Biology subjects

Civit, L.

Publications and source records attributed to Civit, L..

5 recordsLinked to original sources

bioRxiv2026

RNA Organelles in DNA-based Artificial Cells Provide Spatial Aptamer Functions and Enhanced Signal Processing

Naturally occurring biomolecular condensates orchestrate key cellular processes by creating spatially distinct reaction environments, yet engineering synthetic condensates that combine structural programmability with spatioselectively encoded function remains challenging. Here we report multiphase DNA-RNA artificial cells (ACs) that embed functional RNA condensates as organelle-like compartments within programmable DNA core-shell ACs. A single thermal assembly protocol yields three-phase ACs comprising a glassy RNA core organelle embedded in a liquid-like DNA compartment, surrounded by a crosslinked DNA shell. The RNA organelles contain aptamer function, enabling selective protein recruitment and small-molecule activation, while the DNA scaffold provides independent addressability, regulates RNA-condensate size and enhances resistance to serum-mediated degradation. We further show that RNA chemistry can be used to adjust environmental responsiveness: unmodified RNA organelles undergo rapid degradation in serum and release captured protein cargo, whereas 2'-fluoro-modified RNA organelles remain stable for at least 24 h. Finally, by coupling transcriptional modules localized in the DNA core to cell-free protein translation in the surrounding medium, we establish sender-receiver communication between AC populations and self-actuating signal processing within individual DNA-RNA ACs. These results establish hybrid nucleic-acid ACs as programmable, spatially organized systems that couple compartment architecture, RNA molecular recognition and biochemical communication. TOC Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/733869v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@23ee9org.highwire.dtl.DTLVardef@1706336org.highwire.dtl.DTLVardef@181a241org.highwire.dtl.DTLVardef@1c374d8_HPS_FORMAT_FIGEXP M_FIG C_FIG Multiphase DNA-RNA artificial cells integrate a protective DNA shell, a transcriptionally active DNA core, and a functional RNA organelle. Spatial compartmentalization enables signal generation, external protein expression, and selective recapture via RNA aptamers.

Synthetic Biology↗

bioRxiv2026

Expanding the chemical diversity of RNA by transcriptional incorporation of amino acid- and glycosyl-modified nucleotides

With the increasing interest in RNA-based therapies, there is a pressing need to incorporate new chemistries into more complex RNA molecules. These modifications can protect RNA from degradation, improve its pharmacokinetics, and enhance its targeting properties. Here we describe the enzymatic synthesis of chemically modified RNA derivatives using a mutant T7 RNA polymerase to incorporate 23 different base modifications alongside stabilizing ribose modifications, such as 2'-fluoro and 2'-deoxy groups. To investigate the impact on transcription efficiency and fidelity, we employed a pool of 38 template sequences and analyzed the transcripts by next-generation sequencing of the cDNA. Results demonstrated that all modifications were successfully incorporated into RNA, with transcription efficiency influenced by three main factors: type of modification, base modified, and the sequence context. Misincorporation levels during transcription and reverse transcription into cDNA were generally low (<1%) but included noticeable exceptions for some nucleobase-modification combinations. As a robust proof-of-concept we demonstrated the selection of Histidine-U modified aptamer, relying on multiple rounds of transcription and amplification, binding Influenza hemagglutinin protein with low nanomolar KD. We anticipate that this work will significantly contribute to the design and production of chemically modified RNAs with novel functionalities, advancing applications in biomedicine and synthetic biology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/720138v2_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@29b6daorg.highwire.dtl.DTLVardef@a3c425org.highwire.dtl.DTLVardef@1a27a4aorg.highwire.dtl.DTLVardef@ab0e9_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2026

Fluorinated RNA origami enables serum-stable nanodevices for sensing and targeting

