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Frontiers in Chemical Biology ·

Volume 5 - 2026 | https://doi.org/10.3389/fchbi.2026.1914162

Towards programmable metallonucleases via genetic code expansion

  • Eryn Lundrigan1
  • Matthew O’Neill1
  • John Paul Pezacki1*corresponding author
  1. Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, ON, Canada
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Abstract

The rapid advancement of protein engineering and genetic code expansion technologies over the last decade has reshaped how researchers rationally design proteins with novel catalytic functions. Among these approaches, the site-specific incorporation of unnatural amino acids has enabled the introduction of chemical functionalities that are inaccessible to the canonical amino acid space. In this perspective, we highlight the metal-chelating UAA (2,2′-bipyridin-5-yl) alanine (BpyAla) and its emerging utility in mediating nucleic acid cleavage. Multiple studies have demonstrated the successful site-specific incorporation of BpyAla into proteins of interest, where subsequent metal coordination enables catalytic cleavage of DNA and RNA substrates. Here, we discuss the potential of BpyAla-mediated nucleic acid cleavage, with emphasis on the development of next-generation BpyAla analogues, the exploration of alternative metal cofactors, cooperative and multi-residue design strategies, and the expansion of compatible protein scaffolds and nucleic acid substrates. Designable BpyAla-engineered systems represent an emerging frontier in artificial metallonuclease design, with potential long-term relevance to targeted nucleic acid therapeutics.

Keywords

Introduction

The expansion of the genetic code has fundamentally reshaped our ability to interrogate and engineer protein function (Birch-Price et al., 2024; Costello et al., 2024). By enabling the incorporation of unnatural amino acids (UAAs) into proteins in living systems, genetic code expansion (GCE) technologies have propelled protein engineering beyond the constraints of the twenty canonical amino acids. This shift has unlocked access to chemistries and catalytic capabilities that are not naturally encoded, allowing researchers to design proteins with tailored functionalities (Birch-Price et al., 2024; Brouwer et al., 2024; Chemla et al., 2024; Yi et al., 2024; Spina et al., 2026). As a result, UAAs have emerged as powerful tools for developing next-generation protein biologics and research tools with engineered activities.

A defining advantage of GCE is the capacity for precise, site-specific incorporation of UAAs into proteins, affording an unprecedented level of control over protein design. Through this approach, researchers can rationally select a UAA bearing a desired chemical functionality, introduce it at a defined position within a protein of interest, and directly evaluate how that modification confers function (Costello et al., 2024). This workflow effectively transforms proteins into modular, “designer” scaffolds, where chemical reactivity can be installed with residue-level precision (Figure 1). The adaptability of this approach is threefold, arising from the diversity of compatible protein scaffolds, the expanding repertoire of UAAs, and the ability to finely tune activity through precise site-selection. These layers of control provide a highly versatile framework for the rational design of proteins with customizable functions across a wide range of applications.

Workflow for site-specific incorporation of metal-chelating UAA to engineer RNA-cleaving catalysis into protein of interest.
Figure 1. Workflow for site-specific incorporation of metal-chelating UAA to engineer RNA-cleaving catalysis into protein of interest.

In this perspective, we highlight the utility of the bipyridine-containing UAA (2,2′-bipyridin-5-yl) alanine (BpyAla) as a versatile tool for engineering catalytic cleaving function into proteins. BpyAla can coordinate divalent metal ions through its nitrogens, enabling the creation of metallocentres capable of mediating phosphodiester bond cleavage of nucleic acids (Figure 2A). By site-specifically installing BpyAla into selected proteins, one can introduce metal-dependent reactivity in a programmable manner (Klemencic et al., 2024; Lee et al., 2025). In the context of this article, “programmability” is defined as user choice of nucleic acid target-specific protein scaffolds, metal catalyst, rational protein redesign through BpyAla site selection, and or a combination of these factors. This strategy exemplifies how GCE-enabled incorporation of UAAs can be leveraged to endow proteins with non-canonical catalytic functions, opening new avenues for the development of engineered enzymes and protein therapeutics. Despite proof-of-concept studies utilizing BpyAla, the broader design principles and future potential of BpyAla-engineered metallonucleases remains underexplored.

Next-Generation BpyAla Analogue Development. **(A)** Substituent placement on bipyridine of BpyAla. **(B)** Aromatic scaffolds, terpyridinel and phenanthroline. **(C)** Jarvis et al. UAA Trz as a dual function BpyAla derivative.
Figure 2. Next-Generation BpyAla Analogue Development. (A) Substituent placement on bipyridine of BpyAla. (B) Aromatic scaffolds, terpyridinel and phenanthroline. (C) Jarvis et al. UAA Trz as a dual function BpyAla derivative.

Proof of concept studies in BpyAla-driven nucleic acid cleavage

The genetic incorporation of bipyridine-containing UAAs such as BpyAla has emerged as a powerful strategy for installing new catalytic functionality into proteins. By leveraging the metal-chelating properties of the bipyridine moiety, BpyAla enables the formation of designer metallocentres within protein scaffolds, effectively introducing novel reactivity into otherwise non-catalytic systems (Drienovská et al., 2015; Jarvis, 2020).

Importantly, the conceptual foundation for these engineered systems was established decades earlier through studies of synthetic metal-coordination systems. In particular, in vitro work with different metal chelators enabled metal-catalyzed nucleic acid cleavage to move from a chemical curiosity into a targeted catalytic strategy. Research demonstrated that synthetic metal complexes could mimic the reactivity of natural metallonucleases (Schultz and Dervan, 1983; Dervan, 1984; Chin and Banaszczyk, 1989; Young and Chin, 1995).

