Biology subjects

Wu, L.-F.

Publications and source records attributed to Wu, L.-F..

5 recordsLinked to original sources

bioRxiv2025

Intelligent Design of Escherichia coli Terminators

Terminators are specific nucleotide sequences located at the 3 end of a gene and contain transcription termination information. As a fundamental genetic regulatory element, terminators play a crucial role in the design of gene circuits. Accurately characterizing terminator strength is essential for improving the precision of gene circuit designs. Experimental characterization of terminator strength is time-consuming and labor-intensive; therefore, there is a need to develop computational tools capable of accurately predicting terminator strength. Current prediction methods do not fully consider sequence or thermodynamic information related to terminators, lacking robust models for accurate prediction. Meanwhile, deep generative models have demonstrated tremendous potential in the design of biological sequences and are expected to be applied to terminator sequence design. This study focuses on intelligent design of Escherichia coli terminators and primarily conducts the following research: (1) To construct an intrinsic terminator strength prediction model for E. coli, this study extracts sequence features and thermodynamic features from E. coli intrinsic terminators. Machine learning models based on the selected features achieved a prediction performance of R2 = 0.72. (2) This study employs a generative adversarial network (GAN) to learn from intrinsic terminator sequence training data and generate terminator sequences. Evaluation reveals that the generated terminators exhibit similar data distributions to intrinsic terminators, demonstrating the reliability of GAN-generated terminator sequences. (3) This study uses the constructed terminator strength prediction model to screen for strong terminators from the generated set. Experimental verification shows that among the 18 selected terminators, 72% exhibit termination efficiencies greater than 90%, confirming the reliability of the intelligent design approach for E. coli terminators. In sum, this study constructs a terminator strength prediction model and a terminator generation model for E. coli, providing model support for terminator design in gene circuits. This enhances the modularity of biological component design and promotes the development of synthetic biology.

synthetic biology↗

bioRxiv2022

Membrane-remodeling protein ESCRT-III homologs incarnate the evolution and morphogenesis of multicellular magnetotactic bacteria

Endosomal sorting complex required transport (ESCRT) III proteins are essential for membrane remodeling and repair across all domains of life. Eukaryotic ESCRT-III and the cyanobacterial homologs PspA and Vipp1/Imm30 remodel membrane into vesicles, rings, filaments and tubular rods structures. Here our microscopy analysis showed that multicellular bacteria, referred to as magnetoglobules, possess multiple compartments including magnetosome organelles, polyphosphate granules, vesicles, rings, tubular rods, filaments and MVB-like structures. Therefore, membrane remodeling protein PspA might be required for the formation of these compartments, and contribute to the morphogenesis and evolution of multicellularity. To assess these hypotheses, we sequenced nine genomes of magnetoglobules and found a significant genome expansion compared to unicellular magnetotactic bacteria. Moreover, PspA was ubiquitous in magnetoglobules and formed a distinct clade on the tree of eubacterial and archaeal ESCRT-III. The phylogenetic feature suggested the evolution of magnetoglobules from a unicellular ancestor of deltaproteobacterium. Hetero-expression of ellipsoidal magnetoglobule pspA2 gene alone in Escherichia coli resulted in intracellular membrane aggregation. GFP fusion labeling revealed polar location of PspA2 in rod-shaped unicells and regular interval location in filamentous cells. Cryo-electron tomography analysis showed filament bundle, membrane sacculus, vesicles and MVB-like structure in the cells expressing PspA2. Moreover, electron-dense area with a similar distribution as GFP-PspA2 foci in filamentous cells changed the inward orientation of the septum, which might interfere with the cell division. Collectively, these results show the membrane remodeling function of magnetoglobule PspA proteins, which may contribute to morphogenesis and the evolution of multicellularity of magnetotactic bacteria.

microbiology↗

bioRxiv2021

Nonenzymatic loop-closing ligation generates RNA hairpins and is a template-free way to assemble functional RNAs

RNA hairpin loops are the predominant element of secondary structure in functional RNAs. The emergence of primordial functional RNAs, such as ribozymes that fold into complex structures that contain multiple hairpin loops, is generally thought to have been supported by template-directed ligation. However, template inhibition and RNA misfolding problems impede the emergence of function. Here we demonstrate that RNA hairpin loops can be synthesized directly from short RNA duplexes with single-stranded overhangs by nonenzymatic loop-closing ligation chemistry. We show that loop-closing ligation allows full-length functional ribozymes containing a hairpin loop to be assembled free of inhibitory template strands. This approach to the assembly of structurally complex RNAs suggests a plausible pathway for the emergence of functional RNAs before a full-length RNA copying process became available.

synthetic biology↗

bioRxiv2020

Structural basis for the inhibition of the SARS-CoV-2 RNA-dependent RNA polymerase by favipiravir-RTP

The RNA polymerase inhibitor, favipiravir, is currently in clinical trials as a treatment for infection with SARS-CoV-2, despite limited information about the molecular basis for its activity. Here we report the structure of favipiravir ribonucleoside triphosphate (favipiravir-RTP) in complex with the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) bound to a template:primer RNA duplex, determined by electron cryomicroscopy (cryoEM) to a resolution of 2.5 [A]. The structure shows clear evidence for the inhibitor at the catalytic site of the enzyme, and resolves the conformation of key side chains and ions surrounding the binding pocket. Polymerase activity assays indicate that the inhibitor is weakly incorporated into the RNA primer strand, and suppresses RNA replication in the presence of natural nucleotides. The structure reveals an unusual, non-productive binding mode of favipiravir-RTP at the catalytic site of SARS-CoV-2 RdRp which explains its low rate of incorporation into the RNA primer strand. Together, these findings inform current and future efforts to develop polymerase inhibitors for SARS coronaviruses.

molecular biology↗

bioRxiv2018

Efficient genome editing of Magnetospirillum magneticum AMB-1 by CRISPR-Cas9 system for analyzing magnetotactic behavior

Magnetotactic bacteria are a diverse group of microorganisms with the ability to use geomagnetic fields for direction sensing. This magnetotactic behavior can help microorganisms move towards favorable habitats for optimal growth and reproduction. Highly efficient genomic editing is very useful for a comprehensive understanding of the magnetotactic mechanism at the molecular level. In this study, we adapted an engineered CRISPR-cas9 system for efficient inactivation of gene in a widely used magnetotactic bacteria model strain, Magnetospirillum magneticum AMB-1. By combining an engineered nuclease-deficient Cas9 and single-guide RNA, a CRISPR interference system was successfully developed to silence amb0994 expression. More importantly, we succeeded in the construction of a single amb0994 gene deletion mutant using CRISPR-Cas9 with approximate 60-fold high efficiency compared to classical homology double-crossing replacement procedure. This mutant synthesized normally the magnetosomes, but reacted quicker and with less time than the wild-type strain to abrupt magnetic field reversals. A dynamics simulation by modeling M. magneticum AMB-1 cell as an ellipsoid showed that the difference of the motions between wild and{Delta} amb0994 is due to flagellar influence. The behavior observation being consistent with dynamics simulation indicated that Amb0994 is involved in the cellular response to magnetic torque change via controlling flagella. Besides the contribution to a better understanding of the magnetotaxis mechanism, this study demonstrates the CRISPR system as a useful genetic toolbox for high-efficiency genome editing in magnetotactic bacteria.

microbiology↗