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. 2026 Jan 30;6(2):1001–1011. doi: 10.1021/jacsau.5c01369

Site-Directed Modification of mRNA with Functionalized Platinum(IV)–Ammines

Edward Miller †, Charlotte E Farquhar †, Jacob Rodriguez †, Andrei Loas †, Bradley L Pentelute †,‡,§,∥,*
PMCID: PMC12933306  PMID: 41755824

Abstract

Developing chemical toolkits for mRNA modification has remained an immense challenge, driven by the inherent difficulties in targeting mRNA molecules. Antisense oligonucleotides (ASOs) offer a promising framework for targeting specific mRNA sequences, yet they do not possess the capacity to alter the structure of mRNA except through enzyme-mediated hydrolysis. We developed a platinum­(IV)–ASO strategy that combines the sequence specificity of ASOs with the reactivity of platinum to functionalize nucleic acids, including short RNA and mRNA, in a selective enzyme-free manner. Access to Pt­(IV)–ASO constructs was made possible by an equatorial Pt­(IV) ammine derivatization strategy, allowing for the conjugation of carboxylic acids directly to the Pt core. Reactivity with 21-mer RNA and full-length mRNA by Pt–ASO constructs was demonstrated, and the conjugated products were characterized using a suite of orthogonal techniques, such as electrophoretic mobility shift assay, MALDI-TOF MS, temperature-dependent dissociation assay, and RT-qPCR. Constructs were optimized for their reactivity and selectivity, allowing for Pt­(IV)–PMO constructs with subnanomolar IC50 values in an RNA competition assay. This Pt­(IV)–ASO platform facilitates new avenues for RNA modification by providing a strategy for functionalizing nucleic acids with potential applications in molecular biology research.

Keywords: Pt(IV)−prodrugs, bioconjugation, mRNA, antisense oligonucleotides, coordination chemistry

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Introduction

Extensive work has been dedicated to developing diverse chemical toolkits to attenuate and enhance the function of proteins, including discovery of binders, − development of site- and chemoselective bioconjugation reactions, and designing proteolysis targeting chimeras (PROTACs) to degrade proteins. Despite mRNA being the progenitor of proteins, a similar complementary chemical toolkit for modifying native mRNA is not available. This lack of suitable functionalization methods is primarily due to the inherent challenges of targeting mRNA. Unlike proteins, which are built from a diverse set of 20 amino acid monomers and have a wide range of structural complexities, mRNAs consist of only four nucleotide monomers and exhibit limited diversity in their highly fluxional tertiary structure.

One of the most successful approaches for selectively targeting mRNA is based on antisense oligonucleotides (ASOs, Figure A), which are short single stranded synthetic oligomers designed to bind mRNA through base-pairing. ASOs are an expanding class of FDA-approved therapeutics that bind and interact with RNA to restore proper gene expression through enzyme-mediated hydrolysis. − Complementary to this class of RNA-hydrolyzing ASOs are those that exert their biological effect through steric hindrance. Included in this class are peptide nucleic acids (PNAs) , and phosphorodiamidate morpholino oligomers (PMOs), − which are charge-neutral and feature high binding affinity to their complementary strands. As a result of their nonbiological structure, they are proteolytically stable and their adducts with RNA are not recognized by native enzymes. Thus, PNAs and PMOs generally cannot affect mature mRNA structure.

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ASO architectures and Pt­(IV) prodrug synthesis/reactivity. (A) Prevalent ASO architectures. (B) Established “dual-threat” design of Pt­(IV)–prodrugs. (C) Development of a late-stage equatorial Pt­(IV)–ammine functionalization strategy for accessing Pt­(IV)–ASOs that can functionalize RNA in a site-directed manner. Half-maximal inhibitory concentration (IC50) and maximum drug inhibition (I max) determined via competition assay.

Despite the variety in ASO structure and therapeutic applications, the inability to use these molecules to perform covalent modifications of mRNA was identified as an unmet challenge. We proposed to address this challenge by tethering an electrophilic warhead to an ASO, which would allow the construct to functionalize the mRNA after sequence-specific binding. A platinum-based warhead was identified as the ideal electrophile to use for a nonreversible modification strategy of mRNA. Platinum’s inherent reactivity toward nucleic acids, specifically DNA, has led to platinum-containing drugs becoming a crucial class of chemotherapies. Among this class, cisplatin, carboplatin, and oxaliplatin are the most prescribed, and are approved worldwide to treat multiple forms of cancer. These compounds react with DNA to form intrastrand adducts between two neighboring guanosines. Based on this reactivity, it was hypothesized that similar linkages could be formed with RNA, paving the way for this approach to be used for mRNA bioconjugation.