Endonucleases, and more specifically metallonucleases, are the natural catalysts of nucleic acid cleavage, with their activity dependent on coordinated metal ion cofactors such as Zn2+, Mg2+ or Ca2+. The ability of divalent metal ions to promote directed phosphodiester bond cleavage was first recognized through foundational studies in enzymology and RNA chemistry during the mid-20th century. Building on these observations, Chin and colleagues developed synthetic metal complexes capable of recapitulating nuclease-like activity through metal-assisted cleavage mechanisms, including the well-characterized two-metal-ion pathway (Young and Chin, 1995). Importantly, phosphodiester hydrolysis can proceed through multiple catalytic mechanisms, ranging from cooperative two-metal systems to single-metal-ion catalysis, the latter of which has become particularly attractive in the design of artificial metalloenzymes and BpyAla-based catalytic platforms. (Aboelnga and Wetmore, 2019; Pecina et al., 2021; Nikkel et al., 2025). These foundational studies established that metal-coordinated catalytic centers could mediate efficient nucleic acid cleavage, but the next major advance came with the ability to genetically encode these chemistries directly into proteins.

Early foundational work by Peter G. Schultz and colleagues established both the feasibility and utility of this strategy in biological systems through the first site-specific incorporation of BpyAla into proteins using GCE technologies. Their early studies demonstrated the development of an tRNA/aminoacyl-tRNA synthetase pair specific for BpyAla that allowed for the selective incorporation in E. coli, establishing that complex metal-chelating UAAs could be genetically encoded with high fidelity (Xie et al., 2007). Subsequent work by Shultz and colleagues evaluated the incorporation of BpyAla into the transcriptional activator DNA-binding protein CAP (catabolite activator protein) (Hyun and Schultz, 2008). The CAP K26BpyAla construct showed Cu(II)- and Fe(II)-dependent double-stranded DNA cleavage at its consensus sequence with high specificity. These studies provided the first evidence that installing a synthetic metal-chelating residue into a protein scaffold could confer entirely new catalytic activity, engineering a DNA-binding protein into a functional metallonuclease-like system.

Subsequent work has extended BpyAla-enabled catalysis to RNA-targeting proteins. Notably, Ahmed and colleagues demonstrated the incorporation of BpyAla into the RNA-binding protein p19 (Ahmed et al., 2023). This Tombusvirus viral suppressor of RNA silencing binds and sequesters RNA duplexes, effectively inhibiting the RNA silencing pathway and enabling immune evasion in infected plants. To enhance this endogenous activity of RNA sequestering, Ahmed and colleagues converted p19 into an artificial metallonuclease with construct T111BpyAla, capable of promoting RNA cleavage across both siRNA and miRNA targets, further underscoring the generalizability of this approach across nucleic acid substrates (Ahmed et al., 2023). Together, these studies establish a foundation for the use of BpyAla as a modular catalytic UAA that can be deployed in diverse protein contexts. A key frontier in the field will be translating these engineered systems into practical tools, by exploring next-generation BpyAla derivatives, expanding metal cofactor usage, developing cooperative active site design strategies, diversifying protein scaffold and nucleic acids substrate scope.

Expanding the chemical space: BpyAla analogue development

While previous work using BpyAla to cleave nucleic acid substrates has established this UAA as a powerful tool, the next advancement lies in transforming it into a tunable chemical platform. A library of BpyAla analogues with diversified coordination environments, redox properties, and reactivities could enable control over catalysis and broaden its utility across biological contexts.

Firstly, there is significant room for developing BpyAla derivatives with tuned electronic and geometric properties. The electronics of the bipyridyl moiety could be modulated by introducing electron-withdrawing or electron-donating substituents, potentially enhancing metal-binding affinity and improving catalysis. Similarly, altering the substituent positioning on the bipyridyl ring could lead to altered metal-binding activity and influencing redox potential (Figure 2A). Structural tuning could also be inspired by enforcing specific coordination geometries. This could be achieved through introducing rigidified or extended aromatic scaffolds, such as phenanthroline- or terpyridyl-like derivatives respectively (Figure 2B) (Muth and Hill, 2001; Maity et al., 2010). These strategies may create stronger and more defined metal coordination, more closely emulating the preorganized active metalloenzymes (Mcwhinnie and Miller, 1970; Radford et al., 2010).

Secondly, we can imagine the development of BpyAla analogues with expanded side-chain chemistry. For example, Jarvis and colleagues demonstrated the incorporation of the UAA 5-pyridyl-1,2,4-triazine (Trz), which coordinates IrIII through both the triazine and nitrogens, while the triazine also serving as a handle for bioorthogonal click chemistry with bicyclononyne reagents (Figure 2C) (Day et al., 2024). Such dual function designs exemplify that metal coordination can be coupled with targeted chemical modification. In addition to click chemistry moieties being incorporated into BpyAla analogues, one could incorporate photoactivatable or switchable moieties to enable light-activated or temporally controlled nucleic acid cleavage.

Thirdly, future BpyAla analogues could introduce secondary donor groups to emulate the complex coordination environments of natural metalloenzymes. Secondary donors such as imidazole (as in histidine), thiol (as in cysteine) or carboxylate groups (as in aspartate and glutamate) could be integrated into side chain design to stabilize specific metal oxidation states and geometries, ultimately enabling more precise control over catalytic mechanism and efficiency. Taken together, the development of BpyAla analogues designed with tuned electronic and geometric properties, expanded side chain chemistry, and secondary donor groups inspired by natural metalloenzymes, is critical for the expanding the functional scope of genetically encoded metallocentres for nucleic acid cleavage.

