Biology subjects

Ahern, C. A.

Publications and source records attributed to Ahern, C. A..

17 recordsLinked to original sources

bioRxiv2026

Developmental Cerebellar Pathology in Mouse Models of SCN2A Premature Termination Codon Variants

Autism spectrum disorder is a neurodevelopmental disorder with both genetic and environmental contributors. SCN2A, the gene which encodes the alpha subunit of the voltage-gated sodium channel Nav1.2, is a known monogenetic risk factor for autism spectrum disorder. The cerebellum is frequently implicated in autism spectrum disorder and other neurodevelopmental disorders but has largely been unexplored in relation to SCN2A variants, especially from a developmental perspective. Nav1.2 is highly expressed within the cerebellum, specifically within cerebellar granule neurons, where it helps to drive action potential generation and propagation. Cerebellar granule neuron activity and maturation is crucial for shaping the development and morphology of the rest of the cerebellar cortex. Here we investigated early-postnatal cerebellar development in two mouse models carrying patient-derived SCN2A premature termination codon variants, Scn2a-p.Y84X and -p.R1627X. Overall, both Scn2a premature termination codon variant mouse lines displayed largely normal physical development and unaffected non-cerebellar developmental milestones. Scn2aY84X/+ mice, but not Scn2aR1627X/+ mice, demonstrated alterations in cerebellar-driven motor behaviors, specifically faster performance in the surface righting reflex and cliff avoidance compared to wildtype littermates. Coinciding with the behavioral findings, Scn2a variants had divergent and age-dependent effects on cerebellar glutamatergic presynaptic marker expression, indicating that some, but not all, Scn2a premature termination codons impair glutamatergic synapse development. Both Scn2a variants exhibited changes to cerebellar cytoarchitecture, such as reductions in Purkinje cell density and soma size, which was more prominent in Scn2aY84X/+ mice, and faster migration of cerebellar granule neurons from the external granule layer to the internal, indicating a possible shift in the timing of cerebellar maturation. Our results situate the cerebellum as an early site for SCN2A pathophysiology and establish that cerebellar consequences of SCN2A premature termination codon variants may be position- and age-dependent, suggesting that influences beyond simple heterozygous loss of Nav1.2 drive phenotypes. Our characterization of how Scn2a premature termination codon variants differentially impair cerebellar development may help explain the heterogeneity of clinical presentations of SCN2A loss of function variants and potentially inform upon the timing of therapeutic intervention.

neuroscience↗

bioRxiv2026

An atomic interaction conserved for over 600 million years gates inhibitory neurotransmission

Pentameric ligand-gated ion channels (pLGICs) mediate fast inhibitory neurotransmission critical for neuronal network stability. A tyrosine residue in the M2-M3 linker of inhibitory pLGICs, conserved for over 600 million years, is positioned where it could hydrogen bond (H-bond) to the backbone of the neighboring Cys-loop. Given the pathogenic effects of variants of this tyrosine, we hypothesized that this H-bond stabilizes extracellular-to-transmembrane domain coupling essential for channel gating. To test this hypothesis, we used site-directed mutagenesis, noncanonical amino acid incorporation, and electrophysiological recordings in Xenopus laevis oocytes to disrupt this hydrogen bond in GABAA and glycine receptors. Loss of this interaction via tyrosine substitutions or backbone amide modifications that ablate the acceptor or donor, respectively, markedly decrease agonist sensitivity and maximal channel activation, with effects localized to specific subunits. Molecular dynamics simulations support a role for this H-bond in channel gating. These findings reveal a critical atomic interaction underlying a shared mechanism for inhibitory receptor gating and provide a mechanistic explanation for disease-associated mutations linked to epilepsy, neurodevelopmental disability, and hyperekplexia.

