L-lactate is a key metabolite indicative of physiological states, glycolysis pathways, and various diseases such as sepsis, heart attack, lactate acidosis, and cancer. Detection of lactate has been relying on a few enzymes that need additional oxidants. In this work, DNA aptamers for L-lactate were obtained using a library-immobilization selection method and the highest affinity aptamer reached a Kd of 0.43 mM as determined using isothermal titration calorimetry. The aptamers showed up to 50-fold selectivity for L-lactate over D-lactate and had little responses to other closely related analogs such as pyruvate or 3-hydroxybutyrate. A fluorescent biosensor based on the strand displacement method showed a limit of detection of 0.55 mM L-lactate, and the sensor worked in 90 % serum. Simultaneous detection of L-lactate and D-glucose in the same solution was achieved. This work has broadened the scope of aptamers to simple metabolites and provided a useful probe for continuous and multiplexed monitoring.
With the growing consumption of caffeine-containing beverages, detection of caffeine has become an important biomedical, bioanalytical, and environmental topic. We herein isolated four high-quality aptamers for caffeine with dissociation constants ranging from 2.2 to 14.6 μM as characterized using isothermal titration calorimetry. Different binding patterns were obtained for the three single demethylated analogues: theobromine, theophylline, and paraxanthine, highlighting the effect of the molecular symmetry of the arrangement of the three methyl groups in caffeine. A structure-switching fluorescent sensor was designed showing a detection limit of 1.2 μM caffeine, which reflected the labeled caffeine concentration within 6.1% difference for eight commercial beverages. In 20% human serum, a detection limit of 4.0 μM caffeine was achieved. With the four aptamer sensors forming an array, caffeine and the three analogues were well separated from nine other closely related molecules.
Over the last three decades, numerous aptamer-based biosensors have been reported. The basis of these sensors is the selective binding of target analytes by aptamers. In the last few years, a number of papers have been published questioning the binding ability of some popular aptamers such as those documented for As(III), ampicillin, chloramphenicol, isocarbophos, phorate and dopamine. In this article, these papers are reviewed, and the binding assays are described, which may provide possible reasons for obtaining false positive aptamers. Additionally, relevant aptamer selection methods and typical characterization steps are described. It is found that for small molecular targets, using an immobilized library might result in better aptamers. Furthermore, the importance of carefully designed controls to ensure the quality of binding assays is discussed, especially in the case of mutated nonbinding aptamers. Only then, with fully validated aptamers, can subsequent biosensor design bring about meaningful results. • The first critical review of the literature on aptamers that were proven to be non-binding sequences. • Five different aptamers for various small molecules reviewed. • Possible reasons for the generation of such non-binding aptamer sequences proposed and methods to avoid them described.
Various functional DNA molecules have been used for the detection of environmental contaminants in water, but their practical applications have been limited. To address this gap, this review highlights the efforts to develop field-deployable water quality biosensors. The biosensor devices include microfluidic, lateral flow and paper-based devices, and other novel ideas such as the conversion of glucometers for the detection of environmental analytes. In addition, we also review DNA-functionalized hydrogels and their use in diffusive gradients in thin films (DGT) devices. We classify the sensors into one-step and two-step assays and discuss their practical implications. While the review is focused on works reported in the last five years, some classic early works are cited as well. Overall, most of the existing work only tested spiked water samples. Future work needs to shift to real environmental samples and the comparison of DNA-based sensors to standard analytical methods. • Recent development in field-deployable functional DNA based biosensors for environmental water monitoring reviewed. • Articulated the concept of one-step and two-step assays. • Microfluidic device, lateral flow device, paper, hydrogel, and glucose meter based examples reviewed.
Since the report of the RNA aptamer for theophylline, theophylline has become a key molecule in chemical biology for designing RNA switches and riboswitches. In addition, theophylline is an important drug for treating airway diseases including asthma. The classic RNA aptamer with excellent selectivity for theophylline has been used to design biosensors, although DNA aptamers are more desirable for stability and cost considerations. In this work, we selected DNA aptamers for theophylline, and all the top sequences shared the same binding motifs. Binding was confirmed using isothermal titration calorimetry and a nuclease digestion assay, showing a dissociation constant (Kd) around 0.5 μM theophylline. The Theo2201 aptamer can be truncated down to 23-mer while still has a Kd of 9.8 μM. The selectivity for theophylline over caffeine is around 250,000-fold based on a strand-displacement assay, which was more than 20-fold higher compared to the classic RNA aptamer. For other tested analogs, the DNA aptamer also showed better selectivity. Using the structure-switching aptamer sensor design method, a detection limit of 17 nM theophylline was achieved in the selection buffer, and a detection limit of 31 nM was obtained in 10% serum.
