Acid phosphatases, classified under the enzyme commission number EC 3.1.3.2, are orthophosphoric monoester phosphohydrolases that hydrolyze phosphate esters and carry out transphosphorylation reactions in acidic environments. In plants, they are indispensable players in phosphate metabolism, signal transduction, nutrient mobilization during germination, and the acquisition of phosphorus when soils are depleted. Beyond plant physiology, these enzymes serve as clinical markers for human diseases, including prostate cancer and infertility, which gives any new insight into their active-site architecture a relevance that stretches far beyond the botanical world. Yet for many plant acid phosphatase isoenzymes, the structural basis of catalysis remains poorly understood, and Erythrina indica, a leguminous tree of medicinal and ecological importance, had never had its seed phosphatases characterized at this level of detail.

The investigative strategy rested on a classic but powerful technique: residue-specific chemical modification. The team treated purified AP-II with three targeted reagents, each designed to attack one class of amino acid. Dicyclohexylcarbodiimide, abbreviated DCHC, was deployed against carboxylate groups; N-bromosuccinimide, or NBS, against tryptophan; and phenylmethylsulfonyl fluoride, PMSF, against serine. The results were unambiguous. Modification with NBS at 5 millimolar concentration produced complete loss of enzymatic activity, PMSF treatment destroyed more than 90 percent of activity, and DCHC treatment caused a roughly 90 percent decline. Because each reagent is selective for a different residue type, the near-total inactivation pointed directly to the participation of tryptophan, serine, and carboxylate in the enzyme’s active site.

Crucially, the researchers then ran substrate protection experiments to confirm that these residues were not merely incidental casualties of the reagents. When 2 millimolar p-nitrophenyl phosphate, the enzyme’s model substrate, was added before the modification reactions, the loss of activity caused by all three modifying agents was significantly prevented. The substrate, in effect, occupied the active site and shielded the vulnerable residues from chemical attack, exactly as would be expected if those residues were genuinely part of the catalytic machinery rather than bystanders elsewhere on the protein. This protective effect provided strong evidence that all three residue classes are directly involved in the catalytic process.

Kinetic analysis of the partially inactivated enzyme added a subtle but important layer of interpretation. When AP-II was partially inactivated with NBS, PMSF, or DCHC, the maximum reaction velocity, Vmax, decreased while the Michaelis constant, Km, remained essentially unchanged. That dissociation is telling: Km reflects substrate binding affinity, while Vmax reflects the rate of catalytic turnover. A drop in Vmax with stable Km means the modified residues contribute primarily to the chemistry of catalysis rather than to substrate recognition. The enzyme could still grab its substrate, but it could no longer efficiently execute the hydrolysis reaction, implicating the three residue classes in the bond-breaking step itself.

The pH dependence of activity supplied independent confirmation of the carboxylate’s role. Measuring kinetic parameters across the pH range from 3 to 6 and plotting log(Vmax/Km) against pH yielded pKa values of 4.6 and 5.25 for AP-II, values squarely consistent with the ionization behavior of a carboxylate group participating in the reaction. Meanwhile, Lineweaver-Burk plots showed that Km for p-nitrophenyl phosphate stayed nearly constant across pH 3.0 to 6.0 while Vmax changed, demonstrating that pH alters the rate of reaction without altering substrate affinity. The stoichiometry of modification told a similar story of economy: although two tryptophan residues were modified by NBS, extrapolating the plot of residual activity against modified residues to zero activity revealed that only a single tryptophan is directly involved in catalysis. Likewise, a log-log plot of inactivation rate against PMSF concentration gave a slope of one, indicating a single serine residue participates, and the same slope-of-one analysis for DCHC indicated that binding of one molecule of reagent suffices to inactivate the enzyme.

Fluorescence spectroscopy illuminated the physical environment of the catalytic tryptophan. Excited at 280 and 295 nanometers, AP-II emitted with a maximum at 313 nanometers, a blue-shifted signature indicating that the fluorescent tryptophans are buried in the hydrophobic interior of the protein rather than exposed to solvent. Quenching experiments with three agents of different charge, neutral acrylamide, positively charged cesium chloride, and negatively charged potassium iodide, revealed an unusual microenvironment. Acrylamide quenched the fluorescence completely, and remarkably, cesium ions achieved full accessibility as well, while iodide quenched far less effectively. The researchers concluded that the tryptophan microenvironment is strongly negatively charged, which explains why the positively charged cesium ion penetrates so readily while the negatively charged iodide is repelled. Stern-Volmer plots for acrylamide were linear, characteristic of purely dynamic quenching, whereas the upward-curving plots for cesium chloride and potassium iodide indicated a sphere-of-action static component or two populations of tryptophan residues quenched at different rates.

