Biology subjects

Luptak, A.

Publications and source records attributed to Luptak, A..

9 recordsLinked to original sources

bioRxiv2026

A miniature bioluminescent platform for imaging structured RNAs

A broader understanding of RNA biology requires methods for visualizing transcript dynamics in native environments. Common imaging approaches rely on fluorescent probes that require excitation light and often depend on large aptamer arrays, limiting sensitivity and compatibility with many non-coding RNAs, particularly highly structured folds. To address these challenges, we developed a miniaturized bioluminescent platform for RNA imaging. This system employs a compact RNA tag that assembles luciferase fragments upon binding of monomeric RNA-binding proteins (RNA lanterns). Systematic optimization of the RNA component yielded a structured 24-nucleotide tag, representing greater than 3-fold reduction in size relative to the original C-11-B RNA tag, placing it among the smallest RNA imaging tags reported to date. The optimized tag enabled RNA-dependent bioluminescence detection in live cells, notably as a small, single insert within highly structured non-coding RNAs, including the MALAT1 ENE domain and a small HDV-like ribozyme. Collectively, this work establishes a miniaturized bioluminescent platform for imaging a broad range of RNA targets in living systems.

molecular biology↗

bioRxiv2025

An open, integrated platform for multiplexed bioluminescence microscopy

We report an imaging package to democratize all-in-one bioluminescence and fluorescence microscopy. The platform comprises three tools: PhasorViewer, a visualization suite to design experiments and identify optimal probe combinations; PhasorScope, an open-source, cost-effective microscopy framework to upgrade conventional microscopes; and PhasorAnalysis, a dedicated, user-friendly analysis pipeline to quantify phasor imaging data. We demonstrate the utility of the platform for multiplexed, simultaneous fluorescence and bioluminescence imaging with readily accessible optical reporters.

bioengineering↗

bioRxiv2024

Engineering a ribozyme with aminoacyl-tRNA synthetase activity

A ribozyme that can charge a tRNA with amino acids and discriminate between cognate and non-cognate tRNAs is of interest because an RNA with this ability may have been a critical for translation in the transition from the RNA world. In addition, it could provide a tool for incorporating non-canonical amino acids for biotechnology applications. Here, we rationally engineer a ribozyme by fusing a tRNA binding module derived from a T-box riboswitch with a catalytic module (a flexizyme) to generate a ribozyme that can amino acylate a target tRNA. We demonstrate that this ribozyme be readily redesigned to alter tRNA specificity. This ribozyme is compatible with an in vitro translation system and could be used to recode a protein sequence to site-specifically incorporate a non-canonical amino acid.

biochemistry↗

bioRxiv2024

Bioorthogonal cyclopropenones for investigating RNA structure

RNA sequences encode secondary and tertiary structures that impact protein production and other cellular processes. Misfolded RNAs can also potentiate disease, but the complete picture is lacking. To establish more comprehensive and accurate RNA structure-function relationships, new methods are needed to interrogate RNA and trap native conformations in cellular environments. Existing tools primarily rely on electrophiles that are constitutively "on" or triggered by UV light, often resulting in high background reactivity. We developed an alternative, chemically triggered approach to crosslink RNAs using bioorthogonal cyclopropenones (CpOs). These reagents selectively react with phosphines to provide ketenes--electrophiles that can trap neighboring nucleophiles to forge covalent crosslinks. As proof-of-concept, we synthesized a panel of CpOs and appended them to thiazole orange (TO-1). The TO-1 conjugates bound selectively to a model RNA aptamer (Mango) with nanomolar affinity, confirmed by fluorescence turn-on. After phosphine administration, covalent crosslinks were formed between the CpO probes and RNA. The degree of crosslinking was both time and dose-dependent. We further applied the chemically triggered tools to model RNAs in biologically relevant conditions. Collectively, this work expands the toolkit of probes for studying RNA and its native conformations.

biochemistry↗

bioRxiv2023

Inhibition of CPEB3 ribozyme elevates CPEB3 protein expression and polyadenylation of its target mRNAs, and enhances object location memory

A self-cleaving ribozyme that maps to an intron of the cytoplasmic polyadenylation element binding protein 3 (CPEB3) gene is thought to play a role in human episodic memory, but the underlying mechanisms mediating this effect are not known. We tested the activity of the murine sequence and found that the ribozymes self-scission half-life matches the time it takes an RNA polymerase to reach the immediate downstream exon, suggesting that the ribozyme-dependent intron cleavage is tuned to co-transcriptional splicing of the CPEB3 mRNA. Our studies also reveal that the murine ribozyme modulates maturation of its harboring mRNA in both cultured cortical neurons and the hippocampus: inhibition of the ribozyme using an antisense oligonucleotide leads to increased CPEB3 protein expression, which enhances polyadenylation and translation of localized plasticity-related target mRNAs, and subsequently strengthens hippocampal-dependent long-term memory. These findings reveal a previously unknown role for self-cleaving ribozyme activity in regulating experience-induced co-transcriptional and local translational processes required for learning and memory. Significance StatementCytoplasmic polyadenylation-induced translation is one of the key steps for regulating protein synthesis and neuroplasticity in the hippocampus. The CPEB3 ribozyme is a highly conserved mammalian self-cleaving catalytic RNA with unknown biological roles. In this study, we investigated how the intronic ribozyme affects the CPEB3 mRNA maturation and translation, and its subsequent effect on memory formation. Our findings show that the ribozyme activity is anti-correlated with CPEB3 mRNA splicing: inhibition of the ribozyme results in higher mRNA and protein levels, which contribute to long-term memory. Our studies offer new insights into the role of the CPEB3 ribozyme in neuronal translational control for the activity-dependent synaptic functions that underlie long-term memory and demonstrate a novel biological role for self-cleaving ribozymes.

