“Printing” DNA Strand Patterns on Small Molecules with Control of Valency,Directionality, and Sequence

The incorporation of synthetic molecules as corner units in DNA structures has been of interest over the last two decades. In this work, we present a facile method for generating branched small molecule‐DNA hybrids with controllable valency, different sequences, and directionalities (5′–3′) using a “printing” process from a simple 3‐way junction structure. We also show that the DNA‐imprinted small molecule can be extended asymmetrically using polymerase chain reaction (PCR) and can be replicated chemically. This strategy provides opportunities to achieve new structural motifs in DNA nanotechnology and introduce new functionalities to DNA nanostructures.     

“Post‐It” Type Connected DNA Created with a Reversible Covalent Cross‐Link

We report the development of a new heterobase that is held together through reversible bonding. The so‐formed cross‐link adds strong stabilization to the DNA duplex. Despite this, the cross‐link opens and closes through reversible imine bonding. Moreover, even enzymatic incorporation of the cross‐link is possible. The new principle can be used to stabilize DNA duplexes and nanostructures that otherwise require high salt concentrations, which may hinder biological applications.     

The Thiophene “Sigma‐Hole” as a Concept for Preorganized,Specific Recognition of G⋅C Base Pairs in the DNA Minor Groove

In spite of its importance in cell function, targeting DNA is under‐represented in the design of small molecules. A barrier to progress in this area is the lack of a variety of modules that recognize G ? C base pairs (bp) in DNA sequences. To overcome this barrier, an entirely new design concept for modules that can bind to mixed G ? C and A ? T sequences of DNA is reported herein. Because of their successes in biological applications, minor‐groove‐binding heterocyclic cations were selected as the platform for design. Binding to A ? T sequences requires hydrogen‐bond donors whereas recognition of the G‐NH₂ requires an acceptor. The concept that we report herein uses pre‐organized N‐methylbenzimidazole (N‐MeBI) thiophene modules for selective binding with mixed bp DNA sequences. The interaction between the thiophene sigma hole (positive electrostatic potential) and the electron‐donor nitrogen of N‐MeBI preorganizes the conformation for accepting an hydrogen bond from G‐NH₂. The compound–DNA interactions were evaluated with a powerful array of biophysical methods and the results show that N‐MeBI‐thiophene monomer compounds can strongly and selectively recognize single G ? C bp sequences. Replacing the thiophene with other moieties significantly reduces binding affinity and specificity, as predicted by the design concept. These results show that the use of molecular features, such as sigma‐holes, can lead to new approaches for small molecules in biomolecular interactions.     

A Photo‐responsive Small‐Molecule Approach for the Opto‐epigenetic Modulation of DNA Methylation

Controlling the functional dynamics of DNA within living cells is essential in biomedical research. Epigenetic modifications such as DNA methylation play a key role in this endeavour. DNA methylation can be controlled by genetic means. Yet there are few chemical tools available for the spatial and temporal modulation of this modification. Herein, we present a small‐molecule approach to modulate DNA methylation with light. The strategy uses a photo‐tuneable version of a clinically used drug (5‐aza‐2′‐deoxycytidine) to alter the catalytic activity of DNA methyltransferases, the enzymes that methylate DNA. After uptake by cells, the photo‐regulated molecule can be light‐controlled to reduce genome‐wide DNA methylation levels in proliferating cells. The chemical tool complements genetic, biochemical, and pharmacological approaches to study the role of DNA methylation in biology and medicine.     