Detection of arsenic is a long-standing challenge in environmental analytical chemistry. In recent years, using biomolecules and nanomaterials for sensing arsenic has been growingly reported. In this article, this field is critically reviewed based on some recent fundamental understandings including interactions between arsenic and gold, thiol, and DNA aptamers. First, taking advantage of the adsorption of As(III) on noble metal surfaces such as silver and gold, sensors were developed based on surface enhanced Raman spectroscopy, electrochemistry and colorimetry. In addition, by functionalizing metal nanoparticles with thiol containing molecules, As(III) induced aggregation of the particles based on As(III)/thiol interactions. As(V) interacts with metal oxides strongly and competitive sensors were developed by displacing pre-adsorbed DNA oligonucleotides. A DNA aptamer was selected for As(III) and many sensors were reported based on this aptamer, although careful binding measurements indicated that the sequence has no affinity towards As(III). Overall, bio/nano systems are promising for the detection of arsenic. Future work on fundamental studies, searching for more specific arsenic binding materials and aptamers, incorporation of sensors into portable devices, and more systematic test of sensors in real samples could be interesting and useful research topics.
A self-cleaving ribozyme was obtained from <italic>in vitro</italic> selection, displaying site-specific cleavage activity under various denaturing conditions, such as high temperatures, 20 M formamide, and low salt concentrations.
• Fundamentals of phosphorothioate nucleic acids reviewed from synthesis to metal binding. • Applications of phosphorothioate nucleic acids in developing biosensors and chemical biology reviewed. • Applications of phosphorothioate DNA in assembly and the directed growth of nanomaterials reviewed. Phosphorothioate (PS) modification replaces one of the non-bridging oxygen atoms by sulfur in the phosphate backbone of nucleic acids. While PS DNAs have been traditionally used as nuclease-resistant antisense agents and PS RNA as probe of metal binding in ribozymes, multiple new applications have emerged in recent years. In this review, we start by briefly introducing the structure and synthesis of PS nucleic acids followed by their fundamental chemical and biochemical properties. Further, their recently emerged surface science applications are discussed, such as attachment of DNA to various surfaces and nanomaterials containing thiophilic metals such as gold, silver and cadmium, and templating the growth of these materials. Their role in conferring structural effects in the presence of certain metal ions and in fishing out novel aptamers are also discussed. Covalent chemistry can be performed on the sulfur atom for further grafting functional groups to the backbone of DNA. For PS RNA, we discuss their role as probes for metal binding in ribozymes and DNAzymes, which leads to applications in detection of thiophilic metal ions. Since each PS modification site produces a chiral phosphorus center, the synthesis and purification of diastereomers and their applications are emphasized throughout this review. In the end, a few future research directions are discussed.
The RNA-cleaving 17E DNAzyme exhibits different levels of cleavage activity in the presence of various divalent metal ions, with Pb2+ giving the fastest cleavage. In this study, the metal-phosphate interaction is probed to understand the trend of activity with different metal ions. For the first-row transition metals, the lowest activity shown by Ni2+ correlates with the inhibition by the inorganic phosphate and its water ligand exchange rate, suggesting inner-sphere metal coordination. Cleavage activity with the two stereoisomers of the phosphorothioate-modified substrates, Rp and Sp, indicated that Mg2+, Mn2+, Fe2+, and Co2+ had the highest Sp:Rp activity ratio of >900. Comparatively, the activity was much less affected using the thiophilic metals, including Pb2+, suggesting inner-sphere coordination. The pH-rate profiles showed that Pb2+ was different than the rest of the metal ions in having a smaller slope and a similar fitted apparent pKa and the pKa of metal-bound water. Combining previous reports and our current results, we propose that Pb2+ most likely plays the role of a general acid while the other metal ions are Lewis acid catalysts interacting with the scissile phosphate.
