Improvements to Hybridization-Ligation ELISA Methods to Overcome Bioanalytical Challenges Posed by Novel Oligonucleotide Therapeutics.

As oligonucleotides (ONs) and similar nucleic acid therapeutic modalities enter development pipelines, there is continual need to develop bioanalytical methodologies addressing unique challenges they pose. Novel ONs back bone chemistries, especially those enabling stereochemical control, and base modifications are being exploited to improve pharmacological properties, potency, and increase half-lives. These changes have strained established methods, oftentimes precluding development of assays sensitive and specific enough to meet the needs of preclinical programs. For stereopure ONs representing a single molecular species, nontrivial presence of chain-shortened metabolites in biological samples necessitate assays with high specificity. To meet these needs, this report presents a toolbox of novel techniques, easy to implement for existing hybridization-ligation enzyme-linked immunosorbent assay formats, which address this challenge and yield significant sensitivity and specificity enhancements. Ligation efficiency was improved up to 61-fold through addition of polyethylene glycol, betaine, or dimethylsulfoxide, mitigating major differences among sequence-matched ONs of varying stereopurity, enabling sensitivities below 0.100 ng/mL for quantitation. These improvements enabled further refinement of capture probe designs engendering sufficient specificity to discriminate N-1 chain-shortened metabolites at both the 5' and 3' end of the ONs. These generalizable methods advance the performance of mainstay bioanalytical assays, facilitating research and development of innovative ONs therapeutics.

Precision Electrophile Tagging in Caenorhabditis elegans.

Adduction of an electrophile to privileged sensor proteins and the resulting phenotypically dominant responses are increasingly appreciated as being essential for metazoan health. Functional similarities between the biological electrophiles and electrophilic pharmacophores commonly found in covalent drugs further fortify the translational relevance of these small-molecule signals. Genetically encodable or small-molecule-based fluorescent reporters and redox proteomics have revolutionized the observation and profiling of cellular redox states and electrophile-sensor proteins, respectively. However, precision mapping between specific redox-modified targets and specific responses has only recently begun to be addressed, and systems tractable to both genetic manipulation and on-target redox signaling in vivo remain largely limited. Here we engineer transgenic Caenorhabditis elegans expressing functional HaloTagged fusion proteins and use this system to develop a generalizable light-controlled approach to tagging a prototypical electrophile-sensor protein with native electrophiles in vivo. The method circumvents issues associated with low uptake/distribution and toxicity/promiscuity. Given the validated success of C. elegans in aging studies, this optimized platform offers a new lens with which to scrutinize how on-target electrophile signaling influences redox-dependent life span regulation.

T-REX on-demand redox targeting in live cells.

This protocol describes targetable reactive electrophiles and oxidants (T-REX)-a live-cell-based tool designed to (i) interrogate the consequences of specific and time-resolved redox events, and (ii) screen for bona fide redox-sensor targets. A small-molecule toolset comprising photocaged precursors to specific reactive redox signals is constructed such that these inert precursors specifically and irreversibly tag any HaloTag-fused protein of interest (POI) in mammalian and Escherichia coli cells. Syntheses of the alkyne-functionalized endogenous reactive signal 4-hydroxynonenal (HNE(alkyne)) and the HaloTag-targetable photocaged precursor to HNE(alkyne) (also known as Ht-PreHNE or HtPHA) are described. Low-energy light prompts photo-uncaging (t1/2 <1-2 min) and target-specific modification. The targeted modification of the POI enables precisely timed and spatially controlled redox events with no off-target modification. Two independent pathways are described, along with a simple setup to functionally validate known targets or discover novel sensors. T-REX sidesteps mixed responses caused by uncontrolled whole-cell swamping with reactive signals. Modification and downstream response can be analyzed by in-gel fluorescence, proteomics, qRT-PCR, immunofluorescence, fluorescence resonance energy transfer (FRET)-based and dual-luciferase reporters, or flow cytometry assays. T-REX targeting takes 4 h from initial probe treatment. Analysis of targeted redox responses takes an additional 4-24 h, depending on the nature of the pathway and the type of readouts used.

A generalizable platform for interrogating target- and signal-specific consequences of electrophilic modifications in redox-dependent cell signaling.

