenorhabditis elegans is potentiated by stress.

An animal's behavioral and physiological response to stressors includes changes to its responses to stimuli. How such changes occur is not well understood. Here we describe a Caenorhabditis elegans quiescent behavior, post-response quiescence (PRQ), which is modulated by the C. elegans response to cellular stressors. Following an aversive mechanical or blue light stimulus, worms respond first by briefly moving, and then become more quiescent for a period lasting tens of seconds. PRQ occurs at low frequency in unstressed animals, but is more frequent in animals that have experienced cellular stress due to ultraviolet light exposure as well as in animals following overexpression of epidermal growth factor (EGF). PRQ requires the function of the carboxypeptidase EGL-21 and the calcium-activated protein for secretion (CAPS) UNC-31, suggesting it has a neuropeptidergic mechanism. Although PRQ requires the sleep-promoting neurons RIS and ALA, it is not accompanied by decreased arousability, and does not appear to be homeostatically regulated, suggesting that it is not a sleep state. PRQ represents a simple, tractable model for studying how neuromodulatory states like stress alter behavioral responses to stimuli.