Lead (Pb) is a highly toxic heavy metal of great environmental and health concerns, and interestingly Pb 2+ has played important roles in nucleic acids chemistry. Since 2000, using DNA for selective detection of Pb 2+ has become a rapidly growing topic in the analytical community. Pb 2+ can serve as the most active cofactor for RNA-cleaving DNAzymes including the GR5, 17E and 8–17 DNAzymes. Recently, Pb 2+ was found to promote a porphyrin metalation DNAzyme named T30695. In addition, Pb 2+ can tightly bind to various G-quadruplex sequences inducing their unique folding and binding to other molecules such as dyes and hemin. The peroxidase-like activity of G-quadruplex/hemin complexes was also used for Pb 2+ sensing. In this article, these Pb 2+ recognition mechanisms are reviewed from fundamental chemistry to the design of fluorescent, colorimetric, and electrochemical biosensors. In addition, various signal amplification mechanisms such as rolling circle amplification, hairpin hybridization chain reaction and nuclease-assisted methods are coupled to these sensing methods to drive up sensitivity. We mainly cover recent examples published since 2015. In the end, some practical aspects of these sensors and future research opportunities are discussed. • Fundamentals of Pb 2+ recognition by DNA including DNAzymes and G-quadruplex DNA reviewed • Literature in the last five years summarized • Practical aspect of Pb 2+ detection using DNA discussed
The more the merrier: Better selectivity for Zn2+ ions is shown for DNAzymes that bind more metal ions. This selectivity is exemplified by using a series of in vitro selected DNAzymes that contain a single metal ligand modification at the cleavage junction.
Abstract Nickel is a highly important metal, and the detection of Ni2+ using biosensors is a long-stand analytical challenge. DNA has been widely used for metal detection, although no DNA-based sensors were reported for Ni2+. DNAzymes are DNA-based catalysts, and they recruit metal ions for catalysis. In this work, in vitro selection of RNA-cleaving DNAzymes was carried out using a library containing a region of 50 random nucleotides in the presence of Ni2+. To increase Ni2+ binding, a glycyl–histidine-functionalized tertiary amine moiety was inserted at the cleavage junction. A representative DNAzyme named Ni03 showed a high cleavage yield with Ni2+ and it was further studied. After truncation, the optimal sequence of Ni03l could bind one Ni2+ or two Co2+ for catalysis, while other metal ions were inactive. Its cleavage rates for 100 μM Ni2+ reached 0.63 h−1 at pH 8.0. A catalytic beacon biosensor was designed by labeling a fluorophore and a quencher on the Ni03l DNAzyme. Fluorescence enhancement was observed in the presence of Ni2+ with a detection limit of 12.9 μM. The sensor was also tested in spiked Lake Ontario water achieving a similar sensitivity. This is another example of using single-site modified DNAzyme for sensing transition metal ions.
Since 1994, deoxyribozymes or DNAzymes have been in vitro selected to catalyze various types of reactions. Metal ions play a critical role in DNAzyme catalysis, and Zn2+ is a very important one among them. Zn2+ has good biocompatibility and can be used for intracellular applications. Chemically, Zn2+ is a Lewis acid and it can bind to both the phosphate backbone and the nucleobases of DNA. Zn2+ undergoes hydrolysis even at neutral pH, and the partially hydrolyzed polynuclear complexes can affect the interactions with DNA. These features have made Zn2+ a unique cofactor for DNAzyme reactions. This review summarizes Zn2+ -dependent DNAzymes with an emphasis on RNA-/DNA-cleaving reactions. A key feature is the sharp Zn2+ concentration and pH-dependent activity for many of the DNAzymes. The applications of these DNAzymes as biosensors for Zn2+ , as therapeutic agents to cleave intracellular RNA, and as chemical biology tools to manipulate DNA are discussed. Future studies can focus on the selection of new DNAzymes with improved performance and detailed biochemical characterizations to understand the role of Zn2+ , which can facilitate practical applications of Zn2+ -dependent DNAzymes.
Detection of heavy metal contamination in the environment is an on-going analytical challenge. In effort of developing portable biosensors, deoxyribonucleic acid (DNA)-based designs have gained much attention for their high affinity and specificity to metals, stability, cost-efficiency, ease of modification, and batch-to-batch reproducibility. Specific sequences of DNA aptamers and DNAzymes provide grounds for rational designs of fluorescent, colorimetric, and electrochemical detection methods. Aptamers exert only a binding function, while DNAzymes can use heavy metals to catalyze specific chemical and biological transformations. This article starts with a brief introduction of heavy metals and their interactions with DNA. Then DNA aptamers and DNAzymes are respectively reviewed from their in vitro selection, representative DNA sequences, and design of biosensors. For signal transduction, various fluorescent, colorimetric, and electrochemical examples are described. Finally, future perspectives are discussed.
