Crystal Structure of Metallo DNA Duplex Containing Consecutive Watson–Crick‐like T–HgII–T Base Pairs

The metallo DNA duplex containing mercury‐mediated T–T base pairs is an attractive biomacromolecular nanomaterial which can be applied to nanodevices such as ion sensors. Reported herein is the first crystal structure of a B‐form DNA duplex containing two consecutive T–Hg^II–T base pairs. The Hg^II ion occupies the center between two T residues. The N3‐Hg^II bond distance is 2.0 Å. The relatively short Hg^II‐Hg^II distance (3.3 Å) observed in consecutive T–Hg^II–T base pairs suggests that the metallophilic attraction could exist between them and may stabilize the B‐form double helix. To support this, the DNA duplex is largely distorted and adopts an unusual nonhelical conformation in the absence of Hg^II. The structure of the metallo DNA duplex itself and the Hg^II‐induced structural switching from the nonhelical form to the B‐form provide the basis for structure‐based design of metal‐conjugated nucleic acid nanomaterials.     

Donor–Acceptor Heterojunction Configurations Based on DNA–Multichromophore Arrays

Multichromophore arrays of bis(2‐thienyl)diketopyrrolopyrrole (DPP) and naphthalenediimide (NDI) with two Zn^II‐cyclens were constructed using thymidine DNA as a scaffold through the binding of the Zn^II‐cyclens with thymine bases. We demonstrate photocurrent generation in a donor–acceptor heterojunction configuration consisting of the DPP (donor) and NDI (acceptor) arrays co‐immobilized on an Au electrode. The co‐immobilized electrode exhibited good photocurrent responses because of the efficient charge separation between the DPP and NDI arrays. In contrast, an immobilized electrode consisting of randomly assembled DPP‐NDI arrays generated no photocurrent response because DPP formed ground‐state charge‐transfer complexes with NDI in the randomly assembled arrays. Therefore, our approach to generate donor–acceptor heterojunctions based on DNA–multichromophore arrays is a useful method to efficiently generate photocurrent.     

Preparation of Organometallic Ruthenium–Arene–Diaminotriazine Complexes as Binding Agents to DNA

The reactions of two diaminotriazine ligands 2,4‐diamino‐6‐(2‐pyridyl)‐1,3,5‐triazine (2‐pydaT) and 6‐phenyl‐2,4‐diamino‐1,3,5‐triazine (PhdaT) with ruthenium–arene precursors led to a new family of ruthenium(II) compounds that were spectroscopically characterized. Four of the complexes were cationic, with the general formula [(η⁶‐arene)Ru(κ²‐N,N‐2‐pydaT)Cl]X (X=BF₄, TsO; arene=p‐cymene: 1.BF4 , 1.TsO arene=benzene: 2.BF4 , 2.TsO ). The neutral cyclometalated complex [(η⁶‐p‐cymene)Ru(κ²‐C,N‐PhdaT*)Cl] ( 3 ) was also isolated. The structures of complexes 2.BF4 and 3.H2O were determined by X‐ray diffraction. Complex 1.BF4 underwent a partial reversible‐aquation process in water. UV/Vis and NMR spectroscopic measurements showed that the reaction was hindered by the addition of NaCl and was pH‐controlled in acidic solution. At pH 7.0 (sodium cacodylate) Ru–Cl complex 1.BF4 was the only species present in solution, even at low ionic strength. However, in alkaline medium (KOH), complex 1.BF4 underwent basic hydrolysis to afford a Ru–OH complex ( 5 ). Fluorimetric studies revealed that the interaction of complex 1.BF4 with DNA was not straightforward; instead, its main features were closely linked to ionic strength and to the [DNA]/complex ratio. The bifunctional complex 1.BF4 was capable of interacting concurrently through both its p‐cymene and 2‐pydaT groups. Cytotoxicity and genotoxicity studies showed that, contrary to the expected behavior, the complex species was biologically inactive; the formation of a Ru–OH complex could be responsible for such behavior.     

Sequence‐Specific Incorporation of Enzyme–Nucleotide Chimera by DNA Polymerases

DNA polymerases select the right nucleotide for the growing polynucleotide chain based on the shape and geometry of the nascent nucleotide pairs and thereby ensure high DNA replication selectivity. High‐fidelity DNA polymerases are believed to possess tight active sites that allow little deviation from the canonical structures. However, DNA polymerases are known to use nucleotides with small modifications as substrates, which is key for numerous core biotechnology applications. We show that even high‐fidelity DNA polymerases are capable of efficiently using nucleotide chimera modified with a large protein like horseradish peroxidase as substrates for template‐dependent DNA synthesis, despite this “cargo” being more than 100‐fold larger than the natural substrates. We exploited this capability for the development of systems that enable naked‐eye detection of DNA and RNA at single nucleotide resolution.     