biophysics↗

bioRxiv2026

Programmable Repair of Disease-Causing UGA Stop Codons in Mammalian Brain

Protein truncating variants caused by UGA stop codons are the most prevalent class of rare variant mutations in neurodevelopmental diseases. Suppressor transfer RNA (sup-tRNA) have therapeutic potential for premature termination codon (PTC) repair, but have thus far underperformed by traditional AAV delivery platforms and progress has been hampered by the lack of methods to non-invasively assess in vivo activity in mammalian brain. To fill this material gap, we utilize transcranial in vivo bioluminescence imaging data from a luciferase-UGA mouse model to enable payload optimization. These data demonstrate that U6 promotor and AAV2/9 capsids have the lowest in vivo activity, whereas self-complementary AAV2/9 with the tRNA in a minimal 100bp genomic context provide broad and efficacious PTC rescue. Further, payload tRNA multiplexing and use of tRNA introns enable efficacy of low viral titers and sustained rescue. tRNA sequencing of scAAV delivered ArgUGA sup-tRNA in brain demonstrate no effects on endogenous tRNA levels, their acylation or processing, and these features are also maintained in scAAV delivered ArgUGA sup-tRNA. Collectively, this work defines a scalable strategy for precision UGA stop codon suppression, supporting development of durable genetic rescue therapies for neurodevelopmental disorders in the mammalian brain. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/724978v3_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@d0fefeorg.highwire.dtl.DTLVardef@1150446org.highwire.dtl.DTLVardef@cbb839org.highwire.dtl.DTLVardef@abddfc_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Position-dependent effects of SCN2A premature stop codons on neuronal excitability and behavior

SCN2A encodes the voltage-gated sodium channel NaV1.2, a key determinant of spike initiation and propagation in glutamatergic neurons. Premature termination codons are often assumed to produce uniform haploinsufficiency via nonsense-mediated decay, yet whether distinct SCN2A premature stop codons yield equivalent molecular, cellular, and behavioral outcomes remains unknown. We generated two mouse lines carrying patient mutations--Scn2aY84X/+ (p.Tyr84UAA; early coding sequence) and Scn2aR1627X/+(p.Arg1627UGA; terminal coding exon)--on a C57BL/6J background. Allele-specific expression was quantified by targeted next-generation sequencing of whole-brain reverse transcribed cDNA. NaV1.2 protein was measured in half-brain lysates by automated western blot and ex vivo whole-cell recordings were obtained from layer 5b pyramidal-tract neurons in medial prefrontal cortex. A panel of behavioral assays assessed locomotion/exploration, motor learning, anxiety-like behavior, sociability, sensorimotor gating, and seizure susceptibility. Allele-specific RNA handling diverged by position: mRNA carrying Y84X engaged partial nonsense-mediated decay, whereas R1627X transcripts were at allelic balance. Despite this difference in RNA fate, NaV1.2 protein was comparably reduced in both lines. Electrophysiologically, both premature termination codon mutations slowed the action-potential upstroke, with a larger decrement in Scn2aY84X/+than in Scn2aR1627X/+. Spike threshold was depolarized only in Scn2aY84X/+, whereas Scn2aR1627X/+ remained similar to wild type. Frequency-current relations showed reduced firing at near-rheobase inputs in both mutants, with responses approaching wild type at stronger currents. Behaviorally, locomotion, sociability, and sensorimotor gating were preserved. Both lines exhibited increased grooming--consistent with restrictive, repetitive behavior; Scn2aY84X/+alone showed greater exploration in the elevated-risk context and a male-predominant deficit in rotarod learning. In maximal electroshock testing, mortality was lower in both lines without differences in seizure threshold or severity. Our results show that distinct SCN2A premature termination codons are not equivalent to one another, nor to a uniform haploinsufficient state. An early, nonsense-mediated decay-competent premature stop codon (Y84X) and a terminal-exon one (R1627X) produce partially overlapping yet allele-specific effects on neuronal excitability and behavior. These findings establish premature termination codon position as a determinant of phenotype, supporting allele-tailored mechanistic studies and therapeutic strategies.

neuroscience↗

bioRxiv2025

Non-canonical Sodium Channel Isoforms Underlie Chamber Specific Cardiac Excitability

Voltage-gated sodium (NaV) channels drive cardiac excitability. While NaV1.5 is the primary cardiac isoform, the composition and functional contributions of non-NaV1.5 isoforms in the heart remain unclear. Here, we developed a chemical-genetic mouse model (NaV1.5-GX) in which NaV1.5 can be selectively and reversibly inhibited by acyl- and aryl-sulfonamide compounds (GX drugs). NaV1.5-GX mice exhibited normal cardiac function at baseline, but acute GX drug administration caused profound conduction defects and arrhythmias. Whole-heart optical mapping revealed dose-dependent chamber-specific sensitivity to NaV1.5 inhibition, with the right ventricle (RV) being the most sensitive, followed by the left ventricle (LV), left atrium (LA), and right atrium (RA). Patch-clamp recordings of isolated cardiomyocytes with application of NaV isoform-selective inhibitors showed that NaV1.5 contributed 93% of sodium current in the LV, 81% in the RV and 78% in the LA. Non-NaV1.5 isoforms were differentially enriched across chambers: NaV1.8 in the LV, NaV1.1/1.3 in the RV, and NaV1.2/1.6/1.7 in the atria. These results reveal a surprising chamber-specific isoform landscape of cardiac sodium currents which may underlie the right ventricular predominant phenotype of Brugada syndrome and highlight non-NaV1.5 isoforms as potential mediators of chamber-specific cardiac pathologies and as pharmacological targets.