Abstract Mercury (Hg) is a toxic metal posing major health risks to human beings and wildlife. The characterization of Hg fate and transport in aquatic environments is hindered by a lack of sensitive, selective and easily field-deployable analytical techniques. Here we assess the reliability and performance of a Hg2+ sensor based on the selective binding of Hg2+ to a thymine-rich DNA under environmentally-relevant conditions. Experimental results indicate that the interactions between the DNA and SYBR Green I, which produce the detection fluorescence signal, are significantly impacted by pH, metal ligands and natural dissolved organic matter (NDOM). These interferences are largely eliminated by immobilizing the DNA in a polyacrylamide hydrogel, although high concentrations of NDOM, such as fulvic acids, still affect the sensor’s performance due to competitive binding of Hg2+. The binding of Hg2+ to NDOM, however, can be accounted for via equilibrium speciation calculations, which also yield the complexation constant for Hg2+ binding to the DNA in the hydrogel. The equilibrium calculations reproduce the results for the entire set of experimental conditions, from simple electrolyte solutions to complex aqueous compositions mimicking natural lake waters, and across large ranges of pH (3-10) and temperature (5-50 °C).
Many DNA-functionalized nanomaterials and biosensors have been reported, but most have ignored the influence of DNA on the stability of nanoparticles. We observed that cytosine-rich DNA oligonucleotides can etch silver nanoparticles (AgNPs). In this work, we showed that phosphorothioate (PS)-modified DNA (PS-DNA) can etch AgNPs independently of DNA sequence, suggesting that the thio-modifications are playing the major role in etching. Compared to unmodified DNA (e.g., poly-cytosine DNA), the concentration of required PS DNA decreases sharply, and the reaction rate increases. Furthermore, etching by PS-DNA occurs quite independent of pH, which is also different from unmodified DNA. The PS-DNA mediated etching could also be controlled well by varying DNA length and conformation, and the number and location of PS modifications. With a higher activity of PS-DNA, the process of etching, ripening, and further etching was taken place sequentially. The etching ability is inhibited by forming duplex DNA and thus etching can be used to measure the concentration of complementary DNA.
It has been proposed that Mg2+ and Fe2+ are very similar in interacting with ribozymes and some protein-based enzymes, but their activities with DNAzymes have yet to be studied. Here, the activity of Fe2+ as cofactor for a few RNA-cleaving DNAzymes is investigated. 17E is a well-studied DNAzyme that is active in the presence of many different divalent metal ions; it is highly active with Fe2+ with an apparent Kd of 29.7±2.3 μm and a kobs of 1.12±0.11 min-1 in the presence of 1 mm Fe2+ at pH 7.5. Fe2+ has 21-fold higher activity than Mg2+ . Six different DNAzymes are then tested, and only the DNAzymes active with Mg2+ (17E, 8-17, and E5) are active with Fe2+ . Fe2+ has 25 and one- to twofold higher activity than Mg2+ for the 8-17 and E5 DNAzymes, respectively. In pH>7 buffer and in presence of air, 1 mm Fe2+ results in a nonspecific degradation of the DNA strand due to reactive oxygen species (ROS). Cleavage reactions in anoxic environment and antioxidant ascorbate can be used to overcome the effect of oxidation. The findings provide insights for potential DNAzyme catalysis in the early Earth, and they further support the similarity between Mg2+ and Fe2+ in enzyme catalysis.
Catalysts that can work without the need of light and additional oxidants such as H2O2 to degrade organic pollutants have been long sought. In this work, we report that at acidic condition, CeO2 na...
Fluoride boosts the oxidase-like activity of hydrolyzed Ce(<sc>iv</sc>) but inhibits the activity of Ce(<sc>iv</sc>), allowing intentional hydrolysis to be performed for consistent analysis of Ce(<sc>iv</sc>).
