Biology subjects

Sczepanski, J. T.

Publications and source records attributed to Sczepanski, J. T..

4 recordsLinked to original sources

bioRxiv2026

Glycine detection with a nuclease-stable L-RNA sensor

Glycine is a vital extracellular signal in bacteria, plants, and the brain. Although RNA-based sensors detect glycine in cells, their extracellular application in native biological environments is limited by enzymatic degradation from nucleases. Mirror-image RNA is nuclease-resistant and preserves the tertiary structure required for RNA function, but synthesizing long L-RNAs such as the 170-nt glycine sensor (glyS) remains challenging. Here, we applied cross-chiral ligation with natural D-RNA ribozymes to assemble a mirror-image L-RNA glycine sensor (L-glyS). Optimization of the ligation conditions enabled up to 68% conversion to the full-length sensor. L-glyS displayed nuclease resistance and maintained glycine-dependent fluorescence in serum, where the original D-glyS lost function. These results establish cross-chiral ligation as a strategy for constructing long, functional L-RNAs and broaden the possible applications of RNA-based sensors to extracellular detection of small molecules.

synthetic biology↗

bioRxiv2026

Metallothionein loss in cancer cells contributes to increased mutations through defective DNA repair and metabolic imbalance

Understanding which genes are involved in mutagenesis is essential for developing cancer prevention and treatment strategies; establishing protectors of the genome has revolutionized cancer biology. Here, we describe metallothionein (MT) proteins as previously uncharacterized protectors against mutagenesis. MT is a heavy metal binding protein essential for zinc homeostasis and protection against heavy metal cytotoxicity. Because zinc binds approximately 10-15% of the proteome and is critical for processes such as DNA repair and mitochondrial health, MT loss is expected to disrupt these processes. We hypothesized that MT loss induces genomic instability by impairing DNA repair and mitochondrial function. In this study, the consequences of MT deficiency in high-grade serous ovarian cancer (HGSC) were investigated by knockdown of the most highly expressed MT, MT2A. Loss of MT2A resulted in the impaired DNA repair pathway base excision repair (BER), leading to increased mutagenesis. MT2A deficiency produced mitochondrial dysfunction, characterized by a decrease in mitochondrial membrane potential, glycolysis, oxidative phosphorylation, amino acids, and an imbalance of nucleobases. Together, these defects reflect cellular states associated with increased cancer aggressiveness. These findings identify MT as a fundamental hub maintaining genomic and metabolic integrity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/736843v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1af4adeorg.highwire.dtl.DTLVardef@15c588forg.highwire.dtl.DTLVardef@1ba5181org.highwire.dtl.DTLVardef@13d150b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Thymine DNA Glycosylase Binds to R-Loops and Excises 5-Formyl and 5-Carboxyl Cytosine from DNA/RNA Hybrids

R-loops are three-stranded nucleic acid structures consisting of a DNA/RNA hybrid and a displaced single-stranded DNA. Once considered rare byproducts of transcription, R-loops are now recognized as important regulators of various nuclear processes. In particular, evidence indicates a role for R-loops in regulating DNA methylation dynamics. R-loops have been shown to promote active DNA demethylation--the enzymatic reversal of 5-methylcytosine (5mC) back into cytosine--by recruiting associated proteins, providing an attractive targeting mechanism. Nevertheless, many important aspects of this process, including whether the associated proteins bind to and function on R-loops, remain to be substantiated. In this study, we demonstrate for the first time that thymine DNA glycosylase (TDG), a key enzyme in the active DNA demethylation pathway, binds tightly to R-loops in vitro and can excise DNA demethylation intermediates 5-formylcytosine (5fC) and 5-carboxycytosine (5caC) from DNA in DNA/RNA hybrid duplexes. We also show that R-loops guide the strand-specific activity of TDG at CpG sites, potentially explaining the asymmetric distribution of 5fC/5caC at gene promoters. Furthermore, we provide important mechanistic insights into base excision on DNA/RNA hybrid duplexes using 19F NMR. Finally, our findings suggest that TDG-R-loop interactions may be widespread in human cells. Collectively, our results provide strong evidence that R-loops play a critical role in DNA demethylation and support a mechanism in which 5fC/5caC are directly removed from DNA/RNA hybrids in cells. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/668694v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@1ac2095org.highwire.dtl.DTLVardef@18f4952org.highwire.dtl.DTLVardef@16c403org.highwire.dtl.DTLVardef@9ed96c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

An Expanded Substrate Scope for Cross-Chiral Ligation Enables Efficient Synthesis of Long L-RNAs

Despite the growing interest in mirror-image L-oligonucleotides, both as a robust nucleic acid analogue and as an artificial genetic polymer, their broader adoption in research and medicine remains hindered by challenges associated with the synthesis of long sequences, especially for L-RNA. Herein, we present a novel strategy for assembling long L-RNAs via the joining of two or more shorter fragments using cross-chiral ligase ribozymes together with new substrate activation chemistry. We show that 5'-monophosphorylated L-RNA, which is readily prepared by solid phase synthesis, can be activated by chemical attachment of a 5'-adenosine monophosphate (AMP) or diphosphate (ADP), yielding 5'-adenosyl (di-or tri-) phosphate L-RNA. The activation reaction is performed in mild aqueous conditions, proceeds efficiently with short or large L-RNA, and, yielding few biproducts, requires little or no further purification after activation. Importantly, both groups, when added to L-RNA, are compatible with ribozyme-mediated ligation, with the 5'-adenosyl triphosphate permitting rapid and efficient joining of multiple, long L-RNA strands. This is exemplified by the assembly of a 129-nt L-RNA molecule via a single cross-chiral ligation event. Overall, by relying on ribozymes that can be readily prepared by in vitro transcription and L-RNA substrates that can be activated through simple chemistry, these methods are expected to make long L-RNAs more accessible to a wider range of researchers and facilitate the expansion of L-ON-based technologies.

synthetic biology↗