Exploring other metal cofactors

An important conceptual shift in the design of BpyAla-engineered systems is that the metal is not a passive cofactor, but an active parameter that defines catalytic mechanism and biological compatibility. Most work centered on BpyAla mediated nucleic acid cleavage has utilized copper and iron, largely due to their well-established redox activity and ability to promote efficient strand scission (Xie et al., 2007; Ahmed et al., 2023). However, insights from natural metallonucleases suggest that a far broader range of metal ions, such as magnesium, zinc, nickel, cobalt and bismuth, can be leveraged to diversify reactivity (Mack and Dervan, 1990; Morrow and Kolasa, 1992; Dupureur, 2008; Yu and Cowan, 2018; Kumar et al., 2021) (Table 1). Different metals offer distinct catalytic mechanisms, coordination geometries, substrate preferences, and biological compatibilities as summarized in Table 1. Employing these metals in BpyAla-mediated nucleic acid cleavage can allow for achieving precise activation, turnover, substrate selectivity, and the ability to rationally tune catalytic activity.

TABLE 1. Mechanistic and functional comparison of metal cofactors possible for BpyAla-mediated nucleic acid cleavage.

Metal ionMechanism of cleavageSide product generationBiological relevance/compatibilityCoordination preferencesAdvantagesLimitations
Cu2+Oxidative and hydrolyticHigh; reactive oxygen species (1O2, O2 •-, OH•)Moderate; tightly regulated in cellsSquare planar/distorted octahedralHigh catalytic activity; efficient strand scissionOff-target oxidative damage; cytotoxicity
Ni2+Oxidative and hydrolyticLow – moderateModerate; present but less abundantOctahedralLess ROS than Cu2+Lower catalytic rates, potential toxicity
Zn2+HydrolyticMinimalHigh; widely used in natural nucleasesTetrahedralBiocompatibleTypically slower catalysis
Fe2+/Fe3+Oxidative (fenton-type) and hydrolyticReactive oxygen species (OH•)High; biologically abundantOctahedralStrong oxidative cleavage capability; biologically relevantPoor selectivity; off-target oxidative damage
Co2+/Co3+Oxidative or hydrolytic depending on valenceLow – moderateModerate; present in vitamin B12 systemsOctahedralTunable reactivityLess well-characterized; potential toxicity
Mg2+HydrolyticMinimalHigh; common enzymatic cofactorOctahedralBiocompatible; supports enzymatic-like activityGenerally weaker catalysis
Ir3+Oxidative or photo-catalyticModerate – high (ROS under irradiation)Low; non-biological, requires delivery strategyOctahedralEnables photo-controlled catalysis; unique reactivityPoor biocompat-ibility; complex coordination chemistry
Bi3+HydrolyticMinimalLow; limited biological roleVariableTunable reactivityLimited characterization
Be2+HydrolyticMinimalVery low; highly toxic, non-biologicalTetrahedralHigh charge density; strong phosphate interactionExtremely toxic; poor translational potential

Mechanistically, metal-mediated nucleic acid cleavage proceeds through hydrolysis or oxidation pathways (Copeland et al., 2002; Yu and Cowan, 2018; Jiang et al., 2026). Hydrolytic cleavage, often associated with strong Lewis acid metals such as Zn2+ or Mg2+, involves activation of the phosphate backbone through formation of metal-phosphate intermediate, facilitating ester bond hydrolysis. In contrast, oxidative cleavage relies on redox-active metals such as Cu2+ or Fe2+/Fe3+, which generate reactive oxygen species (ROS) through electron transfer to molecular oxygen or peroxide, resulting in strand scission via radical intermediates (Sakamoto et al., 2003; Piovezan et al., 2010; Soler et al., 2015; Miao et al., 2018; Yu and Cowan, 2018). Numerous artificial metallonuclease systems have successfully exploited both mechanisms, highlighting that the choice of metal can directly correspond to catalytic efficiency as well as resulting products. Matching the metal identity to the desired mechanism offers a rational design principle, where soft, non-redox metals may be opted for Lewis acid hydrolysis and redox-active metals for harsher oxidative cleavage.

Beyond catalytic mechanisms, metal cofactor identity offers the potential to shape reaction selectivity. For instance, different metals may preferentially promote cleavage of RNA versus DNA or exhibit distinct activities towards single-versus double-stranded substrates. Additionally, metal cofactor choice also introduces a key trade-off between catalytic efficiency and biological compatibility, with highly reactive redox metals often presenting challenges related to off-target damage and toxicity (Table 1). This is an important consideration relevant to therapeutic applications. Lastly, there exists the intriguing possibility of switchable activity by changing metal availability, creating an environmentally responsive BpyAla system. Together, these considerations underscore that metal selection provides an extra layer of refinement in BpyAla-mediated nucleic acid cleavage design, enabling the rational matching of metal identity to desired catalytic mechanism, selectivity and application.

Multi-residue and cooperative design

While the development of BpyAla analogues and the expansion of available metal cofactors provide important avenues for tuning reactivity, an additional opportunity lies in the design of more sophisticated catalytic metallocentres within protein scaffolds. To date, incorporation of a single BpyAla residue has proven sufficient to confer nucleic acid cleavage activity; however, advancing towards higher efficiency, selectivity, and mechanistic control will likely require multi-site and cooperative design strategies. Moving beyond single-residue modifications enables the development of more defined active sites, better reflecting the complexity observed in natural metalloenzymes.

One approach involves the incorporation of multiple UAAs at strategically chosen positions within the scaffold to generate well defined coordination spheres with tailored geometries. These sites could be composed of multiple BpyAla residues or combinations of structurally distinct BpyAla analogues designed to enforce specified coordination geometries at the desired active site.

