Shuto, Y. | Search bioRxiv

bioRxiv2026

Oxidized EGCG redirects α-synuclein aggregation through shear-selected interactions

-Synuclein (Syn) aggregation is central to Parkinson's disease pathology, and the green-tea polyphenol epigallocatechin gallate (EGCG) has been studied as an inhibit fibril formation. However, EGCG autoxidation and analyses under static, non-fibrillating conditions have obscured whether activity arises from EGCG itself or oxidized species and which interactions persist during Syn assembly. Here we combine HPLC, LC-MS, TEM and solution NMR to compare unoxidized and oxidized EGCG (oxEGCG), while Rheo-NMR monitors Syn during shear-promoted assembly. Unoxidized EGCG showed no detectable binding under static, reducing conditions, whereas oxEGCG bound Syn, inhibited fibrillation and remodeled fibrils. Under shear flow, many oxEGCG-dependent perturbations partially reverted toward free Syn, whereas interactions near the NAC-C-terminal boundary were preferentially retained, coinciding with formation of oligomers and short, thick fibrils. These findings identify oxidation state and flow as determinants of EGCG action on Syn and show that Rheo-NMR distinguishes interactions that persist during shear-promoted assembly.

biochemistry↗

bioRxiv2026

A cuffed CRISPR guide RNA for microRNA activity-dependent genome editing

Cells in multicellular eukaryotic systems are diverse biological units, with characteristics and functions determined by their molecular profiles. CRISPR-Cas9 genome editing has been widely used across biology to modulate gene expression and study gene function. However, there is currently no versatile and scalable method for editing a cells genome in response to endogenous cellular signals. Here, we report the engineering of a CRISPR guide RNA that efficiently confers genome editing in response to the catalytic activity of a target microRNA (miRNA) within a cell. miRNAs are short non-coding RNAs that are widely conserved across eukaryotes and can cleave their target RNA through almost perfect base pairing. In mammals, miRNAs are largely involved in development and homeostasis as well as disease progression and developmental disorders. To leverage these properties for genome editing, we developed a cuffed guide RNA (cgRNA) which is composed of a permutated order of sequence domains from the commonly used single guide RNA (sgRNA). These permutated domains were then concatenated with a miRNA target sequence, yielding a warped guide RNA that is inactive until cleaved by a complementary miRNA. We demonstrated that cgRNA enabled efficient miRNA activity-dependent genome editing in human and mouse cell lines. Biochemical and structural analyses revealed three stages of inhibition of the CRISPR genome-editing pathway for unprocessed cgRNA. Utilizing a lentiviral library of cgRNAs containing miRNA targets covering mouse genome-wide miRNAs, we identified miRNA cleavage activities and their sequence specificities in mouse embryonic stem cells and during smooth muscle cell differentiation. Furthermore, we showed that endogenous mRNA expression could be irreversibly recorded into a DNA sequence using a cgRNA targeted by a synthetic miRNA repeat. cgRNA is a simple, robust, miRNA activity-gated genome editing system that could facilitate the development of cell state-specific genome editing, the mapping of miRNA activity and gene expression landscapes, and the recording of molecularly determined cell states during the long-term progression of multicellular systems.

bioengineering↗