A Push–Pull Mechanism Helps Design Highly Competent G-Quadruplex-DNA Catalysts

Open AccessCCS ChemistryRESEARCH ARTICLE1 Aug 2021A Push–Pull Mechanism Helps Design Highly Competent G-Quadruplex-DNA Catalysts Jielin Chen†, Jiawei Wang†, Stephanie C. van der Lubbe†, Mingpan Cheng†, Dehui Qiu, David Monchaud, Jean-Louis Mergny, Célia Fonseca Guerra, Huangxian Ju and Jun Zhou Chen† State Key Laboratory of Analytical Chemistry for Life Science, School Chemical Engineering, Nanjing University, 210023 †J. Chen, J. Wang, S.C.C. Lubbe, M. Cheng contributed equally to this work.Google Scholar More articles by author , Wang† Lubbe† Theoretical Chemistry, Department Pharmaceutical Sciences, AIMMS, Vrije Universiteit Amsterdam, Amsterdam 1081 HV Cheng† Qiu Google Monchaud Institut de Chimie Moléculaire (ICMUB), CNRS, Université Bourgogne Franche-Comté, Dijon 21078 Mergny Guerra *Corresponding authors: E-mail Address: [email protected] Leiden Institute Gorlaeus Laboratories, 2333 CD https://doi.org/10.31635/ccschem.020.202000473 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Massive efforts are currently being invested improve the performance, versatility, scope applications nucleic acid catalysts. G-quadruplex (G4)/hemin DNAzymes particular interest owing their structural programmability chemical robustness. However, optimized catalytic efficiency is still bottleneck activation mechanism unclear. Herein, we have designed a series parallel G4s with different proximal cytosine (dC) derivatives fine-tune hemin-binding pocket G4-DNAzymes. Combining theoretical experimental methods, assessed dependence enhancement on electronic properties dCs demonstrated how activate proficiency. These results provide interesting clues in recapitulating push–pull as basis peroxidase activity help devise new strategy design highly competent DNA catalysts whose performances same order protease. Download figure PowerPoint Introduction The capability acids was first reported study self-splicing RNAs, or ribozymes, Kruger et al.1 Guerriertakada,2 RNA-cleaving enzymes, DNAzymes, Breaker Joyce.3 Afterward, both ribozymes were implemented efficient biodevices fields including biosensing, transformations, biology.4–7 (G4)-DNAzymes represent class which four-stranded G4-DNA interacts cofactor hemin perform hemoenzyme-type reactions.8–12 current popularity G4-DNAzymes stems from versatile designability13–17 excellent biocompatibility G4 precatalysts.18–21 Unfortunately, below those corresponding leading research strategies improved performance. To date, optimization can be divided into two types: addition exogenous activators,22–29 such spermine,22 cytidine triphosphate (CTP),23 adenine (ATP),23–25 template-assembled synthetic G-quartet (TASQ),26 self-assembling peptides27–29 one side, modification structure30–35 more defined suited binding pocket31,32 itself36,37 other side. latter mostly focuses so-called structure, all constitutive strands codirectionally oriented structure displays fairly accessible G-quartets, privileged site.38 Efforts been structures performances, notably modifying sequences flanking external G-quartet,31,34,35,39 well loops bulges core.33 As an example, it that polyadenine polycytosine tails positively impact performance.31,33,35 also better understand mechanisms behind these improvements. Travascio al. UV–vis9 electron paramagnetic resonance (EPR)10 spectroscopies convincing model G4/hemin system. Yamamoto al.36,40 scrutinized coordination iron atom gain reliable insights hemin/G-quartet interactions vibrational nuclear magnetic (NMR) spectroscopies. We others exploited Lewis acid-base theory demonstrate synergistic cooperation between its nucleotides.31,34 take further leap toward elucidation actual G4-DNAzyme mechanism, demonstrating axial dC activates peroxidase-mimicking manner. Our approach nature-driven: cycle horseradish (HRP), central.41–44 shown Supporting Information Figure S1, push effect provided histidine 170 (His170) stabilizes higher oxidation states during catalysis, pull mediated His42 acting together Arg38 facilitates heterolytic O–O bond cleavage iron-bound hydrogen peroxide (the stoichiometric oxidant). Thus, decided modulate density investigate whether extent influences activity. electron-rich strengthen H2O2 boost Compound 0 ? 