To circumvent premature self-quenching between the ASO and the tethered electrophile, a Pt­(IV)–prodrug approach was pursued to mask the reactivity of platinum. Pt­(IV)-containing coordination complexes are stable toward ligand substitution and are readily reduced to bioactive Pt­(II) compounds. Additionally, engineering a Pt­(IV) reagent for an RNA functionalization strategy is streamlined by the modularity of its primary coordination sphere, which allows for tuning of physical and chemical properties. Numerous applications have been explored with the Pt­(IV) architecture via installation of bioactive ligands to the reductively labile axial positions (Figure B). − While this “dual-threat” strategy is well-established, an mRNA functionalization approach would require that the targeting ASO remains attached upon reduction in order to position the Pt­(II) core for targeted reactivity with mRNA. To accomplish this feature, the ASO would need to be appended to a reductively nonlabile (L-type) coordination site of the Pt­(IV). This “synergistic” configuration is significantly less studied than the “dual-threat” configuration, due to the lack of methods to functionalize these equatorial L-type ligands in Pt­(IV) compounds in a late-stage manner.

Here, we report a coupling strategy between activated carboxylic acids and Pt­(IV)–ammines to generate coordination complexes with a “synergistic” configuration. This method tolerates a variety of Pt­(IV)–prodrugs and biologically relevant carboxylic acids (Figure C), enabling access to Pt­(IV)–PNA and Pt­(IV)–PMO constructs that can site-selectively modify mRNA. We demonstrate that this linkage can be visualized through a reverse-transcription assay, allowing quantification of reactivity and enabling exploration of the structure–activity relationship of Pt­(IV)–ASOs.

Results and Discussion

Taking inspiration from the reported reactivity of transition metal–ammine complexes with electrophiles, − we hypothesized that treatment of a Pt­(IV)–ammine with an activated carboxylic acid could be a suitable approach to functionalize the Pt-core. To explore this hypothesis, the reactivity of Pt­(IV) complex 1a with O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU)-activated acetic acid under basic conditions was examined (Figure A). Under these reaction conditions, formation of functionalized product 2a was observed in good yield (58%). Functionalization of the ammine was supported by 195Pt–1H HMQC NMR spectroscopy, which displayed a downfield shift in the 195Pt signal of 205 ppm upon acylation (Figure S1). This large shift indicates a large electronic change at the Pt center, supporting the modification of the equatorial ammine.

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Pt­(IV)–ammine functionalization is successful across a broad scope of biologically relevant coupling partners. (A) Solution-phase coupling of Pt­(IV)–compounds with carboxylic acids. (B) Two-step procedure for accessing Pt­(IV)–amide complexes with pendant carboxylic acids. (C) Solid-phase coupling of Pt­(IV)–compounds with carboxylic acids (RCO2H). aConditions: 1 (0.10 mmol, 1.0 equiv), RCO2H (2.0 equiv), HATU, 2.0 equiv), N,N-diisopropylethylamine (DIEA, 4.0 equiv), and N,N-dimethylformamide (DMF, 1.25 mL), room temperature (rt), 1 h. bConditions: 1 (0.10 mmol, 1.0 equiv), RCO2H (2.0 equiv), HATU (2.0 equiv), DIEA (8.0 equiv), DMF (625 μL), rt, 1 h. cReaction performed using standard conditions “a” on a 0.5 mmol scale. dConditions: (1) 1 (0.15 mmol, 1.5 equiv), 1,1′-carbonyldiimidazole (CDI, 1.5 equiv), Sieber Amide Resin (1.0 equiv), imidazole·trifluoroacetic acid (0.25 equiv), DMF (920 μL), rt, 16 h. (2) 5-Azidopentanoic acid (5.0 equiv), HATU (5.0 equiv), DIEA (260 μL), DMF (1.3 mL), rt, 30 min. Yields are those of isolated 2a–i, 3a–g, and 4a–b.