Distinguishing Individual DNA Bases in a Network by Non‐Resonant Tip‐Enhanced Raman Scattering

The importance of identifying DNA bases at the single‐molecule level is well recognized for many biological applications. Although such identification can be achieved by electrical measurements using special setups, it is still not possible to identify single bases in real space by optical means owing to the diffraction limit. Herein, we demonstrate the outstanding ability of scanning tunneling microscope (STM)‐controlled non‐resonant tip‐enhanced Raman scattering (TERS) to unambiguously distinguish two individual complementary DNA bases (adenine and thymine) with a spatial resolution down to 0.9 nm. The distinct Raman fingerprints identified for the two molecules allow to differentiate in real space individual DNA bases in coupled base pairs. The demonstrated ability of non‐resonant Raman scattering with super‐high spatial resolution will significantly extend the applicability of TERS, opening up new routes for single‐molecule DNA sequencing.     

One‐Step Formation of “Chain‐Armor”‐Stabilized DNA Nanostructures

DNA‐based self‐assembled nanostructures are widely used to position organic and inorganic objects with nanoscale precision. A particular promising application of DNA structures is their usage as programmable carrier systems for targeted drug delivery. To provide DNA‐based templates that are robust against degradation at elevated temperatures, low ion concentrations, adverse pH conditions, and DNases, we built 6‐helix DNA tile tubes consisting of 24 oligonucleotides carrying alkyne groups on their 3′‐ends and azides on their 5′‐ends. By a mild click reaction, the two ends of selected oligonucleotides were covalently connected to form rings and interlocked DNA single strands, so‐called DNA catenanes. Strikingly, the structures stayed topologically intact in pure water and even after precipitation from EtOH. The structures even withstood a temperature of 95 °C when all of the 24 strands were chemically interlocked.     

Simultaneously Sizing and Quantitating Zeptomole‐Level DNA at High Throughput in Free Solution

Determining the sizes and measuring the quantities of DNA molecules are fundamental tasks in molecular biology. DNA sizes are usually evaluated by gel electrophoresis, but this method cannot simultaneously size and quantitate a DNA at low zeptomole (zmol) levels of concentration. We have recently developed a new technique, called bare‐narrow‐capillary/hydrodynamic‐chromatography or BaNC‐HDC, for resolving DNA based on their sizes without using any sieving matrices. In this report, we utilize BaNC‐HDC for measuring the sizes and quantities of DNA fragments at zmol to several‐molecule levels of concentration. DNA ranging from a few base pairs to dozens of kilo base pairs are accurately sized and quantitated at a throughput of 15 samples per hour, and each sample contains dozens of DNA strands of different lengths. BaNC‐HDC can be a cost‐effective means and an excellent tool for high‐throughput DNA sizing and quantitation at extremely low quantity level.     

Single‐Molecule Mechanochemical Sensing Using DNA Origami Nanostructures

While single‐molecule sensing offers the ultimate detection limit, its throughput is often restricted as sensing events are carried out one at a time in most cases. 2D and 3D DNA origami nanostructures are used as expanded single‐molecule platforms in a new mechanochemical sensing strategy. As a proof of concept, six sensing probes are incorporated in a 7‐tile DNA origami nanoassembly, wherein binding of a target molecule to any of these probes leads to mechanochemical rearrangement of the origami nanostructure, which is monitored in real time by optical tweezers. Using these platforms, 10 pM platelet‐derived growth factor (PDGF) are detected within 10 minutes, while demonstrating multiplex sensing of the PDGF and a target DNA in the same solution. By tapping into the rapid development of versatile DNA origami nanostructures, this mechanochemical platform is anticipated to offer a long sought solution for single‐molecule sensing with improved throughput.     

Enzymatic Synthesis,Amplification, and Application of DNA with a Functionalized Backbone

The ability to amplify DNA along with its unprecedented sequence control has led to its use for different applications, but all are limited by the properties available to natural nucleotides. We previously reported the evolution of polymerase SFM4‐3, which better tolerates 2′‐modified substrates. To explore the utility of SFM4‐3, we now report the characterization of its recognition of substrates with 2′‐azido, 2′‐chloro, 2′‐amino, or arabinose sugars. We find that SFM4‐3 can efficiently synthesize polymers composed of these nucleotides, and most interestingly, that SFM4‐3 can also PCR amplify these modified oligonucleotides. When combined with post‐amplification modification, the latter allows for the exponential amplification of polymers that may be functionalized with desired moieties arrayed in a controlled fashion, the utility of which we demonstrate with extensive small molecule functionalization and the production and initial characterization of a novel DNA hydrogel.     