A complementary strategy is the incorporation of BpyAla with canonical residues to create hybrid active sites. Natural metalloenzymes frequently rely on residues such as histidine, cysteine, aspartate, and glutamate to coordinate metal ions and modulate catalytic activity (Yang, 2011). Incorporating these residues alongside BpyAla offers a route to mimic these native coordination environments while retaining the unique chemical functionality of the UAA. This hybrid design strategy may be particularly effective for constructing mimicked natural metallonuclease active sites with structural stability and catalytic versatility.

Finally, consideration of engineering in secondary coordination spheres provides an additional layer of design control. While the primary coordination sphere consists of the ligand directly covalently bound to the central metal atom, the secondary coordination sphere consists of surrounding residues and solvent molecules interacting electrostatically with the ligand, influencing metal redox reactivity, substrate positioning and stabilization of the complex (Drover, 2022). Engineering these secondary spheres into protein scaffolds could further enhance catalytic efficiency and selectivity, enabling more precise and directed nucleic acid cleavage.

Collectively, multi-residue and cooperative design strategies represent a key step towards transitioning from simple metal-binding proteins to fully engineered metalloenzymes with increasingly sophisticated catalytic systems.

Expanding substrate scope and protein scaffolds

To date, the application of BpyAla-mediated nuclease-like activity has been demonstrated in a limited set of model systems, most notably the RNA-binding protein p19 and the DNA-binding protein CAP. New horizons and applications lie in expanding this technology to a diverse range of protein scaffolds and nucleic acid substrates. In principle, any protein capable of binding DNA or RNA could be engineered to incorporate BpyAla-mediated cleavage functionality. In addition to variation on the protein scaffold, we can also imagine control of substrate scope, with targeted cleavage of a wide array of nucleic acids species.

DNA-binding proteins are particularly attractive candidates due to their inherent sequence specificity and central roles in the regulation of transcription, DNA replication, repair, and chromatin organization (Zheng and Wang, 2011; Hudson and Ortlund, 2014). Classes of DNA-binding proteins that could be engineered to have BpyAla-mediated cleavage activity include transcription factors, DNA replication factors, and DNA repair factors. Many of these proteins contain structurally defined DNA-binding motifs, such as helix-turn-helix, leucine zipper, zinc finger and HMG-box domains, which serve as ideal sites for BpyAla incorporation (Harrison, 1991). Strategic placement of BpyAla within or adjacent to these recognition domains may enable localized, sequence-specific cleavage of target DNA substrates.

Similarly, RNA-binding proteins provide a versatile and largely unexplored platform for engineering sequence specific RNA cleavage. These proteins play critical roles in RNA splicing, stability, transport, and translation, and often recognize highly defined RNA sequences or structural motifs (Hentze et al., 2018). Common RNA-binding domains, including RNA recognition motifs and double-stranded RNA-binding motifs, provide attractive platforms for targeting diverse RNA structures (Corley et al., 2020). By leveraging these existing recognition elements, BpyAla incorporation could enable the direct conversion of binding proteins into catalytic effectors, coupling target specificity with controlled nucleic acid cleavage.

Interestingly, several families of RNA-binding proteins encode sequence specificity directly within the amino acid sequence itself. Pumilio and FBF (PUF) domains are a conserved single stranded RNA-binding domain in eukaryotic organisms whose crescent shaped domains contain tandem repeats along the RNA-binding surface capable of recognizing an eight-nucleotide element of single stranded RNA (Wang et al., 2002; Zhou et al., 2021; Han et al., 2022). These repeats make individual base or whole domain retargeting possible in a predictable manner. Furthermore, PUF fusions have been reported with numerous cleavage domains to achieve sequence specific cleavage and base editing (Wang et al., 2009; Cookea et al., 2011; Mori et al., 2016; Han et al., 2025).

Similarly, pentatricopeptide repeat (PPR) domains are also used widely in engineering efforts for sequence recognition of single-stranded RNA, and can have variable lengths, allowing for more specific targeting than PUF (Barik, 2020). Select RNases utilize PPRs for recognition, known as Protein-only Ribonuclease P (PRORP), found in eukaryotic systems involved with regulation of diverse RNA transcripts (Rackham and Filipovska, 2012; Pinker et al., 2013). Despite successful engineering of PPR domains, these scaffolds present much more complex systems compared to PUFs (Colas des Francs-Small et al., 2018). In the context of expanding protein scaffolds used for BpyAla-mediated cleavage, PUF and PPR domains serve as an interesting platform to be explored.

In parallel, the nucleic acid substrate scope of BpyAla-mediated cleavage systems can be significantly broadened. While initial studies have focused on dsDNA, siRNA, and miRNA, any protein could be redesigned with BpyAla to target a wide range of nucleic acid substrates, including single-stranded RNA, structured RNAs, and other regulatory elements such as lncRNAs, mRNAs and circRNAs. The ability to selectively cleave distinct nucleic acid substrates opens new applications in gene regulation, antiviral strategies, and RNA-targeted therapeutics. Together, expanding both the protein scaffold and substrate repertoire will be essential for establishing BpyAla-based systems as versatile and targeted tools for therapeutic applications.