Despite the known propensity of small-molecule electrophiles to react with numerous cysteine-active proteins, biological actions of individual signal inducers have emerged to be chemotype-specific. To pinpoint and quantify the impacts of modifying one target out of the whole proteome, we develop a target-protein-personalized "electrophile toolbox" with which specific intracellular targets can be selectively modified at a precise time by specific reactive signals. This general methodology, T-REX (targetable reactive electrophiles and oxidants), is established by (1) constructing a platform that can deliver a range of electronic and sterically different bioactive lipid-derived signaling electrophiles to specific proteins in cells; (2) probing the kinetics of targeted delivery concept, which revealed that targeting efficiency in cells is largely driven by initial on-rate of alkylation; and (3) evaluating the consequences of protein-target- and small-molecule-signal-specific modifications on the strength of downstream signaling. These data show that T-REX allows quantitative interrogations into the extent to which the Nrf2 transcription factor-dependent antioxidant response element (ARE) signaling is activated by selective electrophilic modifications on Keap1 protein, one of several redox-sensitive regulators of the Nrf2-ARE axis. The results document Keap1 as a promiscuous electrophile-responsive sensor able to respond with similar efficiencies to discrete electrophilic signals, promoting comparable strength of Nrf2-ARE induction. T-REX is also able to elicit cell activation in cases in which whole-cell electrophile flooding fails to stimulate ARE induction prior to causing cytotoxicity. The platform presents a previously unavailable opportunity to elucidate the functional consequences of small-molecule-signal- and protein-target-specific electrophilic modifications in an otherwise unaffected cellular background.

Temporally controlled targeting of 4-hydroxynonenal to specific proteins in living cells.

In-depth chemical understanding of complex biological processes hinges upon the ability to systematically perturb individual systems. However, current approaches to study impacts of biologically relevant reactive small molecules involve bathing of the entire cell or isolated organelle with excess amounts, leading to off-target effects. The resultant lack of biochemical specificity has plagued our understanding of how biological electrophiles mediate signal transduction or regulate responses that confer defense mechanisms to cellular electrophilic stress. Here we introduce a target-specific electrophile delivery platform that will ultimately pave the way to interrogate effects of reactive electrophiles on specific target proteins in cells. The new methodology is demonstrated by photoinducible targeted delivery of 4-hydroxynonenal (HNE) to the proteins Keap1 and PTEN. Covalent conjugation of the HNE-precursor to HaloTag fused to the target proteins enables directed HNE delivery upon photoactivation. The strategy provides proof of concept of selective delivery of reactive electrophiles to individual electrophile-responsive proteins in mammalian cells. It opens a new avenue enabling more precise determination of the pathophysiological consequences of HNE-induced chemical modifications on specific target proteins in cells.

Histone acetyl transferase (HAT) HBO1 and JADE1 in epithelial cell regeneration.

HBO1 acetylates lysine residues of histones and is involved in DNA replication and gene transcription. Two isoforms of JADE1, JADE1S and JADE1L, bind HBO1 and promote acetylation of histones in chromatin context. We characterized the role of JADE1-HBO1 complexes in vitro and in vivo during epithelial cell replication. Down-regulation of JADE1 by siRNA diminished the rate of DNA synthesis in cultured cells, decreased endogenous HBO1 protein expression, and prevented chromatin recruitment of replication factor Mcm7, demonstrating that JADE1 is required for cell proliferation. We used a murine model of acute kidney injury to examine expression of HBO1-JADE1S/L in injured and regenerating epithelial tissue. In control kidneys, JADE1S, JADE1L, and HBO1 were expressed in nuclei of proximal and distal tubular epithelial cells. Ischemia and reperfusion injury resulted in an initial decrease in JADE1S, JADE1L, and HBO1 protein levels, which returned to baseline during renal recovery. HBO1 and JADE1S recovered as cell proliferation reached its maximum, whereas JADE1L recovered after bulk proliferation had ceased. The temporal expression of JADE1S correlated with the acetylation of histone H4 on lysines 5 and 12, but not with acetylation of histone H3 on lysine 14, demonstrating that the JADE1S-HBO1 complex specifically marks H4 during epithelial cell proliferation. These data implicate JADE1-HBO1 complex in acute kidney injury and suggest distinct roles for JADE1 isoforms during epithelial cell recovery.