Allylic Amination by a DNA–Diene–Iridium(I) Hybrid Catalyst

DNA hybrid catalysis goes organometallic : A DNA strand functionalized with diene ligands forms iridium(I) complexes that can efficiently catalyze an allylic amination in aqueous medium (see scheme). The DNA‐based complexes show high stability and activity, and their secondary structure influences the stereoselectivity of the reaction.

     

Addressable DNA–Myoglobin Photocatalysis

In the spotlight : A hybrid myoglobin, containing a single‐stranded DNA anchor and a redox‐active ruthenium moiety tethered to the heme center can be used as a photocatalyst. The catalyst can be selectively immobilized on a surface‐bound complementary DNA molecule and thus readily recycled from complex reaction mixtures. This principle may be applied to a range of heme‐dependent enzymes allowing the generation of novel light‐triggered photocatalysts.

     

Detecting Counterion Dynamics in DNA–Protein Association

Due to a high density of negative charges on its surface, DNA condenses cations as counterions, forming the so‐called “ion atmosphere”. Although the release of counterions upon DNA–protein association has been postulated to have a major contribution to the binding thermodynamics, this release remains to be confirmed through a direct observation of the ions. Herein, we report the characterization of the ion atmosphere around DNA using NMR spectroscopy and directly detect the release of counterions upon DNA–protein association. NMR‐based diffusion data reveal the highly dynamic nature of counterions within the ion atmosphere around DNA. Counterion release is observed as an increase in the apparent ionic diffusion coefficient, which directly provides the number of counterions released upon DNA–protein association.     

Factors Affecting DNA–DNA Interstrand Cross‐Links in the Antiparallel 3′–3′ Sense: A Comparison with the 5′–5′ Directional Isomer

Reported herein is a study of the unusual 3′–3′ 1,4‐GG interstrand cross‐link (IXL) formation in duplex DNA by a series of polynuclear platinum anticancer complexes. To examine the effect of possible preassociation through charge and hydrogen‐bonding effects the closely related compounds [{trans‐PtCl(NH₃)₂}₂(μ‐trans‐Pt(NH₃)₂{NH₂(CH₂)₆NH₂}₂)]⁴⁺ (BBR3464, 1 ), [{trans‐PtCl(NH₃)₂}₂(μ‐NH₂(CH₂)₆NH₂)]²⁺ (BBR3005, 2 ), [{trans‐PtCl(NH₃)₂}₂(μ‐H₂N(CH₂)₃NH₂(CH₂)₄)]³⁺ (BBR3571, 3 ) and [{trans‐PtCl(NH₃)₂}₂{μ‐H₂N(CH₂)₃‐N(COCF₃)(CH₂)₄}]²⁺ (BBR3571‐COCF₃, 4 ) were studied. Two different molecular biology approaches were used to investigate the effect of DNA template upon IXL formation in synthetic 20‐base‐pair duplexes. In the “hybridisation directed” method the monofunctionally adducted top strands were hybridised with their complementary 5′‐end labelled strands; after 24 h the efficiency of interstrand cross‐linking in the 5′–5′ direction was slightly higher than in the 3′–3′ direction. The second method involved “postsynthetic modification” of the intact duplex; significantly less cross‐linking was observed, but again a slight preference for the 5′–5′ duplex was present. 2D [¹H, ¹⁵N] HSQC NMR spectroscopy studies of the reaction of [¹⁵N]‐ 1 with the sequence 5′‐d{TATACATGTATA}₂ allowed direct comparison of the stepwise formation of the 3′–3′ IXL with the previously studied 5′–5′ IXL on the analogous sequence 5′‐d(ATATGTACATAT)₂. Whereas the preassociation and aquation steps were similar, differences were evident at the monofunctional binding step. The reaction did not yield a single distinct 3′–3′ 1,4‐GG IXL, but numerous cross‐linked adducts formed. Similar results were found for the reaction with the dinuclear [¹⁵N]‐ 2 . Molecular dynamics simulations for the 3′–3′ IXLs formed by both 1 and 2 showed a highly distorted structure with evident fraying of the end base pairs and considerable widening of the minor groove.     

Four‐Dimensional Deoxyribonucleic Acid–Gold Nanoparticle Assemblies

Organization of gold nanoobjects by oligonucleotides has resulted in many three‐dimensional colloidal assemblies with diverse size, shape, and complexity; nonetheless, autonomous and temporal control during formation remains challenging. In contrast, living systems temporally and spatially self‐regulate formation of functional structures by internally orchestrating assembly and disassembly kinetics of dissipative biomacromolecular networks. We present a novel approach for fabricating four‐dimensional gold nanostructures by adding an additional dimension: time. The dissipative character of our system is achieved using exonuclease III digestion of deoxyribonucleic acid (DNA) fuel as an energy‐dissipating pathway. Temporal control over amorphous clusters composed of spherical gold nanoparticles (AuNPs) and well‐defined core–satellite structures from gold nanorods (AuNRs) and AuNPs is demonstrated. Furthermore, the high specificity of DNA hybridization allowed us to demonstrate selective activation of the evolution of multiple architectures of higher complexity in a single mixture containing small and larger spherical AuNPs and AuNRs.     

Redesigning the DNA‐Targeted Chromophore in Platinum–Acridine Anticancer Agents: A Structure–Activity Relationship Study

Platinum–acridine hybrid agents show low‐nanomolar potency in chemoresistant non‐small cell lung cancer (NSCLC), but high systemic toxicity in vivo. To reduce the promiscuous genotoxicity of these agents and improve their pharmacological properties, a modular build–click–screen approach was used to evaluate a small library of twenty hybrid agents containing truncated and extended chromophores of varying basicities. Selected derivatives were resynthesized and tested in five NSCLC cell lines representing large cell, squamous cell, and adenocarcinomas. 7‐Aminobenz[c]acridine was identified as a promising scaffold in a hybrid agent ( P1–B1 ) that maintained submicromolar activity in several of the DNA‐repair proficient and p53‐mutant cancer models, while showing improved tolerability in mice by 32‐fold compared to the parent platinum–acridine ( P1–A1 ). The distribution and DNA/RNA adduct levels produced by the acridine‐ and benz[c]acridine‐based analogues in NCI‐H460 cells (confocal microscopy, ICP‐MS), and their ability to bind G‐quadruplex forming DNA sequences (CD spectroscopy, HR‐ESMS) were studied. P1–B1 emerges as a less genotoxic, more tolerable, and potentially more target‐selective hybrid agent than P1–A1 .     

DNA Reaction–Diffusion Attractor Patterns

Living systems can form and recover complex chemical patterns with precisely sized features in the ranges of tens or hundreds of microns. We show how designed reaction–diffusion processes can likewise produce precise patterns, termed attractor patterns, that reform their precise shape after being perturbed. We use oligonucleotide reaction networks, photolithography, and microfluidic delivery to form precisely controlled attractor patterns and study the responses of these patterns to different localized perturbations. Linear and “hill”‐shaped patterns formed and stabilized into shapes and at time scales consistent with reaction–diffusion models. When patterns were perturbed in particular locations with UV light, they reliably reformed their steady‐state profiles. Recovery also occurred after repeated perturbations. By designing the far‐from‐equilibrium dynamics of a chemical system, this study shows how it is possible to design spatial patterns of molecules that are sustained and regenerated by continually evolving towards a specific steady state configuration.     

Modelling CreA protein–DNA recognition determinants. A molecular dynamics study of fully charged CreA–DNA model in water

CreA, the negative regulator mediating carbon catabolism repression in Aspergillus nidulans, is a protein that contains a DNA-binding domain comprising two zinc finger motifs. A 3D model for the CreA–G4 (5′-GCGGGGGCGT-3′) complex is constructed on the basis of the structure of the Zif268–DNA crystal complex [Science 252 (1991) 809] and using similarity analysis and computer assisted modelling techniques. The CreA–G4 model was then subjected to a set of molecular dynamics (MD) studies. Based on our previous nano second long Zif268–DNA MD simulation, a 170 ps long trajectory was deemed sufficient to test possible DNA–protein interactions. A screening of the static model and of the trajectory was performed for protein amino acids, nucleotide bases, phosphates backbone and water molecules mediating protein–DNA contacts. Energy, root mean square deviation (RMSD), principal inertial moment and distances were analysed. For this time span, the stability shown in fluctuation patterns reveals the presence of a complex behaving in a manner similar to Zif268–DNA.

An unambiguous characterisation of the amino acids involved in DNA-binding was obtained. These results could contribute towards the establishment of a code of protein–DNA recognition for this class of DNA-binding motifs.