neuroscience↗

bioRxiv2022

Pyrophosphate-Mediated Repair of Damaged and Mismatched RNA by a Polymerase Ribozyme

Prior to the emergence of the contemporary biosphere, the first replicating systems are thought to have progressed through an RNA-based stage. Such an evolving world would likely have transferred heritable information during replication using RNA polymerase ribozymes. Though substantial effort has been put forth towards evolving RNA polymerases, many variants suffer from premature termination and low fidelity, resulting in low yields of full-length or active sequences. Replication of longer sequences requires a sufficiently high fidelity to lend an evolutionary advantage to an evolvable system. Here we demonstrate ribozyme-mediated repair of mismatched and damaged RNA sequences. Under conditions of saturating pyrophosphate concentrations, we show that a polymerase ribozyme can repair RNA sequences terminated in a mismatch, a non-extendable 2'-3' cyclic phosphate, or both, to generate a triphosphorylated nucleotide. This repair step increases the fidelity and allows polymerization along an extended template, including the ribozyme itself. This increase of copying fidelity advances the longstanding goal of developing a self-replicating polymerase ribozyme.

biochemistry↗

bioRxiv2022

A modular platform for bioluminescent RNA tracking

A complete understanding of RNA biology requires methods for tracking transcripts in vivo. Common strategies rely on fluorogenic probes that are limited in sensitivity, dynamic range, and depth of interrogation, owing to their need for excitation light and tissue autofluorescence. To overcome these challenges, we developed a bioluminescent platform for serial imaging of RNAs. Small RNA tags were engineered to recruit light-emitting luciferase fragments (termed RNA lanterns) upon transcription. Robust photon production was observed for RNA targets both in cells and in live animals. Importantly, only a single copy of the tag was necessary for sensitive detection, in sharp contrast to fluorescent platforms requiring multiple repeats. Overall, this work provides a foundational platform for visualizing RNA dynamics from the micro to the macro scale.

molecular biology↗

bioRxiv2021

The CPEB3 ribozyme modulates hippocampal-dependent memory

A self-cleaving ribozyme mapping to an intron of the cytoplasmic polyadenylation element binding protein 3 (CPEB3) gene has been suggested to play a role in human episodic memory, but the underlying mechanisms mediating this effect are not known. The ribozymes self-scission half-life matches the time it takes an RNA polymerase to reach the immediate downstream exon, suggesting that the ribozyme-dependent intron cleavage is tuned to co-transcriptional splicing of the CPEB3 mRNA. Here we report that the murine ribozyme modulates its own host mRNA maturation in both cultured cortical neurons and the hippocampus. Inhibition of the ribozyme using an antisense oligonucleotide leads to increased CPEB3 protein expression, which enhances polyadenylation and translation of localized plasticity-related target mRNAs, and subsequently strengthens hippocampal-dependent long-term memory. These findings reveal a previously unknown role for self-cleaving ribozyme activity in regulating experience-induced co-transcriptional and local translational processes required for learning and memory.

molecular biology↗

bioRxiv2019

Regulation of mRNA translation by a photoriboswitch

Optogenetic tools have revolutionized the study of receptor-mediated biological processes, but such tools are lacking for the study of RNA-controlled systems. To fill this gap, we used in vitro selection to isolate a novel RNA that selectively binds the trans isoform of a stiff-stilbene (amino-tSS), a rapidly and reversibly photoisomerizing small molecule. Structural probing revealed that the RNA binds amino-tSS about 100-times stronger than amino-cSS, giving the system robust selectivity for the trans isomer. In vitro and in vivo functional analysis showed that the riboswitch, termed Werewolf-1 (Were-1), inhibits translation of a downstream open reading frame when bound to amino-tSS and photoisomerization of the ligand with a sub-millisecond pulse of light induced the protein expression. Similarly, bacterial culture containing the cis isoform (amino-cSS) supported protein expression, which was inhibited upon photoisomerization to amino-tSS. Reversible regulation of gene expression using a genetically encoded light-responsive RNA will broaden the analysis of complex RNA processes in living cells.

synthetic biology↗