NAD⁺ analogues for decoding the biological chemistry of PARP-mediated ADP-ribosylation

Abstract

ADP-ribosylation is a NAD⁺-dependent post-translational modification that governs DNA repair, transcription, antiviral defense, and stress adaptation. Its transient and heterogeneous nature, however, has limited mechanistic understanding. Chemically modified NAD⁺ analogues have become indispensable chemical biology tools to overcome these barriers. By functionalizing the NAD⁺ scaffold at positions tolerated by PARP enzymes, these probes enable capture and enrichment of ADP-ribosylated substrates, writer-specific labeling, PAR-chain architecture control, reader identification, and real-time imaging. This Review summarizes recent progress in NAD⁺ analogue development, organized by probe function and biological application. We highlight how these chemical tools have transformed ADP-ribosylation research from descriptive detection toward mechanistic interrogation and discuss future directions for the field.

Functionalized NAD⁺ analogues enable PARP substrate identification, spatiotemporal imaging of ADP-ribosylation, and profiling of PAR-interacting proteins.

1. Introduction

ADP-ribosylation is a chemically intricate post-translational modification (PTM) that regulates a broad spectrum of cellular processes, including DNA damage repair,¹ cell cycle progression,² antiviral defense,^3,4 and stress adaptation.⁵ Dysregulation of ADP-ribosylation has been increasingly implicated in human diseases. Hyperactivation of poly(ADP–ribose) polymerase (PARP) enzymes in the context of DNA damage response contributes to cancer cell survival and therapy resistance, whereas impaired ADP-ribosylation dynamics are associated with neurodegenerative disorders, inflammatory diseases, and viral infections.^6,7 These pathological connections have established ADP-ribosylation as a key signaling and functional node in disease pathogenesis. Elucidating the complex regulatory networks that govern ADP-ribosylation, including the coordinated actions of writers, erasers, and readers, is therefore essential not only for understanding fundamental cell biology but also for identifying therapeutic vulnerabilities and developing novel treatment strategies, as exemplified by the clinical success of PARP inhibitors in treating BRCA-mutant cancers.^8–11 Recent reviews have further consolidated the molecular logic, enzymatic diversity, and therapeutic implications of mono- and poly-ADP-ribosylation.^12,13

Unlike classical PTMs such as phosphorylation or acetylation, ADP-ribosylation entails the covalent transfer of one or more ADP–ribose units from nicotinamide adenine dinucleotide (NAD⁺) onto target biomolecules, frequently resulting in linear or branched poly(ADP–ribose) (PAR) chains. This inherent chemical complexity, reflected in the distinction between mono- and poly-ADP-ribosylation, the diversity of acceptor residues, and the architectural heterogeneity of PAR chains, confers remarkable regulatory versatility yet poses substantial challenges for mechanistic dissection.¹⁴ Despite decades of investigation, fundamental questions regarding substrate selectivity, PAR-dependent interacting proteins and processes, the structural basis of polymer architecture, and the PAR code that links these features to functional outcomes remain unresolved.¹⁵ A principal obstacle is the lack of effective methodologies and technologies capable of resolving the dynamic and heterogeneous nature and biofunction of ADP-ribosylation.^16,17 Endogenous ADP-ribosylation is transient, with PAR chains undergoing rapid turnover within seconds to minutes following activation, making it difficult to interrogate native modification states.¹⁸ The modification is structurally heterogeneous, encompassing both mono- and poly-ADP-ribosylation across diverse acceptor residues, and different enzyme isoforms often generate chemically indistinguishable products.¹⁹ Conventional genetic perturbations such as PARP knockout or chemical inhibition frequently fail to delineate isoform-specific functions due to functional redundancy and pleiotropic effects.²⁰ Antibody-based approaches, while widely used, are hampered by limited specificity and an inability to discriminate between mono- and poly-ADP-ribosylation or to resolve acceptor residues. These technical limitations underscore the pressing need for novel chemical biology tools and mass spectrometry-based methodologies to enable systematic dissection of the ADP-ribosylome.

The covalent transfer of the ADP–ribose from the NAD⁺ to protein substrates constitutes the central biochemical event of ADP-ribosylation. Based on this reaction principle, functionalization of the NAD⁺ scaffold has become a critical approach for in-depth investigation of this post-translational modification. By chemically modifying NAD⁺ to endow it with diverse functions, researchers have systematically addressed many challenges in the field. Strategic chemical modification of NAD⁺ at sterically and electronically permissive positions has enabled the generation of functional NAD⁺ analogues that retain catalytic competence while introducing orthogonal reactivity, selectivity, or reporting capabilities.^21–27 These engineered NAD⁺ analogues serve as functional probes, facilitating the discovery of novel substrates, precise modulation of enzyme activity, and the proteome-wide identification of ADP-ribosylation reader proteins, and notably the spatiotemporal visualization of intracellular ADP-ribosylation.²⁸ This chemical toolkit provides unprecedented capabilities for systematically dissecting the ADP-ribosylation regulatory network.

In this Review, we frame NAD⁺ analogues as tools for biological chemistry rather than as a catalogue of modified nucleotides. We first define the PARP-centred writer–product–reader–eraser cycle that gives ADP-ribosylation its macromolecular meaning.²⁹ We then connect NAD⁺ recognition to analogue design and organize the major probe classes according to the mechanistic questions they answer: where ADP-ribosylation occurs, which writer produces it, what PAR architecture is formed, which readers engage the signal and how these events can be followed in cells. This organization is intended for a broad biochemical readership and emphasizes representative studies that combine enzymology, structural biology, chemical synthesis, proteomics, imaging and functional validation. This question-led framework and the major ways in which NAD⁺ analogues link probe chemistry to ADP-ribosylation biology are summarized (Fig. 1).

Fig. 1. Conceptual overview of chemically modified NAD⁺ analogues as tools for ADP-ribosylation research. NAD⁺-derived probes enable writer assignment, substrate mapping, PAR-structure decoding, reader identification, spatiotemporal imaging, and functional interrogation of ADP-ribosylation signaling.

2. PARP-catalyzed ADP-ribosylation: writers, products, readers, and erasers

PARP-catalyzed ADP-ribosylation is best understood as a dynamic writer–product–reader–eraser cycle driven by NAD⁺ chemistry. PARP enzymes use NAD⁺ as the immediate ADP–ribose donor and transfer ADP–ribose to proteins or other biomolecules, thereby generating mono-ADP-ribosylated products or, in the case of polymer-forming enzymes, poly(ADP–ribose) chains (Fig. 2a).³⁰ These MAR and PAR products are interpreted by ADP–ribose-binding reader proteins and are rapidly remodeled or removed by eraser enzymes (Fig. 2b). This cycle explains why ADP-ribosylation is difficult to analyze, a conserved NAD⁺-dependent chemical reaction is diversified through distinct writers, substrates, acceptor residues, polymer architectures, reader interactions, and signal lifetimes, while MARylation, PARylation, and nicotinamide-dependent NAD⁺ salvage remain metabolically coupled (Fig. 2c).³¹

Fig. 2. Mechanistic overview of PARP-mediated ADP-ribosylation and NAD⁺ recycling. (a) DNA strand breaks activate PARP1 through DNA damage recognition, conformational rearrangement, NAD⁺ binding, nicotinamide release, and HPF1-dependent ADP–ribose transfer, predominantly onto serine residues of substrate proteins. (b) PARylation of PARP1 and chromatin-associated proteins recruits PAR-binding factors such as ALC1 and XRCC1, thereby coordinating DNA damage response signaling, chromatin remodeling, repair complex assembly, and cell-cycle control. PAR chains are removed mainly by PARG and ARH3, generating ADP–ribose monomers or short oligomers. (c) PARP enzymes use NAD⁺ as an ADP–ribose donor to catalyze MARylation or PARylation. PARP3 and PARP7 mainly mediate MARylation, whereas PARP1, PARP2, and tankyrases generate PAR chains to different extents. Nicotinamide released during catalysis is recycled through the NAMPT- and NMNAT-dependent salvage pathway to regenerate NAD⁺.