Investigation of the substrate scope revealed that the reaction tolerates a variety of Pt­(IV)–ammine and carboxylic acid coupling partners. Modification of the Pt core from cisplatin to carboplatin (2b, 37%) or oxaliplatin (2c, 65%) was well tolerated, demonstrating the applicability of this method across therapeutically relevant Pt core scaffolds. Additionally, a variety of carboxylic acids could be coupled onto the Pt­(IV) core, including those with valuable conjugation handles such as maleimide (2c, 65%), azide (2d, 63%), and alkyne (2e, 46%). Moreover, a protected glutamic acid for solid-phase peptide synthesis (2f, 50%) was successfully incorporated. Directly bioactive fragments were also successfully coupled to Pt­(IV), including biotin (2g, 73%) for affinity capture studies, 5-carboxytetramethylrhodamine (5-TAMRA, 2h, 30%) for fluorescence imaging applications, and small molecule ligands, such as JQ1 (2i, 69%).

Incorporation of a 9-fluorenemethyl ester at the apical position of Pt­(IV) complexes facilitated a two-step functionalization and deprotection protocol, resulting in Pt­(IV)–amide complexes featuring an axial carboxylic acid suitable for subsequent amide coupling (Figure B). Complexes with various axial ligands (F–, Cl–, ArCO2 –, and 4-tBuPy), , as well as various Pt cores (cisplatin, carboplatin, and oxaliplatin) were accessed in moderate to good yields (3a–g, 18–75%). In addition, functionalization of the Pt core could be accomplished after loading onto a solid support (Sieber amide resin), yielding 4a and 4b in moderate yields (Figure C, 33% and 37%, respectively) after cleavage from the resin. The ability to tolerate various axial ligands and platinum cores as coupling partners is important for the broad application of the method and utility of the functionalized products. Modulating the primary coordination sphere has a profound impact on the electronic parameters of the Pt­(IV) center, which enables fine-tuning of the reduction kinetics of the functionalized products. −

A solid-state structure of 2i, obtained via single-crystal X-ray diffraction, provided definitive evidence for modification of the equatorial Pt­(IV)–NH3 ligand (Figure A, Figure S2, and Table S1). The Pt–NHCO moiety exhibited an X-type Pt–amide bonding structure, characterized by a short C=O bond length of 1.23 Å and long NH–CO bond length of 1.33 Å, consistent with established X-type Pt­(IV)–amide complexes. − The anionic charge of this moiety was neutralized by a triazolium on the JQ1 fragment. From the zwitterionic nature of this structure, it was deduced that the protonated, charge neutral Pt­(IV)-complex is relatively acidic and would be deprotonated under neutral pH conditions as the pK a of 1,2,4-triazolium is 2.2. Conversely, other derivatives resulting from ammine functionalization lack an intramolecular base, necessitating protonation of the Pt­(IV)–amide for charge balance. Two potential tautomers, L-type amide or imidine configurations, were considered for representing these products. Ground state density functional theory (DFT) calculations indicated that the Pt­(IV)–imidine was 3.8 kcal/mol more stable than the corresponding L-type Pt­(IV)–amide (Figure B and Tables S2–S25). This outcome, in combination with the prevalence of Pt­(IV)–imidines characterized crystallographically, − led to the Pt­(IV)–imidine being the preferred tautomer for representation of the protonated, charge-neutral structures.

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Structural analysis of Pt­(IV) complexes by X-ray crystallography and DFT calculations elucidates the bonding geometry of products from Pt­(IV)–ammine functionalization. (A) Solid-state structure of 2i from single-crystal X-ray diffraction analysis. Thermal ellipsoids are plotted at 50% probability level; solvent omitted for clarity. (B) Optimization of ground-state structures by DFT reveals that the Pt­(IV)–imidine tautomer is more stable than the L-type Pt­(IV)–amide tautomer by 2.9–4.2 kcal/mol. Geometry and frequency calculations were performed at the PBE0 level of theory with the SARC-ZORA-TZVP basis set for Pt and ZORA-def2-SVP for remaining elements (C, N, H, O, Cl) with tight convergence criteria.

To demonstrate the utility of these compounds toward mRNA platination, Pt­(IV)–ASO constructs targeting enhanced green fluorescent protein (EGFP) mRNA were investigated to optimize the reactivity and selectivity. A sense region in the open reading frame of the mRNA was chosen based on two design criteria dictated by the inherent reactivity of platinum toward guanosine: (1) this region featured a “GGG” motif as the intended site of platination, allowing for multiple sites of functionalization, and (2) the antisense for this region featured few guanosines, mitigating issues of competing ASO platination. Based on this analysis, an antisense 11-mer PNA was synthesized to this target region by automated flow synthesis employing protocols developed in our laboratory. , To attach Pt to the PNA, a strain-promoted azide–alkyne click strategy was pursued, yielding nBuPt-PNA 369–379 (Figure A).