From chemical tools to therapeutics

The long-term aspiration of these developments is the delivery of BpyAla-engineered metallonucleases into nucleic acid-targeted therapeutics. Current endonuclease-based therapeutics, whether approved or in clinical trials, illustrate the diversity of applications which could ultimately be addressed with UAA-enabled catalytic platforms. With the 2020 Nobel Prize in Chemistry awarded for development of CRISPR-Cas gene editing technologies, the rise of programmable nucleic acid therapeutics has been evident. The first FDA-approved gene-editing therapy, Casgevy, employs the RNA-guided S. pyogenes Cas9 endonuclease and is a sickle-cell disease therapy that disrupts the erythroid-specific enhancer of BCL11A, a repressor of fetal hemoglobin (Singh et al., 2024). By contrast, Cas13 systems recognize a single-stranded RNA substrate and cleave it under the direction of an RNA guide. Clinical trials for the first CRISPR-Cas13 therapeutic, targeting VEGF-A mRNA for treatment of Age-Related Macular Degeneration (AMD) are currently underway (Barthelemy et al., 2025). The ease with which CRISPR systems can be directed to a chosen target by changing the gRNA sequence affords them, and similarly engineered modular systems, a distinct advantage.

A complementary approach has emerged through programmable RNA-binding proteins such as PUF domains, where RNA recognition is encoded directly within the protein rather than through a guide RNA. In these systems, catalytic activity is introduced by genetically fusing the PUF scaffold to nuclease or RNA-editing domains, enabling sequence-specific RNA cleavage and base editing (Wang et al., 2009; Cookea et al., 2011; Mori et al., 2016; Han et al., 2025). This separation of RNA recognition and catalytic function provides a useful conceptual framework for BpyAla-engineered metallonucleases.

Similarly, BpyAla-engineered metallonucleases separate recognition from catalysis. However, rather than introducing a fused catalytic protein domain, catalytic activity is installed chemically through site-specific incorporation of the metal-chelating UAA BpyAla. Recent advances in in vivo RNA base editing using programmable RNA-binding proteins demonstrate that engineered protein-based RNA-targeting systems can be successfully deployed in living organisms, providing encouraging precedents for the development of RNA-targeted catalytic platforms. A distinguishing feature of the BpyAla strategy is its highly configurable design space, in which catalytic function can be independently tuned through the choice of incorporation site, protein scaffold, metal cofactor and UAA chemistry. This modularity offers opportunities to optimize catalytic mechanism, substrate selectivity and biological compatibility beyond what is possible by modifying a catalytic effector domain alone.

Nevertheless, BpyAla-engineered metallonucleases remain an emerging concept that requires substantial validation before therapeutic translation can be achieved. Like other protein-based biologics, BpyAla systems will face challenges related to intracellular delivery, mitigation of off-target cleavage, long-term efficacy and immunogenicity. In addition, the requirement for metal coordination introduces unique considerations, including control of metal availability, catalytic regulation and mitigation of metal-associated toxicity. Many of these challenges may be addressed through continued optimization of UAA design, metal-binding chemistry and protein engineering. However, addressing these challenges in tandem will be critical for the successful deployment of BpyAla-engineered metallonucleases as therapeutic platforms.

Concluding perspective

BpyAla-engineered metallonucleases represent an emerging frontier in biocatalyst design enabled by advances in GCE. Current studies have established that site-specific incorporation of BpyAla can confer Cu(II)- and Fe(II)-dependent cleavage activity toward nucleic acid substrates, including dsDNA, siRNA and miRNA. However, these early proof-of-concept systems represent only a small fraction of the accessible design space. The next steps will involve transforming BpyAla-engineered metallonucleases from isolated engineered proteins into a fully programmable catalytic platform through the integration of multiple design dimensions.

As summarized in Figure 3, this programmability can emerge through combinatorial customization of chemical, functional, architectural and biological design parameters. Chemical diversification through the development of next-generation BpyAla analogues with varying substituents, extended side chains, and modified rigidity may provide increasingly tunable catalytic reactivity. Functional diversification through metal selection will further enable control over cleavage mechanisms, ranging from oxidative pathways mediated by redox-active metals to hydrolytic cleavage promoted by Lewis acid metal cofactors. Architectural diversification through cooperative active site design, including incorporation of multiple BpyAla residues, or canonical metal-chelating residues may enable more sophisticated and enzyme-like catalytic behavior with improved efficiency and selectivity. Finally, biological diversification through protein scaffold selection and expansion of nucleic substrate scope will define target specificity. Together, these design principles establish BpyAla metallonucleases as a modular and highly adaptable framework for engineering catalytic activity into virtually any nucleic acid-binding protein.

Design considerations for BpyAla-engineered programmable metallonucleases. The central panel illustrates the concept of a programmable metallonuclease generated through genetic code expansion. Surrounding panels summarize the major engineering parameters discussed throughout this Perspective: protein scaffold selection, BpyAla analogue design, metal selection, active site design and substrate scope. Protein scaffold selection and Cu/Fe-mediated BpyAla catalysis has been experimentally demonstrated in DNA- and RNA-binding proteins, whereas the development of next-generation BpyAla analogues, alternative metal cofactors, cooperative active site architectures and expanded protein scaffolds and substrate scope represent promising future directions for advancing programmable metallonuclease design.
Figure 3. Design considerations for BpyAla-engineered programmable metallonucleases. The central panel illustrates the concept of a programmable metallonuclease generated through genetic code expansion. Surrounding panels summarize the major engineering parameters discussed throughout this Perspective: protein scaffold selection, BpyAla analogue design, metal selection, active site design and substrate scope. Protein scaffold selection and Cu/Fe-mediated BpyAla catalysis has been experimentally demonstrated in DNA- and RNA-binding proteins, whereas the development of next-generation BpyAla analogues, alternative metal cofactors, cooperative active site architectures and expanded protein scaffolds and substrate scope represent promising future directions for advancing programmable metallonuclease design.