The PARP superfamily illustrates this diversity particularly well. Human cells encode 17 PARP family members, also referred to as ARTD enzymes, which share a related catalytic fold but differ markedly in domain organization, localization, activation mechanism, and substrate-recruitment mode.^32,33 Most PARP family members primarily catalyze mono-ADP-ribosylation, whereas PARP1, PARP2, and tankyrases (PARP5a/5b) can support PAR chain formation. PARP1 and PARP2 are central to DNA damage responses, where DNA binding and allosteric activation drive rapid NAD⁺ consumption and PAR synthesis.^34,35 Tankyrases use ankyrin-repeat clusters to recruit defined substrates and regulate processes such as Wnt signaling, telomere maintenance, and mitotic progression.^36–39 As the final ADP–ribose product usually does not preserve a clear record of the writer that generated it, writer assignment remains a central analytical challenge.

This challenge arises because the final ADP–ribose product carries little information about which enzyme generated it. To overcome this limitation, researchers have turned to NAD⁺ analogues—structurally modified versions of the natural co-substrate.⁴⁰ If a modified NAD⁺ molecule can still be recognized and used by a PARP enzyme, it can serve as a traceable probe rather than just a metabolic fuel. Depending on the modification introduced, these analogues can carry clickable handles, affinity tags, fluorophores, or other functional groups, enabling detection or enrichment of the labeled substrates. More importantly, this design opens the door to a chemical-genetic approach: by engineering a specific PARP variant to preferentially use a non-natural NAD⁺ analogue, researchers can ensure that only the target enzyme incorporates the label. As a result, the labelling event directly reports the identity of the active enzyme, bypassing the need for indirect inferences from inhibitor studies or genetic manipulations.

2.1. Catalytic conversion of NAD⁺ into MAR and PAR signals

At the molecular level, PARP-catalyzed ADP-ribosylation begins with NAD⁺ binding within the catalytic cleft and positioning of an acceptor site for attack on the C1′ position of the nicotinamide ribose. Cleavage of the nicotinamide–ribose bond releases nicotinamide and proceeds through an oxocarbenium-like transition state, after which the ADP–ribose moiety is transferred to the acceptor.⁴¹ This first transfer produces a mono-ADP-ribosylated product, which may either act as the final signaling output or serve as a primer for further polymer elongation. However, the biological outcome of this initial event is not determined solely by which protein is modified. Additional layers of complexity, including the acceptor residue type, the linkage chemistry, the stoichiometry of modification, and the transition from mono-ADP-ribosylation (MAR) to poly-ADP-ribosylation (PAR), all critically influence downstream signaling.⁴² For instance, serine has emerged as the major acceptor residue in HPF1-dependent PARP1/2 signaling, whereas glutamate, aspartate, lysine, tyrosine, cysteine, arginine, and other residues have been reported in distinct contexts.^43,44 Thus, a central question is not simply which proteins are ADP-ribosylated, but rather which writer modifies which residue via which linkage, and whether the modification remains as MAR or develops into PAR.

For polymer-forming PARPs, successive ADP–ribose units are added through ribose–ribose glycosidic linkages, generating PAR chains that vary in length, branching, linkage composition, and local density.^45,46 These structural features are thought to encode biological information, often discussed as the PAR code, because they influence the recruitment and activity of downstream effector proteins. However, endogenous PAR is heterogeneous and rapidly remodeled, making it difficult to determine how a specific chain architecture is generated and how it contributes to function. NAD⁺ analogues provide direct ways to interrogate these catalytic steps. Clickable or affinity-tagged analogues can record newly installed ADP–ribose units and enable enrichment of modified substrates.⁴⁷ Isotopically labeled analogues can distinguish analogue-derived ADP–ribose from endogenous pools. Ribose-modified or non-extendable analogues can limit polymer elongation, allowing initiation to be separated from chain extension. Thus, NAD⁺ analogues do not merely label ADP-ribosylation products; they can also constrain or reshape the products generated by PARP catalysis.

2.2. Reader proteins and propagation of ADP–ribose signals

ADP-ribosylation becomes biologically meaningful when MAR and PAR structures are interpreted by reader proteins. ADP–ribose-binding modules, including macrodomains, WWE domains, PBZ motifs, and other PAR-binding elements, recognize features of ADP-ribosylated substrates or PAR chains and convert these chemical marks into cellular responses.^16,48–51 During DNA damage signaling, for example, PAR formation promotes recruitment of repair factors, chromatin remodelers, and ubiquitin-associated proteins, coupling local NAD⁺ consumption to genome maintenance. Reader engagement is not governed by a uniform ADP–ribose signal. Some proteins preferentially bind MAR, whereas others favor short PAR chains, long polymers, branched structures, or ADP–ribose presented within a particular protein or chromatin environment.⁵² Consequently, it is often unclear whether a candidate reader binds ADP–ribose directly, recognizes a specific PAR architecture, or is recruited indirectly through proximity, electrostatics, or phase-separated assemblies. This uncertainty limits translation of the PAR code from a conceptual model into defined structure–function relationships.

Different NAD⁺ analogue designs address this problem from complementary angles. Chain-terminating analogues can generate restricted PAR structures to test chain-length dependence.⁵³ Clickable analogues introduce enrichment handles into ADP–ribose products and support systematic identification of MAR- or PAR-associated proteins. Photoreactive analogues can capture weak or transient reader interactions by covalent crosslinking. Together, these strategies allow reader proteins to be studied not only as ADP–ribose binders but also as sensors of particular ADP–ribose architectures.

2.3. Eraser enzymes and the lifetime of ADP–ribose signals

ADP-ribosylation is highly reversible. PAR signals are rapidly degraded by poly(ADP–ribose) glycohydrolase (PARG), which hydrolyzes ribose–ribose linkages within PAR, whereas ARH family members, TARG1, MacroD1, and MacroD2 remove or remodel specific ADP–ribose linkages.^54–58 Eraser activity therefore determines which ADP-ribosylated species remain available for capture at a given sampling time.

Probe selection and sampling time should therefore account for the formation and removal kinetics of the modification under study. Rapidly incorporated clickable or affinity-tagged analogues capture nascent ADP-ribosylation before complete turnover.⁵⁹ Chain-terminating analogues stabilize defined intermediates by limiting elongation or remodeling. Metabolic labeling can introduce tagged NAD⁺ derivatives or precursors into cellular nucleotide pools for time-resolved detection. Photoreactive analogues can trap transient enzyme-substrate or reader–PAR interactions before they are lost. Thus, NAD⁺ analogues provide access not only to the presence of ADP-ribosylation but also to its timing, stability, and eraser-sensitive dynamics.

3. NAD⁺ recognition as the chemical basis for probe design

The mechanistic complexity described above makes NAD⁺ a logical starting point for probe engineering. As the immediate ADP–ribose donor used by PARP enzymes, NAD⁺ provides a scaffold for analogue design.⁶⁰ Effective NAD⁺ analogues must balance catalytic compatibility with functional modifications that enable detection, enrichment, crosslinking, imaging, or control of PAR architecture. These structural constraints explain why successful probe development depends on matching the modification site of NAD⁺ to the steric and electronic tolerance of the PARP catalytic pocket (Fig. 3).

Fig. 3. Structural basis of NAD⁺ recognition by the PARP1 catalytic domain. NAD⁺ binding within the catalytic cleft is shaped by interactions with the nicotinamide, ribose, pyrophosphate, and adenine moieties. These structural constraints define which positions of NAD⁺ can tolerate chemical modification and provide the molecular basis for NAD⁺ analogue probe design.

The nicotinamide ring is directly involved in the cleavage step of ADP-ribosylation and is released as nicotinamide during catalysis. For this reason, bulky modification at this region often strongly affects substrate recognition and enzymatic turnover.⁶¹ Introduction of sterically demanding substituents at the nicotinamide C5 position, for example, can limit utilization by wild-type PARPs. This sensitivity can nevertheless be turned into a selectivity principle. In bump-and-hole strategies, engineered mutations within a PARP catalytic pocket generate a complementary cavity that accommodates sterically encumbered NAD⁺ analogues.⁶² Such orthogonal enzyme–analogue pairs allow isoform-selective labeling without broadly perturbing endogenous ADP-ribosylation.