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Reduction of Pt­(IV)–PNAs is influenced by axial group identity. (A) Accessing Pt­(IV)–PNAs via the SPAAC reaction. (B) Effect of axial leaving group identity of Pt­(IV)–ASO on ease of reduction by sodium ascorbate (NaAsc). Ratios were determined by integrating the total ion count area of LC-MS spectra.

Studying the reduction products of nBuPt-PNA 369–379 by liquid chromatography–mass spectrometry (LC-MS) demonstrated that Pt­(II) and PNA remained attached upon reduction, a key requirement for a successful chemical modification strategy. After incubation of nBuPt-PNA 369–379 with 300 μM sodium ascorbate in PBS for 20 h, a new species was observed by LC-MS, corresponding to a Pt­(II)–PNA product, albeit in low conversion (Figure B, Figure S3, 9% integrated product peak area). It was hypothesized that the equatorial X-type amide obtained from deprotonation at neutral pH is a more electron-donating ligand than the starting ammine, resulting in a slow rate of reduction. A series of Pt–PNA constructs were synthesized with electron-withdrawing axial ligands to counteract this increase in the electron density at the platinum center. ArPt-PNA 369–379 and ClPt-PNA 369–379 led to significantly more facile reduction, where ClPt-PNA 369–379 was found to be fully reduced to the Pt­(II)–PNA by LC-MS analysis. The observation of the correct mass/charge ratio of the Pt­(II)–PNA, as well as the agreement between the observed and simulated isotopic splitting patterns for a Pt-containing species (Figure S4) provided definitive evidence for the Pt­(II) remaining attached to the PNA upon reduction. This observation supports the notion that these constructs could be used for the targeted platination of nucleic acids.

To confirm RNA functionalization and characterize the resulting products, the reactivity of nBuPt-PNA 369–379 , ArPt-PNA 369–379 , and ClPt-PNA 369–379 with a 21-mer RNA fragment was examined. This fragment represents a condensed segment of interest within EGFP mRNA (Figure A). After incubation of this series of Pt–PNAs with 21-mer RNA, agarose gel electrophoresis of the reaction mixtures displayed two new species. One species was assigned to be the Pt­(IV)–PNA/RNA duplex due to its similar mobility to the Ac–PNA/RNA duplex. The slowest mobility species was attributed to the Pt­(II)–PNA/RNA duplex, as hydrolysis of the equatorial chloride ions of the Pt­(II) would result in a cationic Pt center, leading to a less negative overall charge compared to the corresponding Pt­(IV)-containing duplex (Figure B). Of note, the more rapidly reducing construct (ClPt-PNA 369–379 ) demonstrated significant reactivity toward forming the Pt­(II)–PNA/RNA duplex. While the lower mobility of this species is consistent with the generation of a cationic platinum complex, an important mechanistic question remained as to whether the Pt­(II) reacted with the RNA or generated undesired intramolecular PNA platination products.

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Pt­(IV)–PNAs functionalize RNA nonreversibly. (A) Overall reaction scheme between 21-mer RNA and Pt­(IV)–PNA constructs under reductive conditions. (B) Examination of the reactivity between RNA (5 μM) and Pt­(IV)–PNAs (10 μM) with varying axial leaving groups. Analysis of reaction mixtures by agarose gel electrophoresis consistent with formation of Pt­(II)–PNA/RNA duplex upon reduction of Pt­(IV)–PNA/RNA. Order of rates of reduction determined from Figure B. (C) MALDI-TOF MS of RNA functionalized by ClPt-PNA 369–379 . (D) Melt curve of the Pt­(IV)–PNA/RNA duplex and Pt­(II)–PNA/RNA duplex obtained from UHPLC by varying the column temperature and integration of free RNA at 280 nm.