Alpha‐Helical Fragaceatoxin C Nanopore Engineered for Double‐Stranded and Single‐Stranded Nucleic Acid Analysis

Nanopores are used in single‐molecule DNA analysis and sequencing. Herein, we show that Fragaceatoxin C (FraC), an α‐helical pore‐forming toxin from an actinoporin protein family, can be reconstituted in sphingomyelin‐free standard planar lipid bilayers. We engineered FraC for DNA analysis and show that the funnel‐shaped geometry allows tight wrapping around single‐stranded DNA (ssDNA), resolving between homopolymeric C, T, and A polynucleotide stretches. Remarkably, despite the 1.2 nm internal constriction of FraC, double‐stranded DNA (dsDNA) can translocate through the nanopore at high applied potentials, presumably through the deformation of the α‐helical transmembrane region of the pore. Therefore, FraC nanopores might be used in DNA sequencing and dsDNA analysis.     

Increasing the Sensitivity and Single‐Base Mismatch Selectivity of the Molecular Beacon Using Graphene Oxide as the “Nanoquencher”

Here, we report a novel, highly sensitive, selective and economical molecular beacon using graphene oxide as the “nanoquencher”. This novel molecular beacon system contains a hairpin‐structured fluorophore‐labeled oligonucleotide and a graphene oxide sheet. The strong interaction between hairpin‐structured oligonucleotide and graphene oxide keep them in close proximity, facilitating the fluorescence quenching of the fluorophore by graphene oxide. In the presence of a complementary target DNA, the binding between hairpin‐structured oligonucleotide and target DNA will disturb the interaction between hairpin‐structured oligonucleotide and graphene oxide, and release the oligonucleotide from graphene oxide, resulting in restoration of fluorophore fluorescence. In the present study, we show that this novel graphene oxide quenched molecular beacon can be used to detect target DNA with higher sensitivity and single‐base mismatch selectivity compared to the conventional molecular beacon.     

Universal pH‐Responsive and Metal‐Ion‐Free Self‐Assembly of DNA Nanostructures

pH‐responsiveness has been widely pursued in dynamic DNA nanotechnology, owing to its potential in biosensing, controlled release, and nanomachinery. pH‐triggering systems mostly depend on specific designs of DNA sequences. However, sequence‐independent regulation could provide a more general tool to achieve pH‐responsive DNA assembly, which has yet to be developed. Herein, we propose a mechanism for dynamic DNA assembly by utilizing ethylenediamine (EN) as a reversibly chargeable (via protonation) molecule to overcome electrostatic repulsions. This strategy provides a universal pH‐responsivity for DNA assembly since the regulation originates from externally co‐existing EN rather than specific DNA sequences. Furthermore, it endows structural DNA nanotechnology with the benefits of a metal‐ion‐free environment including nuclease resistance. The concept could in principle be expanded to other organic molecules which may bring unique controls to dynamic DNA assembly.     

Using the Population‐Shift Mechanism to Rationally Introduce “Hill‐type” Cooperativity into a Normally Non‐Cooperative Receptor

Allosteric cooperativity, which nature uses to improve the sensitivity with which biomolecular receptors respond to small changes in ligand concentration, could likewise be of use in improving the responsiveness of artificial biosystems. Thus motivated, we demonstrate here the rational design of cooperative molecular beacons, a widely employed DNA sensor, using a generalizable population‐shift approach in which we engineer receptors that equilibrate between a low‐affinity state and a high‐affinity state exposing two binding sites. Doing so we achieve cooperativity within error of ideal behavior, greatly steepening the beacon’s binding curve relative to that of the parent receptor. The ability to rationally engineer cooperativity should prove useful in applications such as biosensors, synthetic biology and “smart” biomaterials, in which improved responsiveness is of value.     