0* 1 transfer His42-like manner, leads notable enhancements.23,45 Experimental Methods Materials reagents Oligonucleotides, purified HPLC, purchased Sangon Biotech (Shanghai, China) Takara Biomedical Technology (Dalian, China), dissolved ultrapure water (18.2 M?·cm) used without treatment. concentrations oligonucleotides determined UV–vis absorbance at 260 nm, using molar extinction coef?cients OligoAnalyzer 3.1 ( http://sg.idtdna.com/calc/analyzer). About 100 mM Britton–Robinson buffer (B–R buffer) prepared 0.5 M H3BO3, H3PO4, CH3COOH titrated KOH pH 7.0. All experiments performed 10 B–R (pH 7.0 except pH-dependence experiments) containing 0.05% Triton X-100, 0.1% DMSO, KCl. (10 ?M) buffer, K+, heated 95 °C 5 min, cooled slowly room temperature, stored 4 overnight prior use. Hemin stock solution DMSO. 50 2,2?-azino-bis-(3-ethylbenzothiozoline-6-sulfonic acid) (ABTS), ?-nicotinamide dinucleotide (NADH), freshly every time 3,3?,5,5?-tetramethylbenzidine (TMB) chemicals obtained Sigma (St. Louis, USA). Circular dichroism measurements ?M solutions directly diluting buffer. (CD) spectra collected three Chirascan spectrometer (Applied Photophysics, Leatherhead, UK) wavelength range 220–335 nm 25 °C. Activity 0.4 incubated 0.8 7.0) 2 h. After formation complexes, ABTS (0.6 mM, final concentration) added, then variable 0.1, 0.15, 0.2, 0.3, 0.5, 1, 2.5, 5, start reactions, where ABTS·+ 414 monitored 60 s Cary100 (Agilent, Santa Clara, USA) spectrophotometer. confirm DNAzyme activity, substrates, TMB NADH, chosen. TMB·+ 652 NADH 340 recorded s. coefficient 36,000 M?1 cm?1; 39,000 6220 cm?1.9,34 initial rate (V0, nM s?1) reaction calculated slope plot versus Michaelis constant, Km, Michaelis?Menten model. kinetic repeated times, background alone subtracted. examine pH, measured buffers pHs 3.0 8.0. (0.4 h, 0.6 added initiate reaction. Computational settings calculations functional (DFT)-based program Density Functional (ADF) 2017.208 ZORA-BLYP-D3(BJ)/TZ2P level geometric energies.46–51 Geometries implicit chloroform solvation C1 (i.e., without) symmetry constraints because dielectric constant ? (? = 4.8) close experimentally ? 8).52 verified true minima through analysis (zero imaginary frequencies). energies ?E eq 1: ? E dimer ? monomer (1)where Edimer Emonomer dimers monomers, respectively. Full computational details given Information. Voronoi deformation charges atomic charge distribution analyzed (VDD) method.53 VDD method partitions space cells, nonoverlapping regions closer nucleus A than any nucleus. taking fictitious promolecule reference point, simply superposition densities. change cell when going molecular interacting system associated (QVDD). 2: Q ? voronoi [ ? r ) ] d (2)Therefore, instead computing amount contained volume, computed flow upon formation. Therefore, physical interpretation straightforward. positive corresponds loss electrons, whereas negative electrons A. This effectively means N3 atoms lower negative) charge. Results Discussion Proximal derivative designation It now established while may bind G-quartets G4, acquires atop 3?-terminal G-quartet.31,34 located near efficiently enhances G4.34 optimize F3TC (d[T2G3TG3TG3TG3TC]), tailed substituted variety modified nucleobases (Figure 1a). | Schematic illustration G4-DNAzyme. (a) sequence. (b) Different study. assess consequence efficiency, performing dye light readily monitorable output 2a). modulated introducing groups core, changing H5 OH (OH-dC, F3T-hC), bromine (Br-dC, F3T-brC), iodine (I-dC, F3T-iC), CH2OH (5-OH-Me-dC, F3T-hmC), CH3 (Me-dC, F3T-mC), C5 N5 (5-Aza-2?-dC, F3T-azaC), merging additional pyrrole ring (Pyrrolo-dC, F3T-pC) 1b Table S1). S2) confirmed adopt conformation, indicating did not affect overall topology. Peroxidation facilitated H2O2. Electrostatic potential surface (at 0.005 a.u.) ?0.1 (red) 0.1 (blue) a.u. solvation. (c) QVDD (in me–) solvation, theory. (d) Saturation curves catalyzed presence increasing concentration. consequences studied analyzing electrostatic surfaces (EPSs) 2b). EPSs wild-type dC, accumulation centered atom.46–51 quantify differences various derivatives, possess 2c), line EPSs. (QVDD) most pronounced [QVDD ?305 milli-electrons (me?)], least occurs Br-dC I-dC (?289 me?). Me-dC (?303 me?) 5-Aza-2?