The two ribose units provide a second layer of design logic. The nicotinamide-linked ribose participates in the reactive ADP–ribose unit, whereas the adenosine-linked ribose contributes to NAD⁺ orientation and PAR chain extension.⁶³ Some modifications on the nicotinamide-linked ribose are well tolerated by PARP1 and PARP2 and can yield analogues with near-native catalytic efficiency. By contrast, changes at the adenosine-linked ribose, especially at the 2′ or 3′ hydroxyl positions, often affect ribose–ribose bond formation during polymerization. Deoxy or substituted analogues at these positions can therefore act as chain terminators, supporting initial ADP–ribose transfer while limiting elongation. This property makes ribose-modified analogues useful for separating initiation, short-chain PAR formation, and long-polymer-dependent signaling.

The adenine base is the most versatile site for functional diversification. The N6, C2, and C8 positions can often tolerate azides, alkynes, fluorophores, affinity tags, or photoreactive groups as it occupies a relatively solvent-exposed region of the PARP catalytic cleft.⁶⁴ N6 substitution is commonly used to install small clickable handles that allow downstream fluorescence labeling or biotin-based enrichment after ADP–ribose transfer. C2 substitution can accommodate bulkier groups, including fluorophores and diazirine crosslinkers, making this position especially useful for visualization and reader-capture probes. The adenine ring therefore functions as a modular platform for converting NAD⁺ from a native co-substrate into a multifunctional chemical probe.

The pyrophosphate bridge links the nicotinamide and adenosine ribose units and stabilizes NAD⁺ binding through ionic and hydrogen-bonding interactions. Its high charge density and strict geometric requirements leave relatively little room for major modification.⁶⁵ Even so, pyrophosphate-associated labeling has been valuable for mechanistic studies. Early 14C- or 32P-labeled NAD⁺ analogues, often combined with 2′ or 3′ deoxy modifications, enabled radioactive detection and helped establish protein mono-ADP-ribosylation and PAR synthesis in biochemical systems.⁶⁶ Although these radiolabeled probes are not well suited for modern live-cell imaging or high-throughput proteomics, they provided an important foundation for later probe development.

These structural principles also explain a major practical limitation of NAD⁺ analogues. The pyrophosphate group makes NAD⁺ highly polar and multiply charged, which severely restricts membrane permeability and confines many applications to purified systems, lysates, or permeabilized cells.⁶⁷ Probe design must therefore balance recognition by the target enzyme with access to the relevant intracellular compartment.

4. Major classes of NAD⁺ analogues

The following sections classify probes by the biochemical question they answer. Capture probes identify modified macromolecules; chemical-genetic probes assign writer enzymes; chain terminators test PAR architecture; and visualization probes report spatial and temporal regulation.⁶⁸ This question-led organization helps connect chemical design to interpretable biological mechanism. The structural plasticity of NAD⁺ has produced a chemically diverse set of probes, but their value is best understood in relation to the experimental problems they address. In ADP-ribosylation research, NAD⁺ analogues have been used to capture newly modified substrates, assign modifications to defined ART/PARP writers, restrict or remodel PAR chain architecture, and visualize dynamic ADP-ribosylation events in cells.⁶⁹ (Fig. 5 and Table 1).

Fig. 5. Chemical structures of representative NAD⁺ analogues discussed in this review. (A) Probes for capturing and mapping ADP-ribosylation events. (B) Chemical-genetic NAD⁺ analogues for isoform-selective PARP catalysis. (C) Chain-controlling NAD⁺ analogues for decoding PAR architecture. (D) Visualization-oriented NAD⁺ probes for spatiotemporal analysis. (E) Stable cofactor mimics and modified-ribose analogues. Compound numbers (1)–(18) correspond to those used in the main text and Table 1.

Table 1. Chemical identities, experimentally supported performance and applications of NAD⁺ analogues 1–18. Numbering corresponds to Fig. 5 and Sections 4.1–4.5. For amino-substituted analogues, activity depends on the specific sugar stereochemistry discussed in Section 4.5.

No.	Identity/design	Performance and application	Ref.
1	32P-Labeled adenosyl 2′-deoxy-NAD+	Combines an isotope detection handle with adenosyl 2′-deoxygenation; biochemical rationale from radiolabeling and deoxy-NAD+ studies.	65, 66 and 70
2	32P-Labeled adenosyl 3′-deoxy-NAD+	Adenosyl 3′-deoxygenation probes ribose requirements for ADP–ribose transfer and polymer formation; the isotope supplies a detection handle.	65 and 66
3	C2-Ethynyl–NAD+	Accepted by PARP1/2 and tankyrases; competitive incorporation supports click tagging and biochemical labeling.	22
4	N6-Propargyl–NAD+	Clickable substrate for PARP1 and tankyrase-1; used for affinity enrichment and discovery of candidate PARP1 substrates.	22 and 25
5	C8-(Propargylamino)-NAD+	Weak substrate relative to 4: PARP1 kcat/Km = 0.51 M−1 s−1; no detectable p53/RAP74 labeling in the reported assay. Useful as a structure–activity comparator.	25
6	Nicotinamide–ribose-substituted NAD+ series	The characterized 3′-azido member supports PARP1/2 labeling and yields PARG-resistant PAR; these properties are not established for every depicted substituent.	21
7	ADO-4′-N3–NAD+	Adenosyl 4′-azide broadens compatibility to PAR- and MAR-forming ARTs; general click labeling across enzyme classes.	26
8	C2-Diazirine/3′-azido-NAD+	PARP1-compatible incorporation followed by UV crosslinking and click enrichment of PAR-dependent interacting proteins. The azide is on the nicotinamide ribose.	27
9	N6-Diazirine/3′-azido-NAD+	Companion N6 photoaffinity design; comparison of attachment geometry and substrate acceptance with 8. The azide is on the nicotinamide ribose.	27
10	C5-Substituted/N6-clickable NAD+	Matched to engineered PARPs; orthogonal labeling and enrichment identify writer-dependent protein targets.	61 and 62
11	C8-Butynyl–NAD+	Matched to engineered PARPs for writer-resolved proteomic/site mapping; PARP1 application linked ADP-ribosylation to transcription elongation.	72
12	5-Benzyl-2-ethynyl–NAD+	Efficient labeling with I631G PARP7 rather than wild-type PARP7; discovery of 250 direct targets in HEK293T lysates.	73
13	Clickable adenosyl-deoxy NAD+ series	2′-, 3′- and 2′,3′-deoxy designs combine chain restriction with an alkyne handle; biochemical labeling and simplification of PAR products.	75
14	C2-TMR–NAD+	Fluorescent incorporation following cellular delivery; imaging PAR at DNA damage sites and studying interactions using lifetime/FRET measurements.	28
15	C2-Desthiobiotin NAD+ format	Affinity tag for enrichment of cellular ADP-ribosylated proteins within the delivery strategy; desthiobiotin itself is not fluorescent.	67
16	Cholesterol-/R8K-conjugated TMR–NAD+ formats	Delivery-promoting substituents improve intracellular access of fluorescent NAD+ analogues; live-cell labeling and imaging workflows.	67
17	4′-Thioribose NAD+	Stable to cleavage by CD38/SIRT2 while retaining redox-cofactor activity; CD38 crystallography and mechanistic analysis of NAD+ recognition.	77
18	Amino/azido-substituted NAD+	Dual sugar substitution. The cited study characterizes related amino stereoisomers, not this exact structure; their sirtuin IC50 values cannot be assigned to 18.	78

4.1. Capture probes for mapping ADP-ribosylated biomacromolecules

The first generation of NAD⁺-based probes addressed the most basic analytical problem: how to detect ADP-ribosylated products with sufficient sensitivity. Radiolabeled NAD⁺ and related analogues, including radiolabeled NAD⁺ and deoxy derivatives, enabled autoradiographic detection of MARylated and PARylated proteins in biochemical systems.^65,66,70 These reagents were important for establishing ADP-ribosylation as an enzymatic modification, but they offered limited molecular information. They were not readily compatible with enrichment-based proteomics, did not provide residue-level assignment, and were poorly suited to spatially or temporally resolved experiments in living cells. The capture and enrichment logic of these early probe workflows is illustrated (Fig. 4a). Clickable NAD⁺ analogues were developed to overcome these limitations by installing a small bioorthogonal handle into an NAD⁺ scaffold that can still be processed by PARP enzymes. After enzymatic incorporation of the modified ADP–ribose unit into substrate proteins or PAR chains, azide- or alkyne-bearing products can be coupled to fluorophores or biotin through click chemistry.^25,71 This design converts transient ADP-ribosylation events into chemically enriched species that can be analyzed by fluorescence detection, immunoblotting, affinity purification, or LC–MS/MS-based proteomics. Importantly, the utility of such probes depends on both the position of the chemical handle and the ability of the resulting analogue to preserve relevant PARP turnover and product formation.