Binding‐Induced DNA Nanomachines Triggered by Proteins and Nucleic Acids

We introduce the concept and operation of a binding‐induced DNA nanomachine that can be activated by proteins and nucleic acids. This new type of nanomachine harnesses specific target binding to trigger assembly of separate DNA components that are otherwise unable to spontaneously assemble. Three‐dimensional DNA tracks of high density are constructed on gold nanoparticles functionalized with hundreds of single‐stranded oligonucleotides and tens of an affinity ligand. A DNA swing arm, free in solution, is linked to a second affinity ligand. Binding of a target molecule to the two ligands brings the swing arm to AuNP and initiates autonomous, stepwise movement of the swing arm around the AuNP surface. The movement of the swing arm, powered by enzymatic cleavage of conjugated oligonucleotides, cleaves hundreds of oligonucleotides in response to a single binding event. We demonstrate three nanomachines that are specifically activated by streptavidin, platelet‐derived growth factor, and the Smallpox gene. Substituting the ligands enables the nanomachine to respond to other molecules. The new nanomachines have several unique and advantageous features over DNA nanomachines that rely on DNA self‐assembly.     

Toehold‐Mediated Displacement of an Adenosine‐Binding Aptamer from a DNA Duplex by its Ligand

DNA is increasingly used to engineer dynamic nanoscale circuits, structures, and motors, many of which rely on DNA strand‐displacement reactions. The use of functional DNA sequences (e.g., aptamers, which bind to a wide range of ligands) in these reactions would potentially confer responsiveness on such devices, and integrate DNA computation with highly varied molecular stimuli. By using high‐throughput single‐molecule FRET methods, we compared the kinetics of a putative aptamer–ligand and aptamer–complement strand‐displacement reaction. We found that the ligands actively disrupted the DNA duplex in the presence of a DNA toehold in a similar manner to complementary DNA, with kinetic details specific to the aptamer structure, thus suggesting that the DNA strand‐displacement concept can be extended to functional DNA–ligand systems.     

An Autonomous Bio‐barcode DNA Machine for Exponential DNA Amplification and Its Application to the Electrochemical Determination of Adenosine Triphosphate

A novel autonomous bio‐barcode DNA machine that is driven by template‐dependent DNA replication is developed to exponentially amplify special DNA sequences. Combined with a DNA aptamer recognition element, the DNA machine can be further applied in the aptamer‐based, amplified analysis of small molecules. As a model analyte, adenosine triphosphate (ATP) is determined by using the DNA machine system in combination with a DNA aptamer recognition strategy and differential pulse anodic stripping voltammetry (DPASV). Under the optimum conditions, detection limits as low as 2.8×10^?17 M (3σ) for target DNA and 4.7×10^?9 M (3σ) for ATP are achieved. The satisfactory determination of ATP in K562 leukemia cell and Ramos Burkitt’s lymphoma cell reveal that this protocol possesses good selectivity and practicality. As a promising biomolecular device, this DNA machine may have an even broader application in the rapidly developing field of nanobiotechnology.     

A Programmed DNA Marker Based on Bis(4‐ethynyl‐1,8‐naphthalimide) and Three‐Methane‐Bridged Thiazole Orange