Despite the promise of targeted endonuclease-like therapeutics outlined in this perspective, several challenges remain before larger implementation of these approaches using GCE can be realized. As outlined, managing the cytotoxicity of metal cofactors, minimizing off-target nucleic acid damage, and improved stability of these engineered biologics are limitations currently faced. However, this avenue offers the potential advantages of catalytic turnover and broader target scope. As the discipline matures, continued engineering of configurable protein scaffolds for catalytic activity, particularly through GCE and incorporation of UAAs, may enable increasingly selective, tunable and multifunctional protein scaffolds to be developed for varied nucleic acid targeting approaches. In this context, engineered catalytic systems may present a new paradigm in targeted medicine.

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Footnotes

Edited by: Urszula Katarzyna Komarnicka, University of Wrocław, Poland

Reviewed by: Wenjian Han, Shanghai Jiao Tong University, China

Statements

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding

The author(s) declared that financial support was not received for this work and/or its publication.

Data availability

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

Author contributions

EL: Investigation, Visualization, Validation, Conceptualization, Formal Analysis, Writing – review and editing, Writing – original draft. MO: Visualization, Formal Analysis, Writing – review and editing, Writing – original draft, Investigation. JP: Resources, Writing – review and editing, Conceptualization, Funding acquisition, Supervision, Writing – original draft, Visualization.