Evidence for the nonreversible modification of the RNA was obtained by isolation of the Pt­(II)–PNA/RNA product by ultra-high-performance liquid chromatography (UHPLC), and observation of its corresponding mass by matrix assisted laser desorption/ionization–time of flight (MALDI-TOF) MS (Figure C). Control duplexes of Ac-PNA 369–379 /RNA and unreduced ClPt-PNA 369–379 /RNA readily disassociated in the mass spectrometer (Figures S5 and S6), indicating that the observation of the combined mass was due to an interaction between the Pt­(II)–PNA and RNA that is stronger than base-pairing alone. Additional evidence of functionalized RNA was obtained via a melting temperature study using UHPLC, where the melting temperatures of the two duplexes were determined by varying the column temperature and integrating the free RNA peak (measured at 280 nm) observed with increasing temperature (Figure D). This assay demonstrated that the Pt­(II)–PNA/RNA duplex remained stable upon heating to 85 °C, while control duplex Pt­(IV)–PNA/RNA featured a T m of ∼55 °C (Figure S7). It was further substantiated that this behavior resulted from a Pt–RNA coordination complex by extracting the platinum atom from the Pt­(II)–PNA/RNA duplex via treatment with KCN (Figure S8). This method, known for sequestering platinum lesions from nucleic acids, serves to dissociate any Pt–RNA bonds. , After treatment, the resulting PNA/RNA duplex readily dissociated upon heating, supporting the conclusion that Pt­(II) is acting as a linkage between the PNA and RNA. Reactivity was not observed between ClPt-PNA 369–379 and a 15-mer RNA with a scrambled binding region (Figure S9). This lack of functionalization of the mismatched RNA, as well as the selective functionalization of the matched RNA in a competition assay format, demonstrate that the specificity of the PNA is crucial for selective modification of RNA (Figure S10).

Extension of this reactivity to full-length mRNA (996 nucleotides) demonstrated dose-dependent platination consistent with site-directed functionalization and mRNA silencing (Figure ). After incubation of EGFP mRNA with ClPt-PNA 369–379 (0–8 equiv), agarose gel electrophoresis displayed relatively little change in the mRNA mobility across the series (Figure S11). To differentiate platinated mRNA from unmodified mRNA, reverse-transcription was performed on the reaction mixtures. It was hypothesized that the Pt–PNA lesion would serve to halt reverse-transcription, resulting in truncated cDNA/RNA duplexes as compared to unmodified mRNA. Gel electrophoresis of the reverse-transcribed products demonstrated a dose-dependent decrease in full-length cDNA/RNA duplex and the appearance of truncated cDNA/RNA. This result is consistent with site-directed functionalization of the mRNA and demonstrates that the Pt serves as a lesion, effectively modifying the mRNA. Control complexes of the slower reducing nBuPt-PNA 369–379 , Ac-PNA 369–379 , as well as a scramble ClPt-PNA 369–379 demonstrated no mRNA reactivity (Figures S12 and S13), indicating that the synergy between the platinum reactivity and the specificity of the ASO is critical for mRNA functionalization.

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Reverse transcription reveals the functionalization of mRNA by ClPt-PNA 369–379 . aConditions for functionalizing mRNA: EGFP mRNA (100 nM, 1 equiv), ClPt-PNA 369–379 (0–8 equiv), RNase inhibitor (5%), sodium ascorbate (300 μM), 1x PBS, rt, 20 h.

Sanger sequencing was performed on double-stranded cDNA obtained from the truncated mRNA/cDNA duplex to determine the site of modification. This analysis revealed that the site of modification was ∼120 nucleotides downstream of the PNA’s antisense region and the intended site of modification (Figure S14). To further investigate this discrepancy between the predicted and experimentally determined sites of modification, the structure of the PNA-bound mRNA was simulated using mFold (Figure S15). This structure featured an internal stem loop at the target region, where the intended site of modification is base-paired with a region ∼110 nucleotides downstream, corroborating the sequencing results.

The selectivity of the Pt-mediated mRNA silencing was confirmed and quantified by a competition assay between EGFP mRNA (50 pM) and 1000 equiv of RNA. In this assay, target and off-target modification were quantified by RT-qPCR, where modification of housekeeping genes (GAPDH and β-actin) were used as representative off-target RNA. EGFP mRNA was effectively silenced by ClPt-PNA 369–379 (IC50 = 10.1 ± 6.2 nM) with minimal off-target silencing (Figure A). While ClPt-PNA 369–379 demonstrates good reactivity and selectivity in this assay, the construct exhibited poor water solubility.

