Direct electrical stimulation of the auditory nerve can be used to restore some degree of hearing to the profoundly deaf. Percepts due to electrical stimulation have characteristics corresponding approximately to the acoustic percepts of loudness, pitch, and timbre. To encode speech as a pattern of electrical stimulation, it is necessary to determine the effects of the stimulus parameters on these percepts. The effects of the three basic stimulus parameters of level, repetition rate, and stimulation location on subjects' percepts were examined. Pitch difference limens arising from changes in rate of stimulation increase as the stimulating rate increases, up to a saturation point of between 200 and 1000 pulses per second. Changes in pitch due to electrode selection depend upon the subject, but generally agree with a tonotopic organization of the human cochlea. Further, the discriminability of such place-pitch percepts seems to be dependent on the degree of current spread in the cochlea. The effect of stimulus level on perceived pitch is significant but is highly dependent on the individual tested. The results of these experiments are discussed in terms of their impact on speech-processing strategies and their relevance to acoustic pitch perception.

Methods for rapidly assessing sequence-structure-function landscapes and developing conditional gene-regulatory devices are critical to our ability to manipulate and interface with biology. We describe a framework for engineering RNA devices from preexisting aptamers that exhibit ligand-responsive ribozyme tertiary interactions. Our methodology utilizes cell sorting, high-throughput sequencing, and statistical data analyses to enable parallel measurements of the activities of hundreds of thousands of sequences from RNA device libraries in the absence and presence of ligands. Our tertiary interaction RNA devices exhibit improved performance in terms of gene silencing, activation ratio, and ligand sensitivity as compared to optimized RNA devices that rely on secondary structure changes. We apply our method to building biosensors for diverse ligands and determine consensus sequences that enable ligand-responsive tertiary interactions. These methods advance our ability to develop broadly applicable genetic tools and to elucidate understanding of the underlying sequence-structure-function relationships that empower rational design of complex biomolecules.

Biosensors are key components in engineered biological systems, providing a means of measuring and acting upon the large biochemical space in living cells. However, generating small molecule sensing elements and integrating them into in vivo biosensors have been challenging. Here, using aptamer-coupled ribozyme libraries and a ribozyme regeneration method, de novo rapid in vitro evolution of RNA biosensors (DRIVER) enables multiplexed discovery of biosensors. With DRIVER and high-throughput characterization (CleaveSeq) fully automated on liquid-handling systems, we identify and validate biosensors against six small molecules, including five for which no aptamers were previously found. DRIVER-evolved biosensors are applied directly to regulate gene expression in yeast, displaying activation ratios up to 33-fold. DRIVER biosensors are also applied in detecting metabolite production from a multi-enzyme biosynthetic pathway. This work demonstrates DRIVER as a scalable pipeline for engineering de novo biosensors with wide-ranging applications in biomanufacturing, diagnostics, therapeutics, and synthetic biology.

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Gene expression in Saccharomyces cerevisiae is regulated at multiple levels. Genomic and epigenomic mapping of transcription factors and chromatin factors has led to the delineation of various modular regulatory elements – Enhancers (UAS), core promoters, 5’ untranslated regions (5’ UTR) and transcription terminators/3’ untranslated regions (3’ UTR). However, only a few of these elements have been tested in combinations with other elements and the functional interactions between the different modular regulatory elements remains under explored. We describe a simple and rapid approach to build a combinatorial library of regulatory elements and have used this library to study 26 different enhancers, core promoters, 5’UTRs and transcription terminators/3’ UTRs to estimate the contribution of individual regulatory parts in gene expression. Our combinatorial analysis shows that while enhancers initiate gene expression, core promoters modulate the levels of enhancer mediated expression and can positively or negatively affect expression from even the strongest enhancers. Principal component analysis (PCA) indicate that enhancer and promoter function can be explained by a single principal component while UTR function involves multiple functional components. The PCA also highlights outliers and suggest differences in mechanisms of regulation by individual elements. Our data also identify numerous regulatory cassettes composed of different individual regulatory elements that exhibit equivalent gene expression levels. These data thus provide a catalog of elements that could in future be used in the design of synthetic regulatory circuits.