Biology subjects

Raines, R. T.

Publications and source records attributed to Raines, R. T..

7 recordsLinked to original sources

bioRxiv2026

A peptide catalyst can replace an essential enzyme in a eukaryotic cell

Protein enzymes are central to modern biology, yet how catalysis emerged before the evolution of large, folded proteins remains unresolved. Here we show that a short, genetically encoded peptide can replace an essential enzyme in a living eukaryotic cell. We designed minimal peptides containing a Cys-Xaa-Cys catalytic motif and an endoplasmic reticulum retention signal, and identified variants that rescue the otherwise lethal deletion of protein disulfide isomerase (PDI) in Saccharomyces cerevisiae. Cells relying on these peptides remain viable, though they grow more slowly and adapt by activating stress-response pathways, consistent with PDI being replaced by catalysts of lower intrinsic efficiency. Biochemical analyses show that peptide activity depends on local chemical environment and secondary structure rather than a globular fold. These results demonstrate that short peptides can replace an essential cellular reaction in vivo at the system level, supporting the plausibility of peptide-based catalysis as a precursor to modern protein enzymes.

biochemistry↗

bioRxiv2025

Nuclear Localization Signals Enable the Cellular Delivery of an Anti-CRISPR Protein to Control Genome Editing

Precise regulation of Cas9 activity is essential to minimize off-target effects, mosaicism, chromosomal alterations, immunogenicity, and genotoxicity in genome editing. Although type II anti-CRISPR proteins (Acrs) can inhibit and regulate Cas9, their size and anionic charge generally prevent them from crossing the cell membrane. Existing Acr delivery methods employing vectors or electroporation are either slow and persistent or require external equipment, limiting their therapeutic utility. To address these challenges, we developed a cell-permeable Acr (6xNLS-Acr), which uses nuclear localization signals (NLSs) to cross the cell membrane. We conjugated 6xNLS-Acr to a fluorescent dye to elucidate its cellular entry mechanism and directly visualized its binding to a fluorescent Cas9{middle dot}gRNA complex to study its inhibitory mechanism. 6xNLS-Acr (IC50 = 0.47 {micro}M) directly transduces human cells, including immortalized cell lines, embryonic stem cells, and 3D cell cultures, within 5 min, inhibiting up to 99% of Cas9 activity and increasing genome-editing specificity by nearly 100%. We further compared 6xNLS-Acr with our anthrax-derived Acr delivery platform. Our results demonstrate that 6xNLS-Acr is the most efficacious cell-permeable CRISPR-Cas inhibitor, significantly enhancing the precision and therapeutic potential of CRISPR-based genome editing.

genomics↗

bioRxiv2025

Pseudouridine residues as substrates for serum ribonucleases

In clinical uses, RNA must maintain its integrity in serum that contains ribonucleases (RNases), especially RNase 1, which is a human homolog of RNase A. These omnipresent enzymes catalyze the cleavage of the P-O5'' bond on the 3' side of pyrimidine residues. Pseudouridine ({Psi}) is the most abundant modified nucleoside in natural RNA. The substitution of uridine (U) with {Psi} or N1-methylpseudouridine (m1{Psi}) reduces the immunogenicity of mRNA and increases ribosomal translation, and these modified nucleosides are key components of RNA-based vaccines. Here, we assessed the ability of RNase A and RNase 1 to catalyze the cleavage of the P-O5'' bond on the 3' side of {Psi} and m1{Psi}. We find that these enzymes catalyze the cleavage of UpA up to 10-fold more efficiently than the cleavage of {Psi}pA or m1{Psi}pA. X-ray crystallography of enzyme-bound nucleoside 2',3'-cyclic vanadate complexes and molecular dynamics simulations of enzyme{middle dot}dinucleotide complexes show that U, {Psi}, and m1{Psi} bind to RNase A and RNase 1 in a similar manner. Quantum chemistry calculations suggested that the higher reactivity of UpA is intrinsic, arising from an inductive effect that decreases the pKa of the 2'-hydroxy group of U and enhances its nucleophilicity toward the P-O5'' bond. Experimentally, we found that UpA does indeed undergo spontaneous hydrolysis faster than does m1{Psi}pA. Our findings inform the continuing development of RNA-based vaccines and therapeutic agents.