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Pt­(IV)–ASO constructs selectively functionalize EGFP mRNA in the presence of competing RNA. (A) Effect of Bpep addition and connectivity on reactivity and selectivity. (B) Reactivity of a library of Bpep–[Pt]–ASO constructs. EGFP mRNA (50 pM) and competing RNA (1000 equiv) were incubated with Pt­(IV)–PNA constructs (0–500 nM) in 300 μM sodium ascorbate in PBS for 20 h. For Pt­(IV)–PMO constructs, 5 pM EGFP mRNA and 10,000 equiv of RNA were used due to lower IC50 of Pt­(IV)–PMO constructs. Target (EGFP) vs off-target (β-actin and GAPDH present in competing RNA) modification was measured by RT-qPCR relative to untreated control. Bars represent the standard deviation of the mean based on n = 3 replicates. IC50 and I max values were determined through nonlinear fitting.

To improve the solubility of this construct, Bpep, an arginine rich peptide, was appended to the C-terminus of the PNA. This water-soluble construct (ClPt-PNA 369–379 -Bpep) demonstrated improved activity toward EGFP mRNA knockdown (IC50 = 1.5 ± 0.6 nM) and functionalized off-target genes at higher concentrations (>50 nM). It was hypothesized that the highly cationic Bpep was nonspecifically interacting with RNA, resulting in nonselective reactivity of the Pt­(II) center. To alleviate the off-target effects of the cationic tag, the connectivity of the construct was rearranged to display Bpep on the axial position of the Pt center. This design would take advantage of the inherent reactivity of Pt­(IV) to dissociate its axial ligands upon reduction, thereby decoupling the Pt reactivity from the unselective binding of the Bpep tag. After solution-phase conjugation of 4a to Bpep and subsequent SPAAC reaction with DBCO-PNA, the resulting Bpep-ClPt-PNA 369–379 construct demonstrated superior selectivity for EGFP mRNA, with an IC50 of 4.1 ± 2.4 nM. Importantly, no functionalization of off-target genes was observed with treatment of up to 500 nM construct. Control constructs Ac-PNA 369–379 , Bpep-PNA 369–379 , and Bpep-Pt­(IV) resulted in no mRNA knockdown (Figures S16–S19), further demonstrating that the synergy between the reactivity of platinum and the specificity of the ASO is crucial for mRNA functionalization.

Based on these results, a library of Bpep–Pt–ASOs was synthesized to probe structure–activity relationships (Figure B), with the ASO architecture being the first parameter of examination. Extending the PNA to an 18-mer PNA (Bpep-ClPt-PNA 362–379 ) resulted in a slightly decreased activity, with an IC50 of 8.3 ± 7.8 nM (Figure S20). Modifying the 18-mer PNA to the 18-mer PMO architecture (Bpep-ClPt-PMO 362–379 ) resulted in a significantly improved IC50 of 650 ± 260 pM, while the control compound (Bpep-PMO 362–379 ) exhibited no mRNA functionalization (Figure S21). Importantly, the reactivity of Bpep-ClPt-PMO 362–379 was not significantly impacted by the presence of 1 mM glutathione (Figure S22), a key intracellular reductant and potential competing nucleophile. While the Pt­(IV)–PMO construct demonstrated superior potency for the target sequence than the corresponding Pt­(IV)–PNA, maximum knockdown (I max) of the Pt–PMO construct was significantly lower than that of the Pt–PNA construct (65% vs 84% knockdown, respectively). The effect of the Pt­(II) precursor core structure on reactivity was examined via synthesis of Bpep-CarboPt-PMO 362–379 and Bpep-OxaliPt-PMO 362–379 , although these constructs yielded inferior IC50 and I max values (Figure S23).

A variety of Bpep–ClPt–PMO constructs were synthesized to examine the effect of the mRNA target region on mRNA reactivity. Regions of high binding affinity were selected based on a computational screening approach. , These constructs provided varied IC50 values, with Bpep-ClPt-PMO 191–208 demonstrating an improved IC50 of 420 ± 150 pM. While the range of IC50 values (420 to 3400 pM) obtained by changing the ASO sequence could be explained by an effect of ASO sequence on binding thermodynamics and kinetics, the varying I max (46% to 85%) was not expected (Figures S24 and S25).