Fig. 4. Major classes of NAD⁺-derived chemical probes and their representative workflows. (a)–(d) Clickable or affinity-tagged probes, chemical-genetic probes, chain-terminating probes, and fluorescent or photoaffinity probes provide complementary strategies for substrate enrichment, writer-specific labeling, PAR-architecture control, reader capture, and imaging of ADP-ribosylation dynamics.

Compounds 1 and 2 in Fig. 5, combine phosphate-32P labeling with adenosyl deoxygenation: 1 lacks the 2′-hydroxyl group, whereas 2 lacks the 3′-hydroxyl group. The isotope provides a detection handle without a bulky reporter, while removal of a ribose hydroxyl probes the structural requirements for ADP–ribose transfer and polymer extension. Early radiolabeling and deoxy-NAD⁺ studies established the biochemical rationale for these complementary modifications.^65,66,70 These biochemical tools distinguish incorporation from polymer formation, but isotope counting alone cannot identify the modified amino acid.

The clickable adenine derivatives 3–5 demonstrate how attachment geometry determines probe performance. C2-ethynyl–NAD⁺ (3) is accepted by PARP1, PARP2 and tankyrases and competes with native NAD⁺ in biochemical labeling assays.²² N6-propargyl–NAD⁺ (4) was used to enrich candidate PARP1 substrates from cell lysates, followed by biochemical validation.²⁵ The C8-(propargylamino) analogue 5 is a substantially poorer substrate for PARP1 and tankyrase-1: in the reported PARP1 automodification assay, its catalytic efficiency was 0.51 M⁻¹ s⁻¹, compared with 5.0 × 10³ M⁻¹ s⁻¹ for 4. Probe 5 also failed to detectably label p53 or RAP74 under the tested conditions.²⁵ Thus, 3 and 4 support labeling and enrichment, whereas 5 illustrates a limitation of C8 substitution rather than a generally effective cellular probe.

Several clickable NAD⁺ analogues have been generated through chemoenzymatic routes. In these strategies, modified nicotinamide riboside, NMN, or related precursors bearing azide or alkyne groups are converted into NAD⁺ analogues by enzymes of the NAD⁺ biosynthetic pathway, such as NRK and NMNAT enzymes.^21,26 This approach allows site-defined modification of the NAD⁺ scaffold while avoiding lengthy fully chemical synthesis. To move ADP-ribosylation profiling from purified enzymes or lysates into living cells, later studies used cell-compatible precursor or delivery strategies that enable intracellular generation or uptake of functional NAD⁺ analogues. In this context, the term labeling refers specifically to enzymatic incorporation of analogue-derived ADP–ribose into cellular ADP-ribosylation products, followed by chemical tagging or enrichment. These approaches partially address the poor membrane permeability of native NAD⁺ and allow ADP-ribosylation to be examined under more physiological stress or signaling conditions.

Ribose-functionalized NAD⁺ analogues further illustrate that capture probes must be evaluated as enzymatic substrates, not merely as tagged molecules. The series grouped as 6 in Fig. 5 varies the position and identity of nicotinamide–ribose substituents, including azido and alkynyl-containing groups. Within this series, the nicotinamide–ribose 3′-azido NAD⁺ analogue reported by Zhang and colleagues is efficiently used by PARP1 and PARP2, supports PAR formation, and generates PAR products with increased resistance to PARG-mediated hydrolysis.²¹ This property is useful because it improves recovery of otherwise labile or low-abundance PARylation products. ADO-4′-N3–NAD⁺ (7) was subsequently shown to have broader ART compatibility, including activity with PARP1, PARP2, tankyrases, and mono-ARTs such as PARP10, thereby supporting both PARylation and MARylation workflows.²⁶ These examples emphasize a key design principle: an effective mapping probe should introduce a useful chemical handle while preserving the catalytic and product properties needed for the biological question being asked. The resistance of PAR derived from the characterized 3′-azido member of series 6 to PARG improves recovery but can bias measurements of signal persistence; this property should not be generalized to the other members of series 6 or to 7. The broader substrate scope of 7 supports surveys across ART classes without itself assigning a unique writer.

Some ADP–ribose-dependent interactions are weak, transient, or lost during sample processing. To address this problem, bifunctional NAD⁺ probes combine an enrichment handle with a photoactivatable crosslinker.²⁷ Compound 8 incorporates a diazirine group at the adenine C2 position together with a 3′-azido ribose modification, allowing PARP-dependent incorporation, UV-induced covalent capture of PAR-associated proteins, and subsequent enrichment by click chemistry. Rather than serving only as substrate-labeling reagents, such bifunctional probes connect PAR synthesis to interaction proteomics and are therefore particularly useful for profiling PAR-dependent reader or effector proteins. Compound 9 retains the nicotinamide–ribose 3′-azide while relocating the diazirine-bearing substituent to adenine N6. Comparing 8 and 9 tests how attachment geometry affects enzymatic acceptance and crosslinking. The C2 design 8 supports PARP1-dependent capture of PAR-associated proteins.²⁷ UV-dependent enrichment identifies candidate interactors; competition, enzyme-free and no-UV controls are still needed to distinguish direct readers from proximal proteins.

4.2. Chemical-genetic probes for assigning writer enzymes

A second major application is writer assignment. Because multiple PARP family members use the same native NAD⁺ co-substrate and may generate chemically similar ADP–ribose products, global labeling with a broadly accepted NAD⁺ analogue often cannot reveal which enzyme produced a given modification.^14,61,62 Chemical-genetic strategies address this limitation by pairing an engineered ART/PARP active site with a sterically matched NAD⁺ analogue. The engineered enzyme is designed to accept the modified co-substrate more efficiently than wild-type enzymes, thereby creating an orthogonal writer–probe pair that links product formation to a defined catalytic source. This orthogonal writer–probe strategy is illustrated (Fig. 4b). Compound 10 combines a bulky nicotinamide C5 substituent with an N6-linked alkyne and is paired with engineered PARPs for preferential writer-dependent labeling and click enrichment.^61,62 Compound 11 instead carries a C8-butynyl substituent and is used with complementary engineered PARPs for writer-resolved substrate and modification-site mapping.⁷² It provides a more direct route to writer-specific substrate discovery than inference from inhibitor sensitivity, gene deletion, or changes in global PAR levels, as labeling occurs during catalysis. In the PARP1 system, chemical-genetic labeling enabled proteomic identification of direct targets and helped connect PARP1 catalytic activity to transcriptional regulation, including control of RNA polymerase II elongation.⁷²

The same logic is not restricted to PAR-forming enzymes. Chemical-genetic and proteome-wide site-mapping approaches have also been applied to mono-ARTs such as PARP7, enabling isoform-resolved analysis of MARylation events and identification of cysteine ADP-ribosylation on immune-relevant protein targets.⁷³ This extension is important because MARylation is often less abundant and less easily amplified than PARylation. For mono-PARPs, orthogonal NAD⁺ analogue systems can therefore provide a direct means to connect a specific writer with its substrates, acceptor residues, and pathway context. In this application, 5-benzyl-2-ethynyl–NAD⁺ (12) combines a nicotinamide C5 benzyl group with a C2 ethynyl detection handle. The matched I631G PARP7 variant efficiently uses 12, whereas wild-type PARP7 does not productively use the same orthogonal probe. Labeling in HEK293T lysates identified 250 direct PARP7 targets, connecting cysteine MARylation to immune-relevant proteins, including PARP13.⁷³ Its specificity therefore derives from the engineered enzyme–probe pair, not selective incorporation by endogenous wild-type PARP7.