An arsenic-binding aptamer named Ars-3 was reported in 2009, and it has been used for detection of As(III) in more than two dozen papers. In this work, we performed extensive binding assays using isothermal titration calorimetry, various DNA-staining dyes, and gold nanoparticles. By carefully comparing Ars-3 and a few random control DNA sequences, no specific binding of As(III) was observed in each case. Therefore, we conclude that Ars-3 cannot bind As(III). Possible reasons for some of the previously reported binding and detection were speculated to be related to the adsorption of As(III) onto gold surfaces, which were used in many related sensor designs, and As(III)/Au interactions were not considered before. The selection data in the original paper were then analyzed in terms of sequence alignment, secondary structure prediction, and dissociation constant measurement. These steps need rigorous testing before confirming specific binding of newly selected aptamers. This study calls for attention to the gap between aptamer selection and biosensor design, and the gap needs to be filled by careful binding assays to further the growth of the aptamer field.
Ag10c is a recently reported RNA-cleaving DNAzyme obtained from in vitro selection. Its cleavage activity selectively requires Ag+ ions, and thus it has been used as a sensor for Ag+ detection. However, the previous selection yielded very limited information regarding its sequence requirement, since only ∼0.1% of the population in the final library were related to Ag10c and most other sequences were inactive. In this work, we performed a reselection by randomizing the 19 important nucleotides in Ag10c in such a way that a purine has an equal chance of being A or G, whereas a pyrimidine has an equal chance of being T or C. The round 3 library of the reselection was carefully analyzed and a statistic understanding of the relative importance of each nucleotide was obtained. At the same time, a more active mutant was identified, containing two mutated nucleotides. Further analysis indicated new base pairs leading to an enzyme with smaller catalytic loops but with ∼200% activity of the original Ag10c, and also excellent selectivity for Ag+. Therefore, a more active mutant of Ag10c was obtained and further truncations were successfully performed, which might be better candidates for developing new biosensors for silver. A deeper biochemical understanding was also obtained using this reselection method.
Herein, the excellent Na+ selectivity of a few RNA-cleaving DNAzymes was exploited, where Na+ can be around 3000-fold more effective than K+ for promoting catalysis. By using a double mutant based on the Ce13d DNAzyme, and by lowering the temperature, increased 2-aminopurine (2AP) fluorescence was observed with addition of both Na+ and K+. The fluorescence increase was similar for these two metals at below 10 mM, after which K+ took a different pathway. Since 2AP probes its local base stacking environment, K+ can be considered to induce misfolding. Binding of both Na+ and K+ was specific, since single base mutations could fully inhibit 2AP fluorescence for both metals. The binding thermodynamics was measured by temperature-dependent experiments revealing enthalpy-driven binding for both metals and less coordination sites compared to G-quadruplex DNA. Cleavage activity assays indicated a moderate cleavage activity with 10 mM K+, while further increase of K+ inhibited the activity, also supporting its misfolding of the DNAzyme. For comparison, a G-quadruplex DNA was also studied using the same system, where Na+ and K+ led to the same final state with only around 8-fold difference in Kd. This study provides interesting insights into strategies for discriminating Na+ and K+.
Metal ions play a critical role in the RNA-cleavage reaction by interacting with the scissile phosphate and stabilizing the highly negatively charged transition state. Many metal-dependent DNAzymes have been selected for RNA cleavage. Herein, we report that the Ce13d DNAzyme can use nonmetallic iodine (I2) to cleave a phosphorothioate (PS)-modified substrate. The cleavage yield exceeded 60% for both the Rp and Sp stereoisomers in 10 s, while the yield without the enzyme strand was only ∼10%. The Ce13d cleavage with I2 also required Na+, consistent with the property of Ce13d and confirming the similar role of I2 as a metal ion. Ce13d had the highest yield among eight tested DNAzymes, with the second highest DNAzyme showing only 20% cleavage. The incomplete cleavage was due to competition from desulfurization and isomerization reactions. This DNAzyme was engineered for fluorescence-based I2 detection. With EDTA for masking metal ions, I2 was selectively detected down to 4.7 nM. Oxidation of I- with Fe3+ produced I2 in situ, allowing detection of Fe3+ down to 78 nM. By harnessing nonelectrostatic interactions, such as the I2/sulfur interaction observed here, more nonmetal species might be discovered to assist DNAzyme-based RNA cleavage.
NiO nanoparticles can quickly catalyze oxidation of Amplex red to produce fluorescent products for intracellular imaging, much more efficiently than other types of tested nanozymes.