physiology↗

bioRxiv2025

Prevenying vision loss in a mouse model of Leber Congenital Amaurosis by engineered tRNA

Premature termination codons (PTCs) are associated with rare genetic disorders. Inducing targeted read-through of these nonsense mutations presents a potential therapeutic strategy for modifying disease outcomes. We previously reported that one such PTC, W53X, in the KCNJ13 gene causes blindness and Leber congenital amaurosis type-16 (LCA-16) due to loss of function of the inwardly rectifying potassium channel 7.1 (Kir7.1). Here, we present the proof of concept of a therapeutic approach based on anticodon-engineered transfer RNA (ACE-tRNA). The ACE-tRNA encodes the amino acid tryptophan (Trp) and suppresses the W53X PTC, restoring full-length protein expression. We used helper-dependent adenovirus (HDAd) to deliver the ACE-tRNATrp.UAG (tRNATrp.UAG) and rescue Kir7.1 function and physiology in patient-specific human induced pluripotent stem cell-derived retinal pigment epithelium (hiPSC-RPE) cells. Furthermore, in a W53X mouse model of LCA16, HDAd delivery of tRNATrp.UAG resulted in durable restoration of vision as measured by retinography. This study provides the first example of the therapeutic application of ACE-tRNA for treating an inherited form of blindness.

physiology↗

bioRxiv2025

Molecular Basis of Sodium Channel Inactivation

Voltage-gated sodium channels initiate action potentials and control electrical signaling throughout the animal kingdom. Fast inactivation is an essential auto-inhibitory mechanism and requisite component of sodium channel physiology. Recent structural and electrophysiological results are inconsistent with the canonical "ball and chain" model of fast inactivation thus necessitating an updated theoretical framework. Here, we use encoded fluorescence spectroscopy and high-resolution electrophysiology to capture key steps in the fast inactivation mechanism, from voltage-sensor activation to pore occlusion, an ultra-fast process which occurs in less than 2 milliseconds. Upon depolarization, activation of the domain IV voltage sensor initiates cytoplasmic DIII_DIV linker movement and quickly repositions the IFM motif into a hydrophobic pocket adjacent to the pore. This triggers a structural rearrangement of the pocket. The phenylalanine of the IFM motif contacts the pore-forming helices via a hydrophobic interaction with S6 of DIV and an aromatic/hydrophobic interaction with S6 of DIIII. These two interactions occur only after both S6 segments rotate, thus exposing the hydrophobic gate into the pore producing the fast inactivation. Based on the current results, we propose an alternative "lock and key" model to explain the molecular mechanism of fast inactivation.

biophysics↗

bioRxiv2024

A single main-chain hydrogen bond required to keep GABAA receptors closed

GABAA receptors are the primary inhibitory neurotransmitter receptors throughout the central nervous system. Despite significant progress understanding their three-dimensional structure, a critical gap remains in determining the molecular basis for channel gating. We recently identified M2-M3 linker mutations that suggest linker flexibility has asymmetric subunit-specific correlations with channel opening. Here we use non-canonical amino acids (ncAAs) to investigate the role of main-chain H-hydrogen bonds (H-bonds) that may stabilize the M2-M3 linkers. We show that a single main-chain H-bond within the {beta}2 subunit M2-M3 linker inhibits pore opening and is required to keep the unliganded channel closed. Furthermore, breaking this H-bond during channel opening accounts for approximately one third of the activation energy derived from GABA binding. In contrast, the analogous H-bond in the 1 subunit has no effect on gating. Our observations suggest that channel opening involves state-dependent breakage/disruption of a specific main-chain H-bond within the {beta}2 subunit M2-M3 linker.