4.3. Chain-terminating probes for testing PAR architecture

A third problem concerns the structural heterogeneity of PAR. PARylation is not a single chemical endpoint: PAR chains can differ in length, branching frequency, linkage pattern, and local density, and these features influence reader recruitment and downstream signaling.⁷⁴ Therefore, detecting that a substrate is PARylated does not by itself define the relevant signal. It remains necessary to determine whether a biological response depends on initial ADP–ribose transfer, short oligomers, long linear PAR chains, or branched polymers. The use of chain-terminating analogues to simplify PAR architecture is summarized (Fig. 4c). Chain-terminating NAD⁺ analogues provide a chemical way to simplify this heterogeneity. Because PAR elongation requires ribose hydroxyl groups for ribose–ribose glycosidic bond formation, 2′- or 3′-modified NAD⁺ analogues can permit initial ADP–ribose transfer while restricting subsequent polymer extension.⁷⁵ The deoxy series grouped as 13 combines removal of the adenosyl 2′-OH, 3′-OH or both with an adenine-linked alkyne. These related structures compare hydroxyl requirements for product formation while retaining a handle for fluorescent tagging or affinity enrichment.⁷⁵ Wang and colleagues showed that chain-terminating and clickable NAD⁺ analogues can label ART target proteins and compete with native NAD⁺ in trans-ADP-ribosylation reactions, including reactions on histone substrates. Such analogues are therefore useful for separating initiation from elongation and for reducing the complexity of PAR products before enrichment or mechanistic analysis. In combination with PARP variants that alter chain length or branching, these tools make PAR architecture an experimentally tunable variable rather than an uncontrolled source of heterogeneity.

4.4. Visualization probes for spatiotemporal ADP-ribosylation

A fourth challenge is the need to observe ADP-ribosylation with spatial and temporal resolution. PAR formation can be rapidly induced at sites of DNA damage and then removed by eraser enzymes, so endpoint assays may miss the kinetics and localization of signal formation.⁵⁸ Visualization-oriented NAD⁺ probes are designed to retain sufficient PARP compatibility while introducing fluorophores or bioorthogonal groups that allow PAR formation, turnover, or PAR-associated interactions to be monitored in cells. The imaging-oriented design logic of fluorescent and photoaffinity NAD⁺ probes is illustrated (Fig. 4d). Adenine-modified NAD⁺ analogues have been particularly useful for this purpose because substituents at the adenine C2 position can be tolerated by PARP enzymes while projecting functional groups away from the most reactive portion of NAD⁺. Fluorescent C2-modified NAD⁺ analogues, including C2-TMR-NAD⁺ (14), have been used for real-time cellular imaging of protein poly(ADP-ribos)ylation.²⁸ In this workflow, the fluorescent NAD⁺ analogue reports PARP-dependent PAR formation and, when combined with fluorescence lifetime or FRET-based readouts, can provide information on PAR synthesis and PAR-associated protein interactions. These experiments demonstrate that NAD⁺ analogues can function not only as enrichment reagents but also as probes for dynamic cellular signaling.

The reporter and delivery designs in 15 and 16 provide complementary readouts. C2-desthiobiotin–NAD⁺ (15) supplies an affinity handle for recovering ADP-ribosylated proteins; desthiobiotin is not a fluorescent tag. The cholesterol- and R8K-conjugated TMR analogues grouped as 16 add delivery-promoting substituents to reporter-bearing NAD⁺ scaffolds. These designs enabled cellular uptake and supported labeling and enrichment of cellular ADP-ribosylation under oxidative stress.⁶⁷ Accordingly, 14 illustrates direct PAR imaging after delivery, 15 supports affinity proteomics, and 16 addresses intracellular access. Cellular fluorescence must nevertheless be distinguished from enzymatic incorporation and unreacted probe accumulation.

For live-cell imaging, carrier-assisted delivery, cell-permeable NAD⁺ derivatives and metabolically convertible precursors provide routes to intracellular probe access.^24,67 These approaches must preserve enzymatic incorporation and avoid substantial perturbation of endogenous NAD⁺ metabolism, because both factors affect the interpretation of PAR-associated fluorescence. Fluorescence imaging also connects NAD⁺ probe chemistry with cellular pharmacology. Time-lapse imaging of fluorescent NAD⁺ analogues can follow cellular entry, subcellular distribution and retention, while PAR-associated fluorescence and lifetime/FRET readouts report enzymatic incorporation and downstream interactions.^28,67 Complementary small-molecule, genetically encoded and chemigenetic sensors reveal changes in endogenous pyridine-nucleotide pools and redox state, linking probe exposure to metabolic and cellular responses.⁷⁶ These measurements should be interpreted separately: fluorescence accumulation alone cannot establish uptake of intact analogue or identify its metabolites; pulse–chase experiments, PARP-inhibition controls and LC–MS analysis can help distinguish delivery, conversion and product turnover. When combined with morphology, viability and stress-response measurements in automated high-content imaging, time-resolved signals can support phenotypic drug screening by resolving concentration-dependent responses and cell-to-cell heterogeneity. The growing use of pyridine–nucleotide imaging in phenotypic screening and mechanism-of-action studies therefore offers a route from chemical probes to functional cellular pharmacology, provided that probe loading, phototoxicity and nonspecific metabolic stress are controlled.⁷⁶

4.5. Stable cofactor mimics and amino-substituted analogues

Compound 17 replaces the ring oxygen of the nicotinamide ribose with sulfur. This 4′-thioribose NAD⁺ retains redox-cofactor activity but resists cleavage by human CD38 and SIRT2; crystallographic characterization of its CD38 complex enabled analysis of cofactor recognition without rapid consumption.⁷⁷ It therefore serves structural and mechanistic studies rather than functioning as a demonstrated transferable PARP label. Compound 18 combines an amino substituent on the nicotinamide-linked ribose with an azido substituent on the adenosyl ribose. Related amino-substituted NAD⁺ analogues provide mechanistic comparators for sugar stereochemistry and co-substrate recognition. For the separately characterized 2″-amino ara-β-NAD⁺ stereoisomer, reported SIRT1 and SIRT2 IC50 values were 176.2 ± 9.7 and 153.4 ± 10.1 µM, respectively.⁷⁸ These modest biochemical activities belong to that specific stereoisomer and cannot be assigned to the dual-substituted structure 18. The cited study therefore provides a comparison for amino substitution, rather than direct evidence of the activity or cellular utility of 18.

5. Probe-enabled mechanistic applications

Having established the chemical logic and representative probe classes, the central question becomes how NAD⁺ analogues change biological interpretation. Their value is not simply that they make ADP-ribosylation easier to detect, but that they encode experimental information into the transferred ADP–ribose unit.⁷⁹ The probe is therefore paired with a defined hypothesis: which writer acts, which biomacromolecule is modified, which PAR structure is formed, which reader is recruited and which cellular phenotype follows.

5.1. Proteomic mapping of ADP-ribosylation networks

Proteomic mapping remains the most mature biological application of NAD⁺ probe chemistry. Proteomic workflows use enrichment to recover low-abundance ADP-ribosylated species from complex samples.⁸⁰ Clickable and affinity-tagged NAD⁺ analogues enable this step by installing a chemically tractable handle during PARP catalysis. The labeled proteins can then be visualized, enriched, or analyzed by LC–MS/MS, allowing probe incorporation to be connected with substrate discovery and pathway context (Fig. 6a).^81–83 The value of this strategy is that it shifts ADP-ribosylation analysis from global activity measurement to network-level mapping. Probe-assisted workflows have enabled ADP-ribosylation to be examined in response to DNA damage, oxidative stress⁸⁴ and inflammatory stimulation. Metabolic precursor approaches and cell-compatible NAD⁺ analogue delivery further extend these measurements from purified systems and lysates toward more physiological cellular settings.⁸⁵ These studies indicate that ADP-ribosylation is not restricted to canonical DNA repair factors, but also intersects with transcription, RNA metabolism,⁸⁶ protein quality control, chromatin organization, stress responses, and signal transduction.^87,88

Fig. 6. Proteomic and imaging applications of NAD⁺-derived probes in ADP-ribosylation research. (A) NAD⁺- and ADP–ribose-based probes enable affinity enrichment, quantitative interaction profiling, and LC–MS/MS identification of ADP-ribosylated proteins and PAR/MAR-binding readers. Defined ADP–ribose or PAR probes can further support length-dependent reader profiling and binding-affinity analysis. (B) Fluorescent or cell-compatible NAD⁺ analogues allow visualization of PAR formation and turnover in living cells, providing spatiotemporal information on ADP-ribosylation dynamics.