biochemistry↗

bioRxiv2025

Protective antigen-mediated delivery of an anti-CRISPR proteinfor precision genome editing

Precise control over the dosage of Cas9-based technologies is essential because off-target effects, mosaicism, chromosomal aberrations, immunogenicity, and genotoxicity can arise with prolonged Cas9 activity. Type II anti-CRISPR proteins (Acrs) inhibit and control Cas9 but are generally impermeable to the cell membrane due to their size (6-54 kDa) and anionic charge. Moreover, existing Acr delivery methods are long-lived and operate within hours (e.g., viral and non-viral vectors) or are not applicable in vivo (e.g., nucleofection), limiting therapeutic applications. To address these problems, we developed the first protein-based anti-CRISPR delivery platform, LFN-Acr/PA, which delivers Acrs into cells within minutes. LFN-Acr/PA is a nontoxic, two-component protein system derived from anthrax toxin, where protective antigen proteins bind receptors widespread in human cells, forming a pH-triggered endosomal pore that LFN-Acr binds and uses to enter the cell. In the presence of PA, LFN-Acr enters human cells at concentrations as low as 2.5 pM to inhibit up to 95% of Cas9-mediated knockout, knock-in, transcriptional activation, and base editing. Timing LFN-Acr delivery reduces off-target base editing and increases Cas9 specificity by 41%. LFN-Acr/PA is the most potent known cell-permeable CRISPR-Cas inhibition system, significantly improving the utility of CRISPR for genome editing.

biochemistry↗

bioRxiv2024

Endosomolytic Peptides Enable the Cellular Delivery of Peptide Nucleic Acids

Precision genetic medicine enlists antisense oligonucleotides (ASOs) to bind to nucleic acid targets important for human disease. Peptide nucleic acids (PNAs) have many desirable attributes as ASOs but lack cellular permeability. Here, we use an assay based on the corrective splicing of an mRNA to assess the ability of synthetic peptides to deliver a functional PNA into a human cell. We find that the endosomolytic peptides L17E and L17ER4 are highly efficacious delivery vehicles. Co-treatment of a PNA with low micromolar L17E or L17ER4 enables robust corrective splicing in nearly all treated cells. Peptide-PNA conjugates are even more effective. These results enhance the utility of PNAs as research tools and potential therapeutic agents.

biochemistry↗

bioRxiv2024

Sitetack: A Deep Learning Model that Improves PTM Predictionby Using Known PTMs

Post-translational modifications (PTMs) increase the diversity of the proteome and are vital to organismal life and therapeutic strategies. Deep learning has been used to predict PTM locations. Still, limitations in datasets and their analyses compromise success. Here we evaluate the use of known PTM sites in prediction via sequence-based deep learning algorithms. Specifically, PTM locations were encoded as a separate amino acid before sequences were encoded via word embedding and passed into a convolutional neural network that predicts the probability of a modification at a given site. Without labeling known PTMs, our model is on par with others. With labeling, however, we improved significantly upon extant models. Moreover, knowing PTM locations can increase the predictability of a different PTM. Our findings highlight the importance of PTMs for the installation of additional PTMs. We anticipate that including known PTM locations will enhance the performance of other proteomic machine learning algorithms.

bioinformatics↗

bioRxiv2021

Bifunctional Peptide that Anneals to Damaged Collagen and Clusters TGF-β Receptors Enhances Wound Healing

Transforming growth factor-{beta} (TGF-{beta}) plays important roles in wound healing. The activity of TGF-{beta} is initiated upon binding of the growth factor to extracellular domains of its receptors. We sought to facilitate activation by clustering these extracellular domains. To do so, we used a known peptide that binds to TGF-{beta} receptors without diminishing their affinity for TGF-{beta}. We conjugated this peptide to a collagen-mimetic peptide that can anneal to damaged collagen in a wound bed. We find that the conjugate enhances collagen deposition and wound closure in mice in a manner consistent with the clustering of TGF-{beta} receptors. This strategy provides a means to upregulate the TGF-{beta} signaling pathway without adding exogenous TGF-{beta} and could inspire means to treat severe wounds. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/461420v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@a0c248org.highwire.dtl.DTLVardef@332b3forg.highwire.dtl.DTLVardef@15c3533org.highwire.dtl.DTLVardef@a8b75a_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