General Label-Free Fluorescent Aptamer Binding Assay Using Cationic Conjugated Polymers.

With more and more new aptamers being reported, a general, cost-effective yet reliable aptamer binding assay is still needed. Herein, we studied cationic conjugated polymer (CCP)-based binding assays taking advantage of the conformational change of aptamer after binding with a target, which is reflected by the fluorescence change of the CCP. Poly(3-(3'-N,N,N-triethylamino-1'-propyloxy)-4-methyl-2,5-thiophene hydrochloride) (PMNT) was used as a model CCP in this study, and the optimal buffer was close to physiological conditions with 100 mM NaCl and 10 mM MgCl2. We characterized four aptamers for K+, adenosine, cortisol, and caffeine. For cortisol and caffeine, the drop in the 580 nm peak intensity was used for quantification, whereas for K+ and adenosine, the fluorescence ratio at 580 over 530 nm was used. The longer stem of the stem-loop structured aptamer facilitated binding of the target and enlarged the detection signal. High specificity was achieved in differentiating targets with analogues. Compared with the SYBR Green I dye-based staining method, our method achieved equal or even higher sensitivity. Therefore, this assay is practicable as a general aptamer binding assay. The simple, label-free, quick response, and cost-effective features will make it a useful method to evaluate aptamer binding. At the same time, this system can also serve as label-free biosensors for target detection.

Using the Intrinsic Fluorescence of DNA to Characterize Aptamer Binding.

The reliable, readily accessible and label-free measurement of aptamer binding remains a challenge in the field. Recent reports have shown large changes in the intrinsic fluorescence of DNA upon the formation of G-quadruplex and i-motif structures. In this work, we examined whether DNA intrinsic fluorescence can be used for studying aptamer binding. First, DNA hybridization resulted in a drop in the fluorescence, which was observed for A30/T30 and a 24-mer random DNA sequence. Next, a series of DNA aptamers were studied. Cortisol and Hg2+ induced fluorescence increases for their respective aptamers. For the cortisol aptamer, the length of the terminal stem needs to be short to produce a fluorescence change. However, caffeine and adenosine failed to produce a fluorescence change, regardless of the stem length. Overall, using the intrinsic fluorescence of DNA may be a reliable and accessible method to study a limited number of aptamers that can produce fluorescence changes.

Nanomaterial and Aptamer-Based Sensing: Target Binding versus Target Adsorption Illustrated by the Detection of Adenosine and ATP on Metal Oxides and Graphene Oxide.

Target molecule-induced desorption of aptamer probes from nanomaterials has been a very popular sensing method, taking advantage of the fluorescence quenching or catalytic activity of nanomaterials for signal generation. While it is generally conceived that aptamers desorb due to binding to target molecules, in this work, we examined the effect of competitive target adsorption. From five metal oxide nanoparticles including CeO2, ZnO, NiO, Fe3O4, and TiO2, only ATP was able to induce desorption of its aptamer. Adenosine could not, even though it had an even higher affinity than ATP to the aptamer. The same conclusion was also observed with a random DNA that cannot bind ATP, indicating that the desorption of DNA was due to competitive adsorption of ATP instead of aptamer binding. On graphene oxide, however, adenosine produced slightly more aptamer desorption than ATP under most of the conditions, and this can be partially attributed to the weaker interaction of negatively charged ATP with negatively charged graphene oxide. For such surface-based biosensors, it is recommended that a nonaptamer control DNA be tested side-by-side to ensure the sensing mechanism to be related to aptamer binding instead of target adsorption.

Promotion and Inhibition of the Oxidase-Mimicking Activity of Nanoceria by Phosphate, Polyphosphate, and DNA.

Nanoceria (CeO2 nanoparticles) is an extensively studied nanozyme with interesting oxidase-mimicking activity. As they can work in the absence of toxic and unstable H2 O2 , CeO2 nanoparticles have been widely used in biosensing. CeO2 nanoparticles often encounter phosphate-containing molecules that can affect their catalytic activity, and various reports exist in the literature showing both promoted and inhibited activity. In this work, we systematically studied five types of phosphate: orthophosphate, pyrophosphate, triphosphate, trimetaphosphate, and a polyphosphate with 25 phosphate units (Pi25 ). In addition, DNA oligonucleotides of various length and sequence. DNA was included as they contain a phosphate backbone that can strongly adsorb on nanoceria. We observed that a high concentration of DNA in acetate buffer inhibited activity, whereas a low concentration of DNA in phosphate buffer increased activity. The change of activity was also related to the type of substrate and related to the aggregation of CeO2 . These discoveries provide an important understanding for the further use of CeO2 nanoparticles in biosensor development, materials science, and nanotechnology.