However, proteomic enrichment should be interpreted as discovery rather than proof. A labelled protein may be a direct substrate, a proximal interactor, a reader captured through non-covalent association, or a component of a stress-induced assembly.⁸⁹ This ambiguity is a persistent controversy in ADP-ribosylation proteomics and should be addressed by time-resolved labelling, enzymatic controls, acceptor-site mapping, mutant rescue and functional assays. A remaining challenge is that different proteomic workflows capture different layers of biology. Some probes preferentially record nascent ADP–ribose transfer, whereas others enrich stable products or associated proteins.⁹⁰ Sample lysis, eraser inhibition, click chemistry efficiency, and peptide fragmentation strategy can all bias the apparent ADP-ribosylome. Future applications should therefore report probe incorporation, enrichment specificity, site localization confidence, and functional validation as connected parts of one workflow rather than as separate technical steps.

5.2. Reader discovery and ADP–ribose-dependent interactions

A second major application is the identification of ADP–ribose reader proteins. MAR and PAR signals are interpreted by proteins containing macrodomains, WWE domains, PBZ motifs, and other ADP–ribose-binding regions.⁹¹ NAD⁺-derived and ADP–ribose-based probes have enabled affinity-based discovery of such readers by presenting defined ADP–ribose structures as biochemical baits.^92,93 This is particularly useful because many reader interactions are transient, multivalent, or dependent on polymer length and local concentration. A photo-clickable, chain-terminating NAD⁺ analogue, PCCT–NAD⁺, has recently extended this concept by generating terminally functionalized PAR and enabling bead-immobilized photo-crosslinkable profiling of PAR-interacting proteins under oxidative stress.⁹⁴ Reader profiling has also made the PAR code experimentally approachable. PAR is not a uniform signal: binding can depend on chain length, branching, linkage type, local density, and the chromatin or protein context in which ADP–ribose is displayed.⁹⁵ Defined MAR and PAR probes can therefore separate proteins that preferentially recognize mono-ADP–ribose, short PAR oligomers, longer polymers, or more complex PAR-associated assemblies. Quantitative platforms such as PAQMAN add an important layer by linking probe-defined structures to apparent binding affinity and selectivity.

These approaches support the view that ADP–ribose recognition exists on a spectrum rather than as a simple binder/non-binder classification. Proteomic studies have identified classical PAR-binding modules as well as FHA, BRCT, OB-fold, zinc-finger, RNA-recognition, WD40-containing, and basic residue-rich proteins as potential ADP–ribose-associated factors. Broader reader and disease-oriented datasets further suggest that some factors may engage ADP–ribose indirectly through RNA, chromatin, protein–protein interactions, or condensate-associated recruitment.^96,97 This broader reader landscape is conceptually important. If a protein is enriched by an ADP–ribose probe, the next question should be whether it directly recognizes a defined chemical feature or is recruited by proximity, charge, multivalency, or phase separation.⁹⁸ NAD⁺ and ADP–ribose-based probes are well suited to make this distinction because they can be combined with competition assays, chain-length-controlled baits, photo-crosslinking, quantitative proteomics, and imaging-based validation. In this way, reader studies can move from lists of candidate binders toward structure–function relationships between PAR architecture and biological output.

5.3. Writer-resolved pathway dissection

Global labeling strategies reveal where ADP-ribosylation occurs, but they often cannot identify which PARP enzyme generated a given modification. This limitation is especially important because PARP family members can share substrates, respond to overlapping stimuli, and generate chemically similar ADP–ribose products.⁹⁹ Chemical-genetic NAD⁺ probes address this problem by linking substrate labeling directly to a defined writer. In this strategy, an engineered PARP variant is paired with a sterically matched NAD⁺ analogue that is poorly used by wild-type enzymes but efficiently accepted by the mutant writer. This orthogonal enzyme–probe pair allows substrates generated by a specific PARP to be labeled even in a complex cellular background.¹⁰⁰ Engineered PARP1 systems have, for example, connected PARP1 activity to transcriptional regulation by showing that PARP1-mediated ADP-ribosylation of the negative elongation factor promotes its dissociation from chromatin and facilitates RNA polymerase II elongation.

The conceptual strength of chemical-genetic probes is that they transform ADP-ribosylation from a global stress-associated signal into an enzyme-resolved pathway event. Similar strategies have been extended to mono-ARTs such as PARP7, supporting isoform-specific analysis of MARylation events.¹⁰¹ This is particularly valuable for mono-PARPs, because MARylation is often less abundant, more labile, and less easily amplified than PARylation. Writer-specific labeling can therefore reveal direct substrates that would otherwise be obscured by redundancy, compensatory signaling, or inhibitor pleiotropy. For disease-oriented studies, this distinction matters. Inhibition or deletion of a PARP enzyme can alter NAD⁺ availability, compensatory PARP activity, DNA damage signaling, transcriptional programs, or stress responses.¹⁰² Chemical-genetic labeling provides a more direct route by marking the products generated during catalysis by a selected writer. The most informative future studies will combine these probes with endogenous rescue systems, CRISPR-based editing, quantitative proteomics, and site-specific functional assays so that writer assignment can be linked to biological consequence.

At the same time, chemical-genetic systems require careful controls. Engineered PARP variants may not fully reproduce endogenous regulation, and bulky NAD⁺ analogues may alter catalytic efficiency, substrate preference, product stability, or PAR chain architecture.¹⁰³ Therefore, writer-specific probe data should be interpreted alongside native enzyme perturbation, analogue-compatibility controls, and disease-relevant cellular models. This balanced approach is essential if chemical-genetic labeling is to move from elegant substrate discovery toward mechanistic explanation.

5.4. Functional testing of PAR architecture

Another important application is the functional interrogation of PAR architecture. PARylation should not be treated as a binary modification, because polymer length, branching, linkage composition, and local density can influence reader recruitment, eraser sensitivity, chromatin remodeling, and condensate formation.¹⁰⁴ The biological outcome may therefore differ depending on whether a substrate carries MAR, short PAR oligomers, long linear chains, or branched polymers.

Chain-terminating NAD⁺ analogues provide a practical way to simplify this complexity. By modifying ribose hydroxyl groups involved in ribose–ribose glycosidic bond formation, these probes can permit initial ADP–ribose transfer while limiting subsequent polymer elongation.¹⁰⁵ They are therefore useful for separating initiation-dependent effects from long-chain PAR-dependent processes and for testing whether a response requires extensive polymer formation or can be triggered by MAR or short PAR structures.

This approach is especially relevant for DNA damage signaling, chromatin remodeling, protein recruitment, and phase separation. Long PAR chains may provide multivalent binding platforms for repair factors or chromatin-associated proteins, whereas shorter ADP–ribose structures may be sufficient for local recognition by specific readers.¹⁰⁶ Branching may further alter polymer compactness, reader accessibility, and eraser sensitivity. By constraining PAR synthesis, chain-terminating probes convert PAR architecture from an uncontrolled source of heterogeneity into an experimentally adjustable variable.

The broader opportunity is to make the PAR code testable rather than metaphorical. Instead of asking only whether PAR is present, probe-based studies can ask which structural feature is necessary for a defined outcome: polymer length, branching, linkage pattern, local density, eraser sensitivity or reader selectivity.¹⁰⁷ This is where chain-defined probes, synthetic PAR fragments and reader-profiling proteomics should be integrated with chromatin, DNA-repair and condensate models.