References

  1. Aboelnga M. M., Wetmore S. D. (2019). Unveiling a single-metal-mediated phosphodiester bond cleavage mechanism for nucleic acids: a multiscale computational investigation of a human DNA repair enzyme. J. Am. Chem. Soc. 141, 8646 – 8656. doi:10.1021/jacs.9b03986
  2. Ahmed N., Ahmed N., Bilodeau D. A., Pezacki J. P. (2023). An unnatural enzyme with endonuclease activity towards small non-coding RNAs. Nat. Commun. 14, 3777. doi:10.1038/s41467-023-39105-0
  3. Barik S. (2020). The nature and arrangement of pentatricopeptide domains and the linker sequences between them. Bioinform. Biol. Insights 14, 1177932220906434. doi:10.1177/1177932220906434
  4. Barthelemy N., Sridhar J., Sengillo J. D. (2025). Gene therapy for wet age-related macular degeneration. Bioengineering 12, 1072. doi:10.3390/BIOENGINEERING12101072
  5. Birch-Price Z., Hardy F. J., Lister T. M., Kohn A. R., Green A. P. (2024). Noncanonical amino acids in biocatalysis. Chem. Rev. 124, 8740 – 8786. doi:10.1021/acs.chemrev.4c00120
  6. Brouwer B., Della-Felice F., Illies J. H., Iglesias-Moncayo E., Roelfes G., Drienovská I. (2024). Noncanonical amino acids: bringing new-to-nature functionalities to biocatalysis. Chem. Rev. 124, 10877 – 10923. doi:10.1021/acs.chemrev.4c00136
  7. Chemla Y., Kaufman F., Amiram M., Alfonta L. (2024). Expanding the genetic code of bioelectrocatalysis and biomaterials. Chem. Rev. 124, 11187 – 11241. doi:10.1021/acs.chemrev.4c00077
  8. Chin J., Banaszczyk M. (1989). Highly efficient hydrolytic cleavage of adenosine monophosphate resulting in binuclear Co(III) complex with a novel doubly bidentate Phosphato-Bridge. J. Am. Chem. Soc. 111, 4103 – 4105. doi:10.1021/ja00193a053
  9. Colas des Francs-Small C., Vincis Pereira Sanglard L., Small I. (2018). Targeted cleavage of nad6 mRNA induced by a modified pentatricopeptide repeat protein in plant mitochondria. Commun. Biol. 1 (1), 166. doi:10.1038/s42003-018-0166-8
  10. Cookea A., Priggeb A., Oppermana L., Wickensa M. (2011). Targeted translational regulation using the PUF protein family scaffold. Proc. Natl. Acad. Sci. U. S. A. 108, 15870 – 15875. doi:10.1073/PNAS.1105151108
  11. Copeland K. D., Fitzsimons M. P., Houser R. P., Barton J. K. (2002). DNA hydrolysis and oxidative cleavage by metal-binding peptides tethered to rhodium intercalators. Biochemistry 41, 343 – 356. doi:10.1021/bi011793k
  12. Corley M., Burns M. C., Yeo G. W. (2020). How RNA-binding proteins interact with RNA: molecules and mechanisms. Mol. Cell 78, 9 – 29. doi:10.1016/j.molcel.2020.03.011
  13. Costello A., Peterson A. A., Chen P. H., Bagirzadeh R., Lanster D. L., Badran A. H. (2024). Genetic code expansion history and modern innovations. Chem. Rev. 124, 11962 – 12005. doi:10.1021/ACS.CHEMREV.4C00275
  14. Day G. J., Zaytsev A. V., Brewster R. C., Kozhevnikov V. N., Jarvis A. G. (2024). A dual-purpose non-canonical amino acid for the expanded genetic code: combining metal-binding and click chemistry. Angew. Chem. Int. Ed. 63, e202413073. doi:10.1002/anie.202413073
  15. Drienovská I., Rioz-Martínez A., Draksharapu A., Roelfes G. (2015). Novel artificial metalloenzymes by in vivo incorporation of metal-binding unnatural amino acids. Chem. Sci. 6, 770 – 776. doi:10.1039/c4sc01525h
  16. Drover M. W. (2022). A guide to secondary coordination sphere editing. Chem. Soc. Rev. 51, 1861 – 1880. doi:10.1039/d2cs00022a
  17. Dupureur C. M. (2008). Roles of metal ions in nucleases. Curr. Opin. Chem. Biol. 12, 250 – 255. doi:10.1016/j.cbpa.2008.01.012
  18. Han W., Huang W., Wei T., Ye Y., Mao M., Wang Z. (2022). Programmable RNA base editing with a single gRNA-free enzyme. Nucleic Acids Res. 50, 9580 – 9595. doi:10.1093/NAR/GKAC713
  19. Han W., Yuan B., Fan X., Li W., Yuan Y., Zhang Y. (2025). Effective in vivo RNA base editing via engineered cytidine deaminase APOBECs fused with PUF proteins. Nat. Commun. 16, 9727. doi:10.1038/S41467-025-64748-6
  20. Harrison S. C. (1991). A structural taxonomy of DNA-Binding domains the structures of several classes of DNA-Binding domains reveal a variety of designs for recognizing a specific site on DNA. Nature 353, 6346. doi:10.1038/353715a0
  21. Hentze M. W., Castello A., Schwarzl T., Preiss T. (2018). A brave new world of RNA-Binding proteins. Nat. Rev. Mol. Cell Biol. 19, 327 – 341. doi:10.1038/nrm.2017.130
  22. Hudson W. H., Ortlund E. A. (2014). The structure, function and evolution of proteins that bind DNA and RNA. Nat. Rev. Mol. Cell Biol. 15, 749 – 760. doi:10.1038/nrm3884
  23. Hyun S. L., Schultz P. G. (2008). Biosynthesis of a site-specific DNA cleaving protein. J. Am. Chem. Soc. 130, 13194 – 13195. doi:10.1021/ja804653f
  24. Jarvis A. G. (2020). Designer metalloenzymes for synthetic biology: enzyme hybrids for catalysis. Curr. Opin. Chem. Biol. 58, 63 – 71. doi:10.1016/j.cbpa.2020.06.004
  25. Jiang F., Dong Y., Yu W., Tian H., Yang L., Jia Z. (2026). Metal surface-triggered DNAzyme catalysis for efficient DNA cleavage. Commun. Chem. 9, 91. doi:10.1038/s42004-026-01893-z
  26. Klemencic E., Brewster R. C., Ali H. S., Richardson J. M., Jarvis A. G. (2024). Using BpyAla to generate copper artificial metalloenzymes: a catalytic and structural study. Catal. Sci. Technol. 14, 1622 – 1632. doi:10.1039/d3cy01648j
  27. Kumar M., Lal N., Luthra P. M., Masram D. T. (2021). Exploring the binding and cleavage activities of nickel II complexes towards DNA and proteins. New J. Chem. 45, 6693 – 6708. doi:10.1039/d0nj06210c
  28. Lee J., Moon J., Choi I., Lee Y., Song W. J. (2025). A bipyridyl noncanonical amino acid for expanding the scope of metalloprotein design. Methods Enzym. 720, 141 – 187. doi:10.1016/bs.mie.2025.06.030
  29. Mack D. P., Dervan P. B. (1990). Nickel-mediated sequence-specific oxidative cleavage of DNA by a designed metalloprotein. J. Am. Chem. Soc. 112, 4604 – 4606. doi:10.1021/ja00167a102
  30. Maity B., Roy M., Banik B., Majumdar R., Dighe R. R., Chakravarty A. R. (2010). Ferrocene-promoted photoactivated DNA cleavage and anticancer activity of terpyridyl copper(II) phenanthroline complexes. Organometallics 29, 3632 – 3641. doi:10.1021/OM100524X/SUPPL_FILE/OM100524X_SI_002.CIF