-dC approximately (?305 Catalytic characterization H2O2-mediated dC-modified via time-dependent characteristic formation9 2d S3). found catalytically proficient F3T (d[T2G3TG3TG3TG3T]), but F3T-azaC (C5 substitution) F3T-mC F3TC. exhibited kcat Km values 25.39 ± 0.63 s?1 7.62 0.28 respectively, 12.68 1.58 4.22 1.17 (Table 1), according S4). 3.4- 1.7-fold (kcat), 1.8- 0.97-fold (Km) F3TC, 84.6- 42.3-fold 4.2- 2.3-fold F3T. value native enzymes HRP, varies 800 depending circumstances.54,55 Of note, under conditions F3T-azaC, HRP 578 S5). Interestingly, derivatives-based showed trend S6). Kinetic Parameters Various Derivatives Respect Catalysis ABTS·+a Sequencesb (mM) Vmax (?M (s?1) kcat/Km (s?1·mM?1) kcat/k0c 1.80 0.41 0.12 0.01 0.30 0.02 0.17 1.0 F3T-pC 3.70 0.48 0.42 0.11 1.4 F3T-hC 1.87 0.24 0.15 0.9 F3T-brC 2.26 0.21 0.69 2.3 F3T-iC 2.17 0.25 0.40 1.01 0.04 0.47 3.4 F3T-hmC 3.47 0.13 1.64 0.03 4.09 0.07 1.18 13.6 4.34 0.20 2.97 0.06 7.44 1.72 24.8 5.07 3.00 42.3 10.15 3.33 84.6 aConditions: 0.1–10 H2O2, ABTS, DNAzyme. bSequences S1. ck0 (0.30 0.02) s?1, Vmax/0.4 ?M. Potential next focused stage G4-DNAzyme, is, conversion [coordination Fe(III) atom] (heterolytic H2O2) ultimately produce (vide infra). steps UV?vis absence substrate Figures S7 S8) oxidative degradation complexes. peaks 404, 504, 630 (Soret bands), decreased rapidly after implying decay intermediate, concomitantly band increase 550–620 650–700 regions, S8).10,31 velocity linear correlation 3a), agreement involvement intermediates. exception too fast reliably quantified. 3 Correlation (V0) Note: record, deviation observed one. pHs. Next, produced [which becomes Fe(IV)], porphyrin ring. active Fe(IV) rate-determining step catalysis. In promoted pKa 2.5 (His42.H+/His42), suitable assist since hemin-bound H2O2/HO2? 3.2 4.0. G4-DNAzymes, replaced 4.1 (CTP.H+/CTP), compatible transfer.23 evaluated 8.0 S9). proficiency sensitive F3T-mC, F3T-hmC, relatively high plateaus 5.0 3b). apparent 4.2 4.4 4.5 6.4 (between ?300 me?, confirming induce pKa, making harder deprotonated. thus mimicking role HRP. interaction investigated DFT computations solvation.46–51 energy (?E) ranged ?10.7 ?11.5 kcal·mol?1 4a). For dCs, 4b), simple electronically driven dCs. disconnections Pyrrolo-dC, OH-dC, 5-Aza-2?-dC. (?294 (?10.9 kcal·mol?1) comparison 21.0 s?1. probably caused NH2 group NH incorporated another ring, affected size OH-dC displayed stable) (?11.2 (?296 anticipated, ?303 me? less suggested (682.6 s?1). Optimized structures, kcal·mol–1), N?O distances Å) (red columns) (black line) (blue line). discrepancies V0 explained unique form (H) bonds faces. (with ?E) coordinate (Figures 5a S10), low S11). secondary face. H2O2-binding N3-face (?10.7 only 1.1-fold N5-face (?9.5 kcal·mol?1, S10c), primary face 2.1-fold (?5.4 S11a). equivalence faces explains why nonequivalent OH-dC. addition, involved extended H-bond network surrounding H2O molecules 5b S11b S11c). influence H (two acceptor, donor) density. resulted ?296 ?289 S11c S11d), rationale F3T-hC. representation double formed H2O. Details S10 S11. Blue spheres Fe(III). From point view, indicate must consider possible bias (e.g., nucleobase rotation) environmental effects hydration) accurate predictive models cycles. they cast bright brings ever hemoenzymes thanks quite unique, double, H2O2-coordination site. Overall, based calculations, G4-based DNAzyme, finally continued verify construction our mechanism. has thoroughly studied, central (His) residues, His170, site synergistically interact peroxidation reactions S1).41–44 His, sides hemin, His170 direct atom, distal oxidant stacks plays yet fully understood,30 played 6). Here, surrogates, resulting systems. could controlled introduction either electron-donating electron-withdrawing groups. led (F3T-mC F3T-azaC) 5-Aza-2?-dC, representative examples exhibiting intricate relationship calculations: former, predictions results, slightly stable performances. originated Me-dC. latter, highlighted care taken trying recapitulate silico, bias. indeed multiple anchoring points bolstered second round computation accounted. 6 red Fe(IV), Conclusion Globally speaking, strongly impacted fostering 1. key electron-transfer process, HRP-like combination experiment fine mechanistic inner workings. Each computed, opened broad perspectives computer-aided