5.5. Imaging ADP-ribosylation dynamics in space and time

ADP-ribosylation is highly dynamic, especially during DNA damage responses, where PAR synthesis and degradation can occur within minutes. Endpoint assays may therefore miss the kinetics, localization, and reversibility of the signal.¹⁰⁸ Visualization-oriented NAD⁺ probes address this limitation by converting NAD⁺ analogue incorporation into an optical readout of PAR formation, turnover, or PAR-associated interactions. Fluorophore-modified NAD⁺ analogues are particularly useful when the modification site is compatible with PARP catalysis. Adenine C2-modified probes, including C2-TMR–NAD⁺, can support PARP1-mediated PAR synthesis and have been used to visualize PAR formation at DNA damage sites when combined with appropriate delivery strategies (Fig. 6b).¹⁰⁹ Fluorescence-based approaches, including FLIM–FRET, can further report on covalent PARylation events and non-covalent PAR–protein interactions.

The main biological advantage of imaging is that it reveals information that bulk proteomics cannot provide. The same total level of ADP-ribosylation may correspond to very different spatial patterns: localized PARylation at DNA lesions, signals near replication stress sites, nuclear microdomain formation, or accumulation within transient condensates.¹¹⁰ Imaging-oriented probes can therefore distinguish global increases in ADP-ribosylation from localized signaling events and can compare the timing of PAR synthesis, reader recruitment, chromatin rearrangement, and eraser-mediated signal removal.

A complementary approach is to compare analogue-based imaging with genetically encoded reporters of PAR dynamics. Genetically encoded PAR sensors, including macrodomain-based reporters such as pARS, monitor PAR dynamics without direct delivery of modified NAD⁺.^111,112 Combining fluorescent NAD⁺ probes with genetically encoded sensors may ultimately allow simultaneous monitoring of PAR synthesis, reader engagement, and signal erasure in living cells.

6. From probe-enabled discovery to disease-relevant mechanisms

The next stage of NAD⁺ probe application should move from cataloguing substrates and interactors toward defining disease-relevant mechanisms. ADP-ribosylation is closely linked to cancer, inflammation, viral infection, neurodegeneration, and stress responses, but causal modification events remain unclear in many settings.¹¹³ NAD⁺ probes are well positioned to address this gap because they can connect four levels of evidence: enzyme activity, substrate modification, reader recruitment, and pathway outcome.

In cancer biology, probe-enabled studies can help separate direct PARP-dependent ADP-ribosylation events from the broader cellular consequences of PARP inhibition. Clinical success across BRCA-mutant breast, ovarian, and prostate cancers demonstrates the therapeutic importance of PARP biology. Additional trials in ovarian cancer show that response is shaped by DNA repair genotype, treatment context, and disease state rather than by PARP inhibition alone.^114,115 Maintenance and precision–medicine studies in recurrent ovarian cancer and metastatic prostate cancer further extend this framework across clinically distinct settings.^116,117 Mechanistically, drug response also reflects PARP trapping, chromatin retention, DNA repair pathway rewiring, replication fork protection, PARP1 mutation, and altered PAR turnover.^118,119 PARG loss and replication-fork remodeling further illustrate how ADP–ribose synthesis and removal can reshape therapeutic sensitivity.^120,121 These observations argue that NAD⁺ probes will be most useful in cancer when they are used not only to detect PARP activity, but to define which modification events drive repair, transcriptional adaptation, resistance, or cell death.^122,123

In antiviral and inflammatory signaling, the key question is whether ADP-ribosylation functions as a direct effector mechanism, a scaffold-like recruitment signal, or a secondary marker of cellular stress. Several PARP family members are interferon-stimulated or participate in host–pathogen interactions.^124,125 PARP12, PARP9-DTX3L, and other interferon-linked PARPs can restrict viral replication or shape innate immune outputs, suggesting that writer-specific probes could clarify which antiviral effects require catalytic modification. Viral macrodomains provide the reciprocal view: many viruses encode ADP–ribose-binding or ADP–ribosylhydrolase modules that counter host ADP-ribosylation-dependent immunity.¹²⁶ Coronavirus macrodomain studies, stress-granule biology, and PARP7-dependent interferon control together support ADP-ribosylation as a reversible immune-regulatory axis rather than a simple stress byproduct.

In neurodegeneration and stress-related disease contexts, NAD⁺ probes can help disentangle ADP-ribosylation signaling from broader NAD⁺ metabolic decline. Excessive PARP1 activation has been linked to parthanatos, mitochondrial dysfunction, and neuronal loss.^127,128 Additional studies connect PAR-dependent death signaling to Parkinsonian neurodegeneration, inflammatory tissue injury, and degenerative pathology.¹²⁹ As PARP activity consumes NAD⁺, interpretation of probe-detected ADP-ribosylation must also consider NAD⁺ metabolism, sirtuin activity, mitochondrial function, and age-associated loss of metabolic resilience.^130–132 NAD⁺-boosting strategies and systems-level analyses of NAD⁺ homeostasis provide an important context for deciding whether a detected ADP-ribosylation event is causal, compensatory, or secondary to metabolic stress.

Across these disease settings, the decisive issue is functional validation. Probe labelling should be integrated with structural knowledge of PARP activation, HPF1-dependent residue specificity and chromatin-associated catalysis.¹³³ Site-resolved mass spectrometry can then define acceptor residues, whereas perturbation of the writer, eraser or reader can test whether the labelled event controls the phenotype. Without this sequence, probe discovery risks becoming another catalogue rather than a mechanism.

Clinical and resistance-oriented literature is particularly important when moving from probe-enabled discovery toward therapeutic relevance. Maintenance PARP-inhibitor trials in ovarian cancer, studies in prostate cancer, and broader DNA-damage-response frameworks illustrate how PARP biology intersects with patient stratification and treatment response.¹³⁴ Resistance mechanisms, including restoration of DNA repair capacity, altered replication-fork protection, and PARP1 mutations, emphasize why chemical probes should be integrated with functional assays rather than used only to identify modified proteins.

Therapeutic studies provide a complementary benchmark for evaluating probe-derived mechanisms. The synthetic-lethality principle underlying PARP inhibition was established in BRCA-deficient models and later translated into clinical benefit in BRCA-mutant breast and ovarian cancers.¹³⁵ Viral macrodomain biology and parthanatos studies show that ADP–ribose recognition and hydrolysis can also determine infection outcomes and cell-death phenotypes. Tankyrase biology further connects PARP-family catalysis to Wnt signaling, telomere regulation, and small-molecule drug discovery.

In disease models, probe-enabled experiments should be interpreted in light of established PARP biology rather than treated as stand-alone cataloguing tools. PARP1-dependent cell death and parthanatos are especially relevant in neurological injury and degeneration, while PAR-binding proteins and photoaffinity-based reader probes illustrate how polymer architecture or MAR-specific recognition may influence disease-associated complexes.¹³⁶ Emerging links between mono-ADP–ribose reader proteins, nuclear pore biology, age-related neurological disease, and ADP-ribosylation-associated condensates further broaden the biological contexts in which NAD⁺ probes may be informative.¹³⁷ These connections also extend to age-related neurodegeneration and condensate-dependent stress signaling.¹³⁸

The structural heterogeneity of PAR itself provides another rationale for application-oriented probe design. Chain length, branching, and eraser activity can reshape biological output, so chain-defined probes and eraser-aware workflows are needed to connect PAR architecture with reader recruitment and signal termination. Earlier chemical-reporter and enzymology studies also provide the conceptual basis for modern clickable, fluorescent, and specificity-enhanced NAD⁺ analogues.¹³⁹

Several additional lines of evidence further clarify where NAD⁺-probe chemistry can add mechanistic resolution. PARP1 and PARP2 are dynamically regulated during DNA damage and replication stress, reinforcing the need for probes that distinguish catalytic activity from total protein abundance.¹⁴⁰ Site-level studies have also shown that ADP-ribosylation can occur on serine and arginine residues, among other acceptor chemistries, meaning that probe-based enrichment should be paired with residue-resolved validation whenever possible.

6.1. Connecting probe chemistry with drug discovery

The clinical success of olaparib and niraparib demonstrates the therapeutic value of inhibiting PARP-dependent DNA repair in defined cancer settings.^{11,115,134,141,142} NAD⁺ analogues contribute upstream by identifying which catalytic events and substrates should be perturbed. Clickable probes quantify newly formed ADP-ribosylation, orthogonal enzyme–analogue pairs assign writer-dependent substrates, and fluorescent probes report the spatial and temporal consequences of candidate inhibitors.^{25,28,61,72,73} These measurements can support mechanistic target validation and cellular pharmacodynamic assays, although probe incorporation alone does not establish direct drug binding, therapeutic efficacy or clinical benefit.