  31. McWhinnie W. R., Miller J. D. (1970). The chemistry of complexes containing 2,2′-Bipyridyl, 1, 10-Phenanthroline, or 2,2′,6′,2′′-Terpyridyl as ligands. Adv. Inorg. Chem. Radiochem. 12, 135 – 215. doi:10.1016/S0065-2792(08)60049-7
  32. Miao T., Deng Q., Gao H., Fu X., Li S. (2018). Theoretical studies on DNA-cleavage mechanism of Copper(II) complexes: probing generation of reactive oxygen species. J. Chem. Inf. Model. 58, 859 – 866. doi:10.1021/acs.jcim.8b00055
  33. Mori T., Nakamura K., Masaoka K., Fujita Y., Morisada R., Mori K. (2016). Cleavage of influenza RNA by using a human PUF-Based artificial RNA-Binding protein–staphylococcal nuclease hybrid. Biochem. Biophys. Res. Commun. 479, 736 – 740. doi:10.1016/j.bbrc.2016.09.142
  34. Morrow J. R., Kolasa K. A. (1992). Cleavage of DNA by nickel complexes. Inorganica Chim. Acta 195, 245 – 248. doi:10.1016/S0020-1693(00)85319-0
  35. Muth G. W., Hill W. E. (2001). Phenanthroline-Cu(II) cleavage as a probe of rRNA structure. Methods 23, 218 – 232. doi:10.1006/METH.2000.1133
  36. Nikkel D. J., Kaur R., Wetmore S. D. (2025). How can one metal power nucleic acid phosphodiester bond cleavage by a nuclease? Multiscale computational studies highlight a diverse mechanistic landscape. J. Phys. Chem. B 129, 3 – 18. doi:10.1021/acs.jpcb.4c05875
  37. Pecina A., Rosa-Gastaldo D., Riccardi L., Franco-Ulloa S., Milan E., Scrimin P. (2021). On the metal-aided catalytic mechanism for phosphodiester bond cleavage performed by nanozymes. ACS Catal. 11, 8736 – 8748. doi:10.1021/acscatal.1c01215
  38. Pinker F., Bonnard G., Gobert A., Gutmann B., Hammani K., Sauter C. (2013). PPR proteins shed a new light on RNase P biology. RNA Biol. 10, 1457 – 1468. doi:10.4161/RNA.25273
  39. Piovezan C., Jovito R., Bortoluzzi A. J., Terenzi H., Fischer F. L., Severino P. C. (2010). Heterodinuclear FeIIIZnII-bioinspired complex supported on 3-aminopropyl silica. Efficient hydrolysis of phosphate diester bonds. Inorg. Chem. 49, 2580 – 2582. doi:10.1021/ic902489j
  40. Rackham O., Filipovska A. (2012). The role of Mammalian PPR domain proteins in the regulation of mitochondrial gene expression. Biochimica Biophysica Acta Gene Regul. Mech. 1819, 1008 – 1016. doi:10.1016/J.BBAGRM.2011.10.007
  41. Radford R. J., Nguyen P. C., Tezcan F. A. (2010). Modular and versatile hybrid coordination motifs on α-helical protein surfaces. Inorg. Chem. 49, 7106 – 7115. doi:10.1021/ic100926g
  42. Sakamoto S., Tamura T., Furukawa T., Komatsu Y., Ohtsuka E., Kitamura M. (2003). Highly efficient catalytic RNA cleavage by the cooperative action of two Cu(II) complexes embodied within an antisense oligonucleotide. Nucleic Acids Res. 31, 1416 – 1425. doi:10.1093/nar/gkg238
  43. Schultz P. G., Dervan P. B. (1983). Sequence-specific double-strand cleavage of DNA by penta-N-methylpyrrolecarboxamide-EDTA·Fe(II). Proc. Natl. Acad. Sci. U. S. A. 80, 6834 – 6837. doi:10.1073/pnas.80.22.6834
  44. Schultz P. G., Dervan P. B. (1984). Distamycin and Penta-N-Methylpyrrolecarboxamide binding sites on native DNA A comparison of Methidiumproyl-EDTA-Fe(II) footprinting and DNA affinity cleaving. J. Biomol. Struct. Dyn. 1, 1133 – 1147. doi:10.1080/07391102.1984.10507508
  45. Singh A., Irfan H., Fatima E., Nazir Z., Verma A., Akilimali A. (2024). Revolutionary breakthrough: FDA approves CASGEVY, the first CRISPR/Cas9 gene therapy for sickle cell disease. Ann. Med. Surg. 86, 4555 – 4559. doi:10.1097/MS9.0000000000002146
  46. Soler M., Figueras E., Serrano-Plana J., González-Bártulos M., Massaguer A., Company A. (2015). Design, preparation, and characterization of Zn and Cu metallopeptides based on tetradentate aminopyridine ligands showing enhanced DNA cleavage activity. Inorg. Chem. 54, 10542 – 10558. doi:10.1021/acs.inorgchem.5b01680
  47. Spina S. C., Bailey J. S., Kimmel B. R. (2026). Bind, catalyze, and quantify: a modern protein and enzyme engineering toolbox of genetically encoded non-canonical amino acids. Protein Eng. Des. Sel. 39, gzag007. doi:10.1093/protein/gzag007
  48. Wang X., McLachlan J., Zamore P. D., Hall T. M. T. (2002). Modular recognition of RNA by a human Pumilio-homology domain. Cell 110, 501 – 512. doi:10.1016/S0092-8674(02)00873-5
  49. Wang Y., Cheong C. G., Tanaka Hall T. M., Wang Z. (2009). Engineering splicing factors with designed specificities. Nat. Methods 6, 825 – 830. doi:10.1038/NMETH.1379
  50. Xie J., Liu W., Schultz P. G. (2007). A genetically encoded bidentate, metal-binding amino acid. Angew. Chem. Int. Ed. 46, 9239 – 9242. doi:10.1002/anie.200703397
  51. Yang W. (2011). Nucleases: diversity of structure, function and mechanism. Q. Rev. Biophys. 44, 1 – 93. doi:10.1017/S0033583510000181
  52. Yi H. B., Lee S., Seo K., Kim H., Kim M., Lee H. S. (2024). Cellular and biophysical applications of genetic code expansion. Chem. Rev. 124, 7465 – 7530. doi:10.1021/acs.chemrev.4c00112
  53. Young M. J., Chin J. (1995). Dinuclear copper(II) complex that hydrolyzes RNA. J. Am. Chem. Soc. 117, 10577 – 10578. doi:10.1021/ja00147a002
  54. Yu Z., Cowan J. A. (2018). Metal complexes promoting catalytic cleavage of nucleic acids — biochemical tools and therapeutics. Curr. Opin. Chem. Biol. 43, 37 – 42. doi:10.1016/j.cbpa.2017.10.029
  55. Zheng Z., Wang Y. (2011). DNA binding proteins: outline of functional classification. Biomol. Concepts 2, 293 – 303. doi:10.1515/bmc.2011.023
  56. Zhou W., Melamed D., Banyai G., Meyer C., Tuschl T., Wickens M. (2021). Expanding the binding specificity for RNA recognition by a PUF domain. Nat. Commun. 12 (12), 5107. doi:10.1038/s41467-021-25433-6

Citation

Lundrigan, E., et al. (2026). Towards programmable metallonucleases via genetic code expansion. Frontiers in Chemical Biology, Volume 5. https://doi.org/
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5 - 2026

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Copyright © 2026 Lundrigan, O’Neill and Pezacki.

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*Correspondence: John Paul Pezacki

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