Biology subjects

Sychla, A.

Publications and source records attributed to Sychla, A..

6 recordsLinked to original sources

bioRxiv2026

An RNA Language Model trained on sequence alone reveals the structural logic of Internal Ribosome Entry Sites

Millions of RNA sequences are readily available, but the structures that determine their function are not. Picornaviruses initiate translation through Internal Ribosome Entry Sites (IRESes), RNA elements that recruit ribosomes independent of the 5` cap. These elements are large and highly divergent, and structural understanding remains limited to a handful of cases. We address this bottleneck by introducing an RNA language model (Albatross), trained purely on sequence, that predicts high-quality IRES structures at scale. We collect in cellulo chemical probing data for 96 full-length IRESes from divergent viruses and show that Albatross achieves far higher precision (0.80) than state-of-the-art predictions (0.47). Analyzing 75,000 IRES structures, we discover a novel Type II structural subclass and validate it experimentally. We demonstrate the pipeline broadly generalizes to identify functional structures, including tertiary contacts and alternative riboswitch structures. These findings establish Albatross as a scalable framework that accelerates RNA structure discovery and antiviral targeting.

molecular biology↗

bioRxiv2025

Heterologous expression of microbial nitroreductases for TNT degradation in transgenic animals

TNT (2,4,6-trinitrotoluene) from unexploded ordinances is a common environmental pollutant near munitions factories, military training sites, and areas of armed conflict. As physical and chemical approaches for TNT remediation are costly and difficult to scale, in situ bioremediation is an attractive alternative. TNT is a phytoaccumulative pollutant. Herbivores engineered to detoxify TNT are a potentially cost-effective platform to bioremediate large areas of contaminated sites while grazing or browsing. As a first step towards engineering herbivores with metabolic capabilities for TNT degradation, we engineered the animal genetic model, Drosophila melanogaster, to screen a library of microbial nitroreductases that can catalyse the initial degradation steps required for TNT degradation. We find strong, cofactor-dependent activity in fly lysates engineered to express NsfA from Escherichia coli, and NsfI from Enterobacter cloacae.

synthetic biology↗

bioRxiv2024

High-throughput investigation of genetic design constraints in domesticated Influenza A Virus for transient gene delivery

Replication-incompetent single cycle infectious Influenza A Virus (sciIAV) has demonstrated utility as a research and vaccination platform. Protein-based therapeutics are increasingly attractive due to their high selectivity and potent efficacy but still suffer from low bioavailability and high manufacturing cost. Transient RNA-mediated delivery is a safe alternative that allows for expression of protein-based therapeutics within the target cells or tissues but is limited by delivery efficiency. Here, we develop recombinant sciIAV as a platform for transient gene delivery in vivo and in vitro for therapeutic, research, and manufacturing applications (in vivo antimicrobial production, cell culture contamination clearance, and production of antiviral proteins in vitro). While adapting the system to deliver new protein cargo we discovered expression differences presumably resulting from genetic context effects. We applied a high-throughput screen to map these within the 3'-untranslated and coding regions of the hemagglutinin-encoding segment 4. This screen revealed permissible mutations in the 3'-UTR and depletion of RNA level motifs in the N-terminal coding region.

bioengineering↗

bioRxiv2023

Efficient gene activation in plants by the MoonTag programmable transcriptional activator

CRISPR/Cas-based transcriptional activators have been developed to induce gene expression in eukaryotic and prokaryotic organisms. The main advantages of CRISPR-Cas based systems is that they can achieve high levels of transcriptional activation and are very easy to program via pairing between the guide RNA and the DNA target strand. SunTag is a second-generation system that activates transcription by recruiting multiple copies of an activation domain (AD) to its target promoters. SunTag is a strong activator; however, in some species it is difficult to stably express. To overcome this problem, we designed MoonTag, a new activator that worked on the same basic principle as SunTag, but whose components are better tolerated when stably expressed in transgenic plants. We demonstrate that MoonTag is capable of inducing high levels of transcription in all plants tested. In Setaria, MoonTag is capable of inducing high levels of transcription of reporter genes as well as of endogenous genes. More important, MoonTag components are expressed in transgenic plants to high levels without any deleterious effects. MoonTag is also able to efficiently activate genes in eudicotyledonous species such as Arabidopsis and tomato. Finally, we show that MoonTag activation is functional across a range of temperatures, which is promising for potential field applications.

bioengineering↗

bioRxiv2022

Optimized dCas9 Transcription Activators for Plants

Understanding how gene expression impacts plant development and physiology is important for rationally engineering improved crops. Programmable Transcriptional Activators (PTAs), including CRISPR-Cas activators, have traditionally relied on a limited number of transcriptional activation domains. Usually the VP64 domain, derived from human herpes simplex virus, is fused to a DNA-binding domain to activate target gene expression. We reasoned there was considerable space for PTA improvement by replacing VP64 with a plant-derived activation domain. To address this, we designed, built, and tested a PTA library of 38 putative plant transcriptional activation domains. Domains from HSFA6b, AvrXa10, DOF1, DREB1, and DREB2 genes function as strong activators in Setaria viridis and Arabidopsis thaliana in protoplast assays and in transgenic plants. Overexpression of multiple endogenous genes (FT, PAP1, WUS) reached levels similar to the highly expressed housekeeping gene, PP2A, regardless of basal expression level. Further, these domains were effective in different PTA architectures, including the dCas9-SunTag, dCas9-Moontag, and TALE-SunTag systems. Lastly, we demonstrate the ability of these improved PTAs to map enhancer regions that promote gene expression in plants. This work demonstrates the effective and flexible nature of PTAs to activate target genes in plants, providing tools that can be used to improve agronomically relevant traits of interest.

plant biology↗

bioRxiv2021

Genetically engineered insects with sex-selection and genetic incompatibility enable population suppression

Engineered Genetic Incompatibility (EGI) is a method to create species-like barriers to sexual reproduction. It has applications in pest control that mimic Sterile Insect Technique when only EGI males are released. This can be facilitated by introducing conditional female-lethality to EGI strains to generate a sex-sorting incompatible male system (SSIMS). Here we demonstrate a proof of concept by combining tetracycline-controlled female lethality constructs with a pyramus-targeting EGI line in the model insect Drosophila melanogaster. We show that both functions (incompatibility and sex-sorting) are robustly maintained in the SSIMS line and that this approach is effective for population suppression in cage experiments. Further we show that SSIMS males remain competitive with wild-type males for reproduction with wild-type females, including at the level of sperm competition.

bioengineering↗