To understand the origin of the divergence in I max values, time-dependent reactivity studies of constructs based on PMO178–195, PMO191–208, and PMO362–379 were undertaken. It was observed that the functionalization of mRNA by all constructs followed first-order decay kinetics with varying rate constants (Figure ). These marked differences in rates suggest that the platination of the mRNA by the reduced Pt­(II)–ASO is rate-determining, where the rate of this functionalization is influenced by differences in the microenvironment of the target region in the mRNA, allowing for tuning of the functionalization kinetics. A mechanism where reduction of the Pt­(IV) is rate-determining was rejected by the observation that the reaction is zero-order in reductant (Figure S26). Taken together, the combined proposed mechanism features an initial equilibrium binding event, forming a Pt­(IV)–ASO/mRNA duplex. The Pt­(IV) then undergoes exergonic reduction, furnishing a Pt­(II)–ASO/mRNA duplex. This duplex then undergoes rate-determining cross-linking, resulting in the nonreversible modification of the mRNA.

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Kinetics of EGFP mRNA functionalization by Bpep–ClPt–PMO are affected by the ASO sequence. Functionalization of EGFP mRNA (5 pM) by Pt–PMO constructs (50 nM) in the presence of competing RNA (10,000 equiv) was measured by RT-qPCR (standard conditions from Figure B) relative to t = 0 h. Bars represent the standard deviation of the mean based on n = 3 replicates.

Conclusions

We developed a late-stage functionalization strategy for coupling of carboxylic acids to the ammine position of Pt­(IV)–prodrugs. This reaction was tolerant of numerous coupling partners, including different Pt–cores and varying axial leaving groups. A variety of carboxylic acids could be employed, including those with valuable conjugation handles and bioactive motifs. Functionalized products with pendant azido functional groups could be readily coupled to ASOs via SPAAC to generate Pt­(IV)–ASO constructs, which, after tuning of the reduction rate by varying the axial ligand, could nonreversibly functionalize both short RNA fragments and mRNA. Nonreversible functionalization of nucleic acids was demonstrated via electrophoretic mobility shift assay, temperature-dependent dissociation assay, MALDI-TOF MS, and RT-qPCR.

Pt­(IV)–ASO constructs were selective for the gene of interest in the presence of 1000–10,000 equiv of competing RNA, with an IC50 of 10.1 ± 4.6 nM for the initial construct ClPt-PNA 369–379 . Several rounds of optimization were performed, including (1) addition of Bpep to the axial position of the Pt to solubilize the construct, (2) changing the ASO to a PMO to improve IC50, and (3) optimizing the sequence of the PMO to further improve IC50 and I max. The resulting Bpep-ClPt-PMO 191–208 demonstrated an IC50 of 420 pM with an I max of 85% with no off-target functionalization of competing RNA. Mechanistic studies revealed that the rate-determining step of the reaction was the formation of a coordination complex between the mRNA and the Pt­(II), where the rate of this reaction was strongly affected by the PMO sequence. The Pt­(IV)–ASO platform offers an approach to nonreversibly functionalize nucleic acids, including RNA and mRNA, thereby expanding the chemical space for ASO design and unlocking opportunities in molecular biology research.

Supplementary Material

au5c01369_si_001.pdf (8MB, pdf)

Acknowledgments

We gratefully acknowledge the National Cancer Institute (R01-CA237063) for financial support. E.M. was supported by a postdoctoral fellowship from the Ludwig Center at MIT’s Koch Institute for Integrative Cancer Research. We thank Dr. Peter Mueller for X-ray crystallographic analysis and Dr. Gino Occhialini for assistance with geometry optimization by DFT. We also thank Dr. Simon Rössler, Dr. Nathalie Grob, Ching-Pei Hsu, Dr. Nate Dow, and Dr. Giulio Fittolani for helpful discussions.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacsau.5c01369.

  • General information, experimental procedures, computational methods, and NMR spectra (PDF)

CRediT: Edward Miller conceptualization, data curation, investigation, methodology, software, visualization, writing - original draft, writing - review & editing; Charlotte E. Farquhar formal analysis, methodology, validation, visualization, writing - original draft; Jacob Rodriguez formal analysis, investigation, methodology, validation, visualization, writing - original draft; Andrei Loas data curation, project administration, resources, supervision, writing - original draft, writing - review & editing; Bradley L. Pentelute conceptualization, data curation, formal analysis, funding acquisition, project administration, resources, supervision, writing - original draft, writing - review & editing.

The authors declare no competing financial interest.

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