A key distinction is between PARP catalysis and non-catalytic scaffolding or chromatin-binding functions. NAD⁺-analogue incorporation reports catalytic output, whereas protein depletion removes both catalytic and non-catalytic activities. Comparing labeling with selective inhibitors, wild-type and catalytically impaired rescue constructs, chromatin occupancy and DNA-repair or survival measurements can help separate these contributions.^20,61,72 PARP trapping is a further drug-induced property that must be assessed separately: clinical inhibitors can differ in their ability to stabilize PARP–DNA complexes despite suppressing PAR synthesis.^119,122,123 Probe-defined changes in substrates or reader recruitment can therefore inform inhibitor selectivity and resistance studies, but cannot substitute for trapping assays.

Therapeutic NAD⁺ targeting also extends beyond PARPs. NAMPT inhibition reduces nicotinamide salvage and can expose NAD⁺-dependent metabolic vulnerabilities in cancer, while simultaneously affecting redox reactions and other NAD⁺-consuming enzymes.^105,108 CD38 inhibition offers a different strategy by limiting NAD⁺ consumption and altering ADP–ribose/cyclic ADP–ribose signaling; metabolic effects of CD38 enzyme inhibition should be distinguished from the immune mechanisms of CD38-targeting antibodies.^108,132 Conversely, nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN) supplementation seek to augment NAD⁺ availability. Their effects depend on uptake, conversion and tissue-specific NAD⁺ homeostasis, so benefits in experimental models do not establish efficacy across human diseases.^{106,108,130–132} These strategies may have different, sometimes opposing, consequences in tumor and normal tissues.

In cancer, combining probe-based activity measurements with NAD⁺ metabolomics and repair phenotypes can test whether an intervention acts through PARP signaling or broader metabolic depletion. In inflammation, writer-specific labeling and reader profiling can resolve ADP-ribosylation-dependent immune outputs; in neurodegenerative disease models, analogous measurements can distinguish excessive PARP activation and parthanatos from loss of NAD⁺-dependent metabolic resilience.^{6,7,12,30,129,132} A useful discovery workflow therefore links probe-defined target engagement or catalytic output to an orthogonal functional assay and a disease-relevant phenotype. This connects probe chemistry to drug discovery while keeping biochemical mechanism distinct from therapeutic validation.

7. Unresolved questions and future directions

Several unresolved questions should shape the next generation of NAD⁺ analogue studies. First, it remains unclear how much biological information is encoded directly in PAR architecture and how much is imposed by local protein concentration, chromatin context and phase separation.¹⁴¹ Second, analogue tolerance is not equivalent to native catalysis; modified NAD⁺ molecules may bias chain length, linkage pattern, substrate selection or eraser sensitivity. Third, intracellular delivery strategies can alter NAD⁺ metabolism and stress responses, making cellular interpretation more complex.

Future work should move toward benchmarked probe panels rather than single-probe studies. The most informative experiments will compare structurally related analogues across multiple PARPs, quantify kinetic trade-offs, map acceptor sites, measure PAR architecture and connect these readouts to reader recruitment and cellular phenotypes.¹⁴² Such integration would make NAD⁺ analogues useful not only for detecting ADP-ribosylation, but also for explaining how ADP–ribose chemistry controls macromolecular function.

8. Conclusions and perspectives

Chemically modified NAD⁺ analogues have transformed ADP-ribosylation research from descriptive detection toward mechanism-oriented biological chemistry. Their conceptual power lies in exploiting the dependence of PARP-family enzymes on a shared co-substrate while using chemical modification to resolve writer specificity, substrate identity, PAR architecture, reader engagement and signal dynamics.¹⁴³ In this sense, NAD⁺ analogues do not merely report that ADP-ribosylation occurred; they help explain which catalytic process produced the signal and how that signal is interpreted by biological macromolecules.

For future chemical biology studies, the central challenge is to link probe chemistry to biochemical causality. Each analogue should be evaluated for catalytic efficiency, product structure, cellular delivery, eraser sensitivity and downstream function.¹⁴⁴ The strongest advances will connect synthesis to enzymology, enzymology to proteomics or imaging, and detection to disease-relevant mechanism. This integration will determine whether NAD⁺ probe chemistry can move ADP-ribosylation from an observable modification to a therapeutically interpretable biological process.

Author contributions

Lijuan Gao: conceptualization, investigation, writing – original draft, writing – review & editing, visualization. Yangyang Zhou: writing – original draft, writing – review & editing, visualization. Tianye Dai: writing – review & editing, visualization. Yanhui Xu: writing – review & editing. Jing You: writing – review & editing. Jiahua Jiang: writing – review & editing. Xiao-Nan Zhang: conceptualization, supervision, project administration, funding acquisition, writing – review & editing. Baohua Zhao: supervision, project administration, writing – review & editing.

Conflicts of interest

The authors declare that they have no conflicts of interest with the contents of this article.

Biographies

Biography

Lijuan Gao .

Lijuan Gao completed her undergraduate studies at the School of Food Science and Biology, Hebei University of Science and Technology. She is currently pursuing her master's degree at the College of Life Sciences, Hebei Normal University, and receiving joint research training at the School of Biopharmacy, China Pharmaceutical University. Her research focuses on the chemical biology of ADP-ribosylation, including the development of functional NAD⁺ analogues for investigating poly(ADP–ribose) (PAR)–protein interactions.

Biography

Yangyang Zhou .

Yangyang Zhou is affiliated with the School of Pharmacy, China Pharmaceutical University (CPU). She conducted three years of research on ADP-ribosylation at the CPU School of Biopharmacy and published a research article in Targetome as a co-first author. She is currently a visiting scholar at Harvard University, where her research focuses on the molecular mechanisms of neurodegenerative diseases and targeted therapeutic strategies.

Biography

Tianye Dai .

Tianye Dai is a student in pharmacy at China Pharmaceutical University. His research interests center on the chemical biology of ADP-ribosylation.

Biography

Yanhui Xu .

Yanhui Xu completed her undergraduate studies at Hebei Agricultural University and is currently pursuing her master's degree at the School of Biopharmaceutical Sciences, China Pharmaceutical University. Her research focuses on ADP-ribosylation.

Biography

Jing You .

Jing You is an undergraduate student majoring in biology at the College of Life Sciences, Hebei Normal University. Her research focuses on the design and synthesis of NAD⁺ analogues for studying ADP-ribosylation.

Biography

Jiahua Jiang .

Jiahua Jiang obtained his master's degree in medicinal chemistry from China Pharmaceutical University, conducting his research at the State Key Laboratory of Natural Medicines. He also received joint research training at the university's School of Biopharmaceutical Sciences.

Biography

Xiao-Nan Zhang .

Xiao-Nan Zhang is a professor at the School of Biopharmacy, China Pharmaceutical University. He received his PhD from East China University of Science and Technology in 2014. From 2015 to 2022, he worked as a postdoctoral researcher and subsequently as an assistant researcher at the School of Pharmacy, University of Southern California. He has published over twenty research articles in journals including Nature Communications, Science Advances, Journal of Controlled Release, Angewandte Chemie International Edition, ACS Catalysis, and Chemical Science. His research focuses on protein post-translational modifications and their therapeutic applications, antibody-based drug development, and the design and application of targeted protein degradation strategies.

Biography

Baohua Zhao .

Baohua Zhao is a professor, doctoral supervisor, and university-recognized academic leader at Hebei Normal University. He serves as vice chair of the Hebei Society for Microbiology and vice president of the Inspection and Quarantine Branch of the Hebei Association of Animal Science and Veterinary Medicine. His research focuses on the molecular mechanisms of animal microbial pathogenesis and strategies for disease prevention and control. He has led two projects funded by the National Natural Science Foundation of China and several provincial research projects, and has received two third-class Hebei Provincial Science and Technology Progress Awards.

Data availability

No new data were created or analysed in this review article.