biophysics↗

bioRxiv2023

Beta-subunit-eliminated eHAP expression (BeHAPe) cells reveal new properties of the cardiac voltage-gated sodium channel

Voltage-gated sodium (NaV) channels drive the upstroke of the action potential and are comprised of a pore-forming -subunit and regulatory {beta}-subunits. The {beta}-subunits modulate the gating, trafficking, and pharmacology of the -subunit. These functions are routinely assessed by ectopic expression in heterologous cells. However, currently available expression systems may not capture the full range of these effects since they contain endogenous {beta}-subunits. To better reveal {beta}-subunit functions, we engineered a human cell line devoid of endogenous NaV {beta}-subunits and their immediate phylogenetic relatives. This new cell line, {beta}-subunit-eliminated eHAP expression cells (BeHAPe), were derived from haploid eHAP cells by engineering inactivating mutations in SCN1B, SCN2B, SCN3B, SCN4B, MPZL1, MPZL2, MPZL3, MPZ and JAML. In diploid BeHAPe cells, the cardiac NaV -subunit, NaV1.5, was highly sensitive to {beta}-subunit modulation and revealed that each {beta}-subunit and even MPZ imparted unique gating properties. Furthermore, combining {beta}1 and {beta}2 with NaV1.5 generated a sodium channel with hybrid properties, distinct from the effects of the individual subunits. Thus, this approach revealed an expanded ability of {beta}-subunits to regulate NaV1.5 activity and can be used to improve the characterization of other /{beta} NaV complexes.

biophysics↗

bioRxiv2023

Structural context of homomeric interactions in the Ig domain of the MPZ (P0) myelin adhesion protein and relation to Charcot-Marie-Tooth disease phenotype variants

Mutations in Myelin Protein Zero (MPZ) account for 5% of Charcot-Marie-Tooth cases and can cause demyelinating or axonal phenotypes, reflecting the diverse roles of MPZ in Schwann cells. MPZ holds the apposing membranes of the myelin sheath together, with the adhesion role fulfilled by the extracellular lmmunoglobulin-like domain (lgMPZ), which can oligomerize. Current knowledge for how the lgMPZ might form oligomeric assemblies involving 3 weakly-interacting interfaces has been extrapolated from a protein crystal structure in which individual rat lgMPZ subunits are packed together under artificial conditions. These interfaces include one that organizes the lgMPZ into tetramers, a dimer interface that could link tetramers together, and a third hydrophobic interface that could mediate binding to lipid bilayers or the same hydrophobic surface on another lgMPZ domain. There are at present no data confirming whether the proposed lgMPZ interfaces actually mediate oligomerization in solution, whether they are required for the adhesion activity of MPZ, whether they are important for myelination, or whether their loss results in disease. We performed NMR and SAXS analysis of wild-type lgMPZ as well as mutant forms with amino-acid substitutions designed to interrupt its presumptive oligomerization interfaces. Here, we confirm the interface that mediates lgMPZ tetramerization, but find that dimerization is mediated by a distinct interface that has yet to be identified. We next correlated CMT phenotypes to subregions within lgMPZ tetramers. Axonal late-onset disease phenotypes (CMT2l/J) map to surface residues of lgMPZ proximal to the transmembrane domain. Early-onset demyelinating disease phenotypes (CMT1B/Dejerine-Sottas syndrome) map to two groups: one is described by variants that disrupt the stability of the lg-fold itself and are largely located within the core of the lg domain; whereas another describes a surface on the distal outer surface of lgMPZ tetramers. Computational docking studies predict that this latter disease-relevant subregion may mediate dimerization of lgMPZ tetramers.

neuroscience↗

bioRxiv2022

Backbone amides are key determinants of Cl- selectivity in CLC ion channels

Chloride homeostasis is tightly regulated in cellular compartments by dedicated channels and transporters. Whereas CLC-type channels select for Cl- over other anions, all other chloride channels are indiscriminate in their anionic permeability. Pore-lining side chains are thought to determine Cl- selectivity in CLC channels. However, orientation and functional roles of these side chains is not conserved among CLCs. All CLC pores are lined by backbone amides in a conserved structural arrangement, suggesting a role of mainchain groups in selectivity. We replaced pore-lining residues in the CLC-0 and bCLC-k channels with their respective -hydroxy acid counterparts using nonsense suppression method. This exchanges peptide-bond amides with ester-bond oxygens, incapable of hydrogen-bonding with permeating anions. Backbone substitutions functionally degrade inter-anion discrimination in a site-specific manner. These effects depend on the presence of a glutamate side chain that competes with ions permeating through the pore. Molecular dynamics simulations show that ion energetics within the bCLC-k pore are primarily determined by interactions with backbone amides. Insertion of an -hydroxy acid significantly alters ion selectivity and global pore hydration. We propose that backbone amides are conserved determinants of Cl- specificity in CLC channels in a mechanism reminiscent of that described for K+ channels.