Opposite salt-dependent stability of i-motif and duplex reflected in a single DNA hairpin nanomachine.

We herein report a DNA hairpin structure containing a polycytosine loop region, and this hairpin can operate like a nanomachine allowing independently controlled stability of the i-motif loop and duplex stem region. This was made possible by the opposite salt-dependent stability of DNA duplex and hairpin, thus providing a new method for designing molecular devices or switches design. A singly-labeled fluorescent method was used to measure the stability of an i-motif DNA in the presence of various metal ions. Salt in general destabilizes the i-motif but stabilizes duplex DNA, allowing us to engineer an i-motif containing hairpin for modulating the stability of each secondary structure independently.

Fluorescence Polarization for Probing DNA Adsorption by Nanomaterials and Fluorophore/DNA Interactions.

Fluorescence polarization (FP) is attractive for measuring binding interactions and has been recently used to study DNA adsorption on nanomaterials. Since most nanomaterials are strong fluorescence quenchers, correlations among adsorption efficiency, quenching efficiency, and FP need to be interpreted carefully. In this work, carboxyfluorescein (FAM)-labeled DNA oligonucleotides were studied under various quenching conditions. First, quenching was induced by lowering the pH, taking advantage of the fact that FAM is almost nonfluorescent at a pH below 4. Strong interactions were observed between the FAM label and polyadenine DNA, as judged by the increased FP at low pH, while FAM-labeled polythymine DNA was less affected by the pH. Comparisons were also performed with FAM-labeled poly(ethylene glycol) and bovine serum albumin. An equation was derived to calculate the effect of fluorescence quenching and DNA adsorption by nanomaterials. For strongly quenching nanomaterials, such as graphene oxide, DNA adsorption alone does not change the measured FP. Light scattering and weak fluorescence from graphene oxide increase FP in these cases. For comparison, a strongly adsorbing but weak quenching material, Y2O3, was also studied and the result was consistent with a normal binding reaction. Overall, FP is a powerful technique for binding and adsorption assays, but quenched samples need to be interpreted with care.

Fluorescein-Stabilized i-Motif DNA and Its Unfolding Leading to a Stronger Adsorption Affinity.

Several previous studies have indicated that polydeoxycytidine (poly-C) DNA has an anomalously high affinity for different types of surfaces. It was hypothesized that the formation of an i-motif structure could be a factor responsible for this enhanced affinity, but this is against the notion that a folded molecule should have fewer interactions with a surface. Herein, the properties of poly-C DNA were examined in detail, focusing on the presence or absence of a FAM (carboxyfluorescein) label and its subsequent adsorption on graphene oxide. Fluorescence and CD spectroscopy studies indicated that FAM can stabilize an i-motif structure in C15 DNA. In particular, the fluorescence of FAM is drastically quenched when the DNA is folded. This structure is irreversibly unfolded upon heating. Furthermore, the unfolded structure has an even higher affinity for graphene oxide than the folded structure. Finally, a large portion of the folded C15 unfolds upon desorption from graphene oxide, and unfolding could happen upon adsorption or desorption of the DNA. This study provides a method to further enhance the adsorption stability of poly-C DNA and calls for care when investigating the potential effects of dye labels on DNA.

DNA Oligonucleotide-Functionalized Liposomes: Bioconjugate Chemistry, Biointerfaces, and Applications.

Interfacing DNA with liposomes has produced a diverse range of programmable soft materials, devices, and drug delivery vehicles. By simply controlling liposomal composition, bilayer fluidity, lipid domain formation, and surface charge can be systematically varied. Recent development in DNA research has produced not only sophisticated nanostructures but also new functions including ligand binding and catalysis. For noncationic liposomes, a DNA is typically covalently linked to a hydrophobic or lipid moiety that can be inserted into lipid membranes. In this article, we discuss fundamental biointerfaces formed between DNA and noncationic liposomes. The methods to prepare such conjugates and the interactions at the membrane interfaces are also discussed. The effect of DNA lateral diffusion on fluid bilayer membranes and the effect of membrane on DNA assembly are emphasized. DNA hybridization can be programmed to promote fusion of lipid membranes. Representative applications of this conjugate for drug delivery, biosensor development, and directed assembly of materials are briefly described toward the end. Some future research directions are also proposed to further understand this biointerface.

Adsorption of Phosphate and Polyphosphate on Nanoceria Probed by DNA Oligonucleotides.