Chemically modified RNAs with increased stability and reduced immunogenicity have transformed RNA therapeutics. Rational RNA design methods, including RNA origami, seek to further extend RNA medicine and biotechnology by encoding advanced functions such as signalling, targeting, and controlled release within the RNA polymer. However, current design methods lack the ability to integrate chemical modification or predict how it shapes the structure of large RNA assemblies inhibiting its use in RNA therapeutics. Here we demonstrate that 2-fluoro pyrimidine RNA (FY-RNA) origami structures can be co-transcriptionally folded to generate serum-stable nanodevices. Cryogenic electron microscopy reveals that FY-RNA can alter folding pathways and perturb tertiary motifs, while molecular dynamics simulations show how 2-fluoro modification affects hydrogen bonding, sugar pucker, and helix-helix interactions. Despite these structural perturbations, fluorogenic aptamers embedded within RNA origami retain partial activity and enable logic-based molecular sensing in human serum. Finally, we use an FY-RNA scaffold to determine the structure of an FY-RNA anti-Spike aptamer bound to the Spike protein at 3.4 [A] resolution, uncovering fluorine-specific structural motifs and protein interactions. Together, our results establish design principles for nuclease-resistant RNA architectures and position FY-RNA as a versatile polymer for constructing medical nanodevices and environmental sensors. More broadly, this work provides a framework for systematically exploring the folding landscape of chemically modified RNAs, expanding the chemical and functional diversity accessible to nucleic acid nanotechnology and RNA medicine.

biochemistry↗

bioRxiv2025

Single-Particle Tracking and Positional Phenotyping Reveals Variant-Specific Early Checkpoints in SARS-CoV-2 Cell Entry

SARS-CoV-2 entry is governed by Spike (S) protein-mediated engagement of ACE2 and subsequent activation of either plasma membrane fusion mediated by TMPRSS2 or endocytic uptake. Currently, most insights into these pathways come from bulk assays that obscure the fate of individual virions, thereby concealing intricate mechanistic details that can inform on therapeutic intervention strategies. Here, we applied single-particle fluorescence imaging to directly observe the early checkpoints of SARS-CoV-2 cell entry pathways and separate binding from internalization. Fluorescent virus-like particles (VLPs) pseudotyped with either G614 or Omicron BA.5 S protein variants were imaged on HEK293T-ACE2 (TMPRSS2-negative) and classified at the single-particle level as surface-interacting, crossing, or internal. At baseline, G614 VLPs show higher binding and a larger internalized share than BA.5 VLPs, revealing general divergence in early entry behavior between variants. A trivalent anti-S receptor-binding domain aptamer reduces G614 binding and lowers its internalization. Conversely, the aptamer does not block BA.5 VLP cell binding but increases its internalization efficiency. Pitstop 2, an inhibitor of clathrin-mediated endocytosis, causes no significant change in this observation window, consistent with early clathrin-sensitive events having already progressed. Quantification of trajectories reveals variant-specific mobility: BA.5 displays higher step length than G614, consistent with greater lateral scanning and surface retention. Together, these compact single-particle readouts expose variant-resolved early checkpoints in entry and provide a simple platform to test how ligands and pathway probes shift binding and internalization.

biophysics↗

bioRxiv2025

Lock, Protect, and Bind: In Vitro Selection of LNA-modified Aptamers Using a Mutant T7 RNA Polymerase

RNA therapeutics are powerful tools for gene modulation and targeted therapies, but their clinical application is hindered by nuclease degradation and immunogenicity. Incorporating chemical modifications, like locked nucleic acids (LNAs), can enhance nuclease resistance, targeting properties, and thermal stability. Traditionally, LNA incorporation has relied on solid-phase synthesis of short RNAs. Engineered polymerases capable of incorporating xenonucleic acids (XNAs), including LNA, into longer RNAs have been described. However, their XNA yield is limited by primer and template copy numbers, and the generated DNA-XNA duplexes can be difficult to purify. We present a novel approach for incorporating LNA-ATP and LNA-TTP alongside 2Fluoro (2F)-modified pyrimidines via in vitro transcription using a mutant T7 RNA polymerase. This method enables efficient, primer-independent synthesis and amplification of LNA-modified RNA with low error rates. To demonstrate its utility, we performed in vitro selection (SELEX) of LNA- and 2F-modified aptamers targeting Influenza hemagglutinin (HA) and human CD40 ligand (hCD40L), two therapeutically relevant proteins. Iterative SELEX cycles yielded aptamers with low-nanomolar affinities, high specificity, and high nuclease resistance. Overall, this approach provides a scalable and versatile platform for generating chemically stabilized RNAs, fully compatible with SELEX, and holds potential for developing next-generation RNA-based therapeutics with improved pharmacokinetics.

molecular biology↗