biophysics↗

bioRxiv2022

Mechanosensitive pore opening of a prokaryotic voltage-gated sodium channel

Voltage-gated ion channels orchestrate electrical activities that drive mechanical functions in contractile tissues such as the heart and gut. In turn, contractions change membrane tension and impact ion channels. Voltage-gated ion channels are mechanosensitive, but the mechanisms of mechanosensitivity remain poorly understood. Here, we leverage the relative simplicity of NaChBac, a prokaryotic sodium channel from Bacillus halodurans, to investigate its mechanosensitivity. In whole-cell experiments on heterologously transfected HEK293 cells, shear stress reversibly altered the kinetic properties of NaChBac and increased its maximum current, comparably to the mechanosensitive eukaryotic sodium channel NaV1.5. In single-channel experiments, patch suction reversibly increased the open probability of a NaChBac mutant with inactivation removed. A simple kinetic mechanism featuring a mechanosensitive pore opening transition explained the overall response to force, whereas an alternative model with mechanosensitive voltage sensor activation diverged from the data. Structural analysis of NaChBac identified a large displacement of the hinged intracellular gate, and mutagenesis at the hinge abolished NaChBac mechanosensitivity, further supporting the proposed mechanism. Overall, our results suggest that NaChBac responds to force because its pore is intrinsically mechanosensitive. This mechanism may apply to other voltage-gated ion channels, including NaV1.5.

physiology↗

bioRxiv2022

Tuning aromatic contributions by site-specific encoding of fluorinated phenylalanine residues in bacterial and mammalian cells

The aromatic side-chains of phenylalanine, tyrosine, and tryptophan interact with their environments via both hydrophobic and electrostatic interactions. Determining the extent to which these contribute to protein function and stability is not possible with conventional mutagenesis. Serial fluorination of a given aromatic is a validated method in vitro and in silico to specifically alter electrostatic characteristics, but this approach is restricted to a select few experimental systems. Here, we report a new group of pyrrolysine-based aminoacyl-tRNA synthetase/tRNA pairs that enable the site-specific encoding of a varied spectrum of fluorinated phenylalanine amino acids in E. coli and mammalian (HEK 293T) cells. By allowing the cross-kingdom expression of proteins bearing these unnatural amino acids at biochemical scale, these tools will enable deconstruction of biological mechanisms which utilize aromatic-pi interactions in structural and cellular contexts. Statement of SignificanceThe aromatic side-chains of phenylalanine, tyrosine, and tryptophan are crucial for protein function and pharmacology due to their hydrophobic and electrostatic contributions to catalytic centers and ligand-binding pockets. However, few experimental approaches can chemically assess the functional roles of aromatics in cellular environments. The accepted computational method for aromatic interrogation is via serial fluorination, which lacks an experimental correlate in bacterial or mammalian cell systems. We have identified a family of synthetases to encode multiple different types of fluorinated phenylalanine residues in E. coli and HEK cells via nonsense suppression. The efficiency of these synthetases is sufficient to support biochemical characterization and structural determination of proteins with site-specific incorporation of unnatural phenylalanine analogs.

biochemistry↗

bioRxiv2019

Divergent Cl-and H + pathways underlie transport coupling and gating in CLC exchangers and channels