Structural studies rounded out the picture. Thermal denaturation caused activity loss accompanied by a red shift of the fluorescence maximum from 313 to 333 nanometers, the classic signature of protein unfolding exposing buried tryptophans to water. Chemical denaturation with guanidine hydrochloride abolished activity at concentrations as low as 0.2 molar, further supporting the involvement of carboxylate residues in catalysis. Denatured enzyme titrated with NBS revealed five total tryptophans in AP-II, of which two are surface-exposed and three are buried in the core. Circular dichroism spectroscopy showed that AP-II is composed of roughly 45 percent alpha-helix, 23 percent beta-sheet, and 31 percent random coil, and a sixth-degree polynomial fit of the far-UV CD spectrum achieved a coefficient of determination of approximately 0.89, which the authors interpreted as indicating greater structural stability of the major form. In silico structure prediction went further, suggesting a homodimeric enzyme of 102 kilodaltons with about 5.2 percent N-linked glycosylation and conserved histidine, tryptophan, aspartate, and tyrosine residues, an architecture typical of large plant acid phosphatases.

The authors are candid about the limits of their approach. The identification of active-site residues rests primarily on chemical modification rather than high-resolution crystallography, the three-dimensional structure is computationally predicted and awaits experimental validation, and the entire characterization was performed with the artificial substrate p-nitrophenyl phosphate, which may not mirror the enzyme’s physiological substrate preferences. Future work, they suggest, should include site-directed mutagenesis of the identified residues and testing against natural substrates. Even so, the study delivers a coherent and technically rigorous account of how three amino acids, a carboxylate acting in acid-base catalysis, a buried tryptophan stabilizing the substrate, and a serine participating in phosphoryl transfer, cooperate to hydrolyze phosphate esters in the acidic interior of a seed. It is a reminder that even a tree growing on a residential street in Pune can harbor molecular machinery of elegant precision, and that careful classical biochemistry, bolstered by modern statistics and computation, can still resolve that machinery one residue at a time.

Subject of Research: Identification of the catalytic carboxylate, tryptophan, and serine residues in acid phosphatase form II from Erythrina indica seeds

Article Title: Identification of catalytic residues of acid phosphatase form II from Erythrina indica seeds

Article References: Uzgare, A. S., & Shaikh, F. M. R. (2026). Identification of catalytic residues of acid phosphatase form II from Erythrina indica seeds. Discover Chemistry, 3(1), Article 488. https://doi.org/10.1007/s44371-026-00948-3

Image Credits: AI Generated

DOI: 10.1007/s44371-026-00948-3

Keywords: acid phosphatase, Erythrina indica, active site, chemical modification, tryptophan, serine, carboxylate, enzyme kinetics, fluorescence spectroscopy, circular dichroism, plant biochemistry, catalytic mechanism

Prime editing is often described as a molecular search-and-replace system. It couples a catalytically impaired Cas9 nickase to an engineered reverse transcriptase, and it is programmed by a prime editing guide RNA, or pegRNA, which both locates the genomic target and carries the template encoding the desired edit. Because the method writes new genetic information without making a double-strand break in the DNA and without requiring an external donor template, it has long been viewed as one of the most versatile tools in the genome editing repertoire, capable of installing all twelve possible base-to-base conversions as well as small insertions and deletions. Yet translating that versatility into animals, and ultimately patients, has proved difficult. The prime editor protein is far too large to fit inside a single adeno-associated virus vector, forcing researchers to rely on cumbersome dual-vector systems, while concerns about the long-term expression of an editor in a patient’s tissues have made viral delivery unattractive for clinical development.

Non-viral delivery offers a way around those concerns. Lipid nanoparticles, the same technology that carried mRNA vaccines into billions of arms, can deliver the prime editor in transient mRNA form together with its pegRNA, giving the editor a brief window of activity before it is degraded by the cell. The problem has been efficiency. Earlier attempts to deliver prime editing systemically with lipid nanoparticles produced editing levels far below what would be needed clinically, required repeated injections, or depended on doses of RNA so high that they exceeded what regulators would consider translatable. The fragile nature of long RNA molecules inside the bloodstream and inside cells is a central culprit: unmodified or lightly modified pegRNAs are rapidly chewed apart by nucleases, and even end modifications at the RNA termini, the standard protective measure borrowed from antisense oligonucleotide chemistry, leave long internal stretches of the guide exposed and vulnerable.

The Wuhan-led team’s solution was to extend chemical protection far beyond the ends of the guide RNA. Rather than modifying only the terminal nucleotides, the researchers densely incorporated modified RNA motifs throughout both the fixed regions of the pegRNA, such as the scaffold and the engineered extension motifs like evopreQ1, and the variable regions, including the spacer, the reverse transcriptase template, and the primer binding site. The chemical toolkit relied on well-established modifications from the RNA therapeutics field, principally 2′-O-methyl groups on the ribose sugar and phosphorothioate linkages in the phosphate backbone, both of which are known to shield RNA from nuclease degradation and, in the case of 2′-O-methyl groups, to subtly alter pairing geometry and affinity. The team describes the resulting molecules as super-end-modified RNAs, in which terminal protection and dense internal modification act in combination.

