Cross-Binding of Adenosine by Aptamers Selected Using Theophylline.

We recently reported that some adenosine binding aptamers can also bind caffeine and theophylline with around 20-fold lower affinities. This discovery led to the current work to examine