The CLC family of anion transporting proteins is comprised of secondary active H+-coupled exchangers and of Cl- channels. Both functional subtypes play key roles in human physiology, and mutations causing their dysfunction lead to numerous genetic disorders. Current models suggest that the CLC exchangers do not utilize a classical ping-pong mechanism of antiport, where the transporter sequentially interacts with one substrate at a time. Rather, in the CLC exchangers both substrates bind and translocate simultaneously while moving through partially congruent pathways. How ions of opposite electrical charge bypass each other while moving in opposite directions through a shared permeation pathway remains unknown. Here, we use MD simulations in combination with biochemical and electrophysiological measurements to identify a pair of highly conserved phenylalanine residues that form an aromatic pathway, separate from the Cl- pore, whose dynamic rearrangements enable H+ movement. Mutations of these aromatic residues impair H+ transport and voltage-dependent gating in the CLC exchangers. Remarkably, the role of the aromatic pathway is evolutionarily conserved in CLC channels. Using atomic-scale mutagenesis we show that the electrostatic properties and conformational flexibility of these aromatic residues are essential determinants of channel gating. Our results suggest that Cl- and H+ move through physically distinct and evolutionarily conserved routes through the CLC channels and transporters. We propose a unifying mechanism that describes the gating mechanism of CLC exchangers and channels.

biochemistry↗

bioRxiv2018

Engineered transfer RNAs for suppression of premature termination codons

Premature termination codons (PTCs) are responsible for 10-15% of all inherited disease. PTC suppression during translation offers a promising approach to treat a variety of genetic disorders, yet small molecules that promote PTC read-through have yielded mixed performance in clinical trials. We present a high-throughput, cell-based assay to identify anticodon engineered transfer RNAs (ACE-tRNA) which can effectively suppress in-frame PTCs and faithfully encode their cognate amino acid. In total, we identified ACE-tRNA with a high degree of suppression activity targeting the most common human disease-causing nonsense codons. Genome-wide transcriptome ribosome profiling of cells expressing ACE-tRNA at levels which repair PTC indicate that there are limited interactions with translation termination codons. These ACE-tRNAs display high suppression potency in mammalian cells, Xenopus oocytes and mice in vivo, producing PTC repair in multiple genes, including disease causing mutations within the cystic fibrosis transmembrane conductance regulator (CFTR).

molecular biology↗

bioRxiv2017

Conformational Dynamics In TRPV1 Channels Reported By An Encoded Coumarin Amino Acid

Transient Receptor Potential Vanilloid (TRPV1) channels support the detection and integration of nociceptive input. Currently available functional and structural data suggest that that TRPV1 channels have two potential gates within their cation selective permeation pathway: a barrier formed by a bundle crossing at the intracellular entrance and a second constriction created by the ion selectivity filter. To describe conformational changes associated with channel gating within the pore, the fluorescent non-canonical amino acid (f- ncAA) coumarin-tyrosine was genetically encoded at Y671, a residue proximal to the selectivity filter. TRPV1 channels expressing coumarin at either site displayed normal voltage- and agonist-dependent gating. Next, total internal reflection microscopy (TIRF) was performed to enable ultra-rapid, millisecond imaging of the conformational dynamics in single TRPV1 channels in live cells. Here, the data obtained from channels expressed in human derived cells show that optical fluctuations, photon counts, and variance of noise analysis from Y671 coumarin encoded in TRPV1 tetramers correlates closely with channel activation by capsaicin, thus providing an direct optical marker of channel activation at the selectivity filter. In companion molecular dynamics simulations, Y671 displays alternating solvent exposure between the closed and open states, giving support to the optical data. These calculations further suggest a direct involvement of Y671 in controlling the relative position of the pore helix and its role in supporting ionic conductance at the TRPV1 selectivity filter.

biophysics↗

bioRxiv2016

Engineered tRNA suppression of a CFTR nonsense mutation

Ten percent of human diseases are caused by nonsense mutations that lead to premature truncation of the protein reading frame. Small molecules that promote read-through of such PTC have significant clinical promise but current iterations suffer from low in vivo efficacy and the nonselective amino acid incorporation. Alternatively, while gene-modifying approaches, such as CRISPR/Cas9, represent a long-term solution, such treatments may be far from reaching the clinical setting. Building on previous work by our group and others, we describe a tRNA engineering approach that enables the conversion of an in frame nonsense stop mutation to the naturally occurring amino acid, thus rescuing the full-length wild type protein. Data is presented demonstrating the functionality of the approach with the rescue of CFTR W1282X, a human mutation that causes cystic fibrosis (CF). The stringency of the approach is confirmed by mass spectrometry in a model protein indicating the encoding of only tryptophan at the TGA suppression site. The data describe the first use of an edited tRNA to repair a CF causative mutation and serve a proof of principle for the eventual use of codon-edited tRNA for the therapeutic rescue of PTC disease codons.

cell biology↗