The optimization was systematic. In human cell experiments using electroporated prime editor mRNA, the researchers tested modification patterns in each structural element of the pegRNA in turn, measuring editing outcomes at endogenous loci such as EMX1, where the system installed a three-base deletion, and HEK3, where it installed a single-base insertion. They varied the density of 2′-O-methyl and phosphorothioate modifications at the 5′ end of the spacer, in the primer binding site, in the reverse transcriptase template, and in the extended scaffold motifs, and then combined the best-performing patterns. Analyses of editing byproducts showed that the dense modifications did not increase the proportion of indels or unwanted scaffold-insertion events relative to total editing, and cell viability assays indicated the heavily modified guides were not toxic to the treated cells, an important consideration since excessive chemical modification of guide RNAs has previously been associated with cellular stress.

The in vivo results were the centerpiece. Using lipid nanoparticles to co-deliver prime editor mRNA and the densely modified pegRNA into mice, the team achieved nearly 70 percent prime editing efficiency in the bulk liver, a level indicating that most hepatocytes in the organ had been edited. Perhaps more consequential for clinical translation, a single injection at a lipid nanoparticle dose the authors describe as clinically translatable was sufficient to suppress the expression of the target protein in vivo, and under those dose conditions the densely modified guide produced an approximately 80-fold increase in editing efficiency compared with conventional end-modified pegRNAs. Toxicity assessments, including serum liver enzyme measurements and analysis of editing byproducts, supported the tolerability of the approach, and off-target analyses at predicted sites suggested the chemical modifications did not worsen targeting fidelity.

The generality of the strategy proved equally important. The dense motif modification approach was not confined to prime editing. When the team applied the same modification logic to guide RNAs carrying the widely used MS2 RNA motif, which is exploited in RNA-guided recruitment platforms such as base editor systems that tether an editor protein to a Cas9 nickase through an RNA aptamer interaction, they saw broad improvements across multiple RNA sequences and split RNA-guided genome editing platforms, with base editing efficiencies rising by up to 11-fold. The researchers also combined the modified guides with mRNA encoding ancillary factors known to boost prime editing, such as MLH1-domain constructs, and observed further gains in vivo, indicating that RNA chemistry and protein engineering enhancements can be layered rather than traded off against one another.

The findings arrive at a moment when the field is converging on transient, non-viral delivery as the safest route to in vivo genome editing. Clinical programs using lipid nanoparticle delivery of CRISPR-Cas9 mRNA and guide RNA have already demonstrated that single-dose in vivo gene knockout is feasible in humans, and prime editing has shown curative promise in ex vivo settings such as engineered hematopoietic stem cells for sickle cell disease. What has lagged is in vivo precision editing, where the required correction must be installed in a large fraction of target tissue cells rather than simply disrupting a gene. By raising editing yields at clinically plausible doses, the dense modification strategy narrows the gap between what prime editing can do in a dish and what it can do in a body, and because the modifications are applied to the RNA guide rather than to the editor protein, they can in principle be adopted without redesigning the editing enzymes themselves.

Challenges remain before such a system could reach patients, including extending the approach beyond the liver, where lipid nanoparticles naturally accumulate, to other tissues, scaling the manufacture of long, densely modified pegRNAs under clinical quality standards, and confirming long-term safety in larger animal models. The authors note that the methodology builds on their earlier work developing rapid methods for generating long chemically modified pegRNAs, and patent applications on the pegRNA modifications have been filed through Wuhan University. Still, the study offers a concrete, generalizable recipe: protect the guide RNA densely, deliver the editor transiently, and let chemistry do much of the work that delivery vehicles alone could not. If the efficiency gains hold across tissues and disease targets, the modest RNA molecule at the heart of prime editing may prove to be the lever that finally lifts the technology from laboratory promise toward therapeutic reality.

Subject of Research: Engineered prime editing guide RNAs with densely modified RNA motifs for robust in vivo genome editing

Article Title: Dense RNA motif modifications enable robust in vivo prime editing and enhance efficiencies of diverse editing systems

Article References: Lei, X., Chen, D., Zhang, K., Liu, X., Chen, Q., Zhang, Y., Ji, R., Zhu, J., Zhang, Q., Zhang, Y., & Yin, H. (2026). Dense RNA motif modifications enable robust in vivo prime editing and enhance efficiencies of diverse editing systems. Nature Biomedical Engineering. https://doi.org/10.1038/s41551-026-01787-4

Image Credits: AI Generated

DOI: 10.1038/s41551-026-01787-4

Keywords: prime editing, pegRNA, chemical modification, lipid nanoparticles, in vivo genome editing, base editing, RNA therapeutics, gene therapy, 2'-O-methyl, phosphorothioate, mouse liver, non-viral delivery