Phosphate-containing molecules exist in many forms in biology and the environment, and their interaction with metal oxides is an important aspect of their chemistry and biochemistry. In this work, phosphates with different degrees of polymerization (e.g., orthophosphate, pyrophosphate (PPi), sodium triphosphate (STPP), sodium trimetaphosphate (STMP), and polyphosphate with 25 phosphate units) and phosphates with one or two capping groups were studied. CeO2 nanoparticles (nanoceria) were used as a model metal oxide. DNA is also a polyphosphate, and a fluorescently labeled DNA oligonucleotide was mixed with nanoceria. These phosphate species were individually added to displace the adsorbed DNA. Longer phosphate chains were more efficient when each molecule was used at the same molar concentration, whereas PPi and STPP were most efficient at the same total phosphorus atom concentration. By capping the phosphate with organic groups, the affinity was significantly decreased. Isothermal titration calorimetry (ITC) was also performed to quantitatively measure thermodynamic parameters. Although STMP was very slow at displacing DNA, it was still adsorbed very strongly by nanoceria from ITC, indicating kinetic effects likely due to its ring structure. This observation allowed us to use the DNA as a probe to study the hydrolysis of STMP to form STPP. In summary, this study provides a systematic understanding of phosphate species interacting with metal oxides, and interestingly, it demonstrates an analytical application as well.

Interfacing DNA Oligonucleotides with Calcium Phosphate and Other Metal Phosphates.

Calcium phosphate (CaP) has long been used for DNA delivery, although its fundamental interaction with DNA, especially with single-stranded DNA oligonucleotides, remains to be fully understood. Using fluorescently labeled oligonucleotides, we herein studied DNA adsorption isotherm and the effect of DNA length and sequence. Longer DNAs are adsorbed more strongly, and at neutral pH, poly-C DNAs are adsorbed more than the other three DNA homopolymers. However, at near pH 11, the pH of CaP synthesis, T30 DNA is adsorbed more strongly than C30 or A30. This can explain why T30 and G30 can fully inhibit the growth of CaP, while A30 and C30 only retarded its growth kinetics. DNA adsorption also reduces aggregation of CaP. DNA desorption experiments were carried out using concentrated urea, thymidine, or inorganic phosphate as competitors, and desorption was observed only in the presence of phosphate, suggesting that DNA uses its phosphate backbone to interact with the CaP surface. Desorption was also promoted by raising the NaCl concentration suggesting the electrostatic nature of interaction. Finally, ten different metal phosphate materials were synthesized by co-precipitating each metal ion (Ce3+, Fe3+, Ca2+, Ni2+, Zn2+, Mn2+, Ba2+, Cu2+, Sr2+, Co2+), and DNA adsorption by these phosphate precipitants was found to be related to their surface charge and metal chemistry. This work has revealed fundamental surface science of DNA adsorption by CaP and other metal phosphate salts, and this knowledge might be useful for gene delivery, biomineralization, and DNA-directed assembly of metal phosphate materials.

Probing metal-dependent G-quadruplexes using the intrinsic fluorescence of DNA.

K+ enhanced the intrinsic fluorescence of a series of G-quadruplex DNAs, while Pb2+ quenched the fluorescence. The metals showed interesting quadruplex binding kinetics with various DNA sequences.

Freezing promoted hybridization of very short DNA oligonucleotides.

Shorter DNA probes provide better specificity for hybridization, but they may not form stable duplexes at room temperature. In this study, we used thiazole orange to follow DNA hybridization upon freezing and achieved stable 5-mer duplex DNA. Using multiple short probes in tandem, long DNA could also be studied. This study provides insights into DNA hybridization in the frozen state and expands the application of freezing for nucleic acid chemistry.

Tuning DNA adsorption affinity and density on metal oxide and phosphate for improved arsenate detection.

Arsenic (As) contamination in groundwater presents a major health and environmental concern in developing countries. Typically, As is found in two oxidation states. Most chemical tests for inorganic arsenic are focused on As(III), and few have been developed for As(V). We are interested in developing biosensors for As(V) based on its similarity with phosphate. Building upon previous work involving DNA-capped Fe3O4 nanoparticles for As(V) detection, we investigated two other nanomaterials: CeO2 and CePO4 in terms of DNA adsorption and As(V) induced DNA desorption. Fluorescently labeled DNA is physically adsorbed to the surface sites on the nanoparticle surface via its phosphate backbone. In the cases of CeO2 and Fe3O4, the fluorescence was quenched due to electron transfer, whereas for the insulating CePO4, no quenching was observed. Arsenate, being similar to phosphate, can also bind to the surface of the nanoparticles and displace the DNA, increasing the fluorescence signal. The length and sequence of DNA were systematically studied. Using this method, CeO2 performed significantly better than Fe3O4, lowering the detection limit by almost 10-fold. In addition, for CeO2 and CePO4, using shorter DNA was more effective for As(V) detection than using the longer DNA since they both adsorb DNA more tightly than Fe3O4 does. Overall, CeO2 has the best performance since it has an intermediate adsorption affinity of DNA, while CePO4 adsorbs DNA too strongly.