Two large conjugated naphthalimide derivatives with or without three‐methane‐bridged thiazole orange (TO3; i.e., compounds 1 a and 2 a , respectively) were designed and synthesized. The fluorescence of the naphthalimide group in compound 1 a at λ=532 nm initially decreased and that for the TO3 group at λ=655 nm increased sequentially upon adding Salmon testes (St) DNA. In contrast, without the TO3 group, the fluorescence intensity of compound 2 a monotonously decreased in response to the addition of DNA. The non‐monotonic change in the fluorescence for compound 1 a could be divided into two linear sections with two different wavelengths in the range of 0<R_{b/ 1 a} <1.2 and 1.2 <R_{b/ 1 a} <6.0 (R_{b/ 1 a} =[base pair]/[ 1 a ]). Thus, compound 1 a can be regarded as a programmed responding molecule for DNA, which can semi‐quantitatively determine the concentration of DNA over a large concentration range from the standard fluorescence curve of compound 1 a at different wavelengths when bound with DNA. Furthermore, the binding modes of compounds 1 a and 2 a with StDNA were studied by using CD spectroscopy and melting temperature (T_m) testing. The results showed that compound 1 a interacts with StDNA through multi‐interactions including weak intercalation, weak minor groove binding, and inter‐dye interactions, whereas compound 2 a bound with DNA through simultaneous intercalation and minor groove binding.     

Single Conducting Polymer Nanowire Based Sequence‐Specific,Base‐Pair‐Length Dependant Label‐free DNA Sensor

We have fabricated a highly sensitive, simple and label‐free single polypyrrole (Ppy) nanowire based conductometric/chemiresistive DNA sensor. The fabrication was optimized in terms of probe DNA sequence immobilization using a linker molecule and using gold‐thiol interaction. Two resultant sensor designs working on two different sensing mechanisms (gating effect and work function based sensors) were tested to establish reliable sensor architecture with higher sensitivity and device‐to‐device reproducibility. The utility of the work function based configuration was demonstrated by detecting 19 base pair (bp) long breast cancer gene sequence with single nucleotide polymorphism (SNP) discrimination with high sensitivity, lower detection limit of ∼10⁻¹⁶ M and wide dynamic range (∼10⁻¹⁶ to 10⁻¹¹ M) in a small sample volume (30 µL). To further demonstrate the utility of the DNA sensor for detection of target sequences with different number of bases, targets with 21 and 36 bases were detected. These sequences have implications in environmental sample analysis or metagenomics. Sensor response showed increase with the number of bases in the target sequence. For long sequence (with 36 bases), effect of DNA alignment on sensor performance was studied.     

Determination of genomic N3‐methylthymidine in human cancer cells treated with nitrosamines using capillary electrophoresis with laser‐induced fluorescence

Methylating substances alter DNA by forming N3‐methylthymidine (N3mT), a mutagenic base modification. To develop a sensitive analytical method for the detection of N3mT in DNA based on capillary electrophoresis with laser‐induced fluorescence detection (CE‐LIF), we synthesized the N3mT‐3’‐phosphate as a chemical standard. The limit of detection was 1.9 amol of N3mT, which corresponds to one molecule of N3mT per 1000 normal nucleotides or 0.1%. With this method, we demonstrated that the carcinogenic nitrosamine N’‐nitrosonornicotine (NNN) induced N3mT in the human lung cancer cell line A549. Treatment with NNN also caused an elevated degree of 5‐hydroxymethylcytidine (5hmdC) in DNA, while the methylation degree (i.e. 5‐methylcytidine; 5mdC) stayed constant. According to our data, NNN could, via yet unknown mechanisms, play a role in the formation of N3mT as well as 5hmdC. In this study we have developed a new sensitive analytical method using CE‐LIF for the simultaneous detection of the three DNA modifications, 5mdC, 5hmdC and N3mT.     

Crystallization of DNA‐Capped Gold Nanoparticles in High‐Concentration,Divalent Salt Environments

The multiparametric nature of nanoparticle self‐assembly makes it challenging to circumvent the instabilities that lead to aggregation and achieve crystallization under extreme conditions. By using non‐base‐pairing DNA as a model ligand instead of the typical base‐pairing design for programmability, long‐range 2D DNA–gold nanoparticle crystals can be obtained at extremely high salt concentrations and in a divalent salt environment. The interparticle spacings in these 2D nanoparticle crystals can be engineered and further tuned based on an empirical model incorporating the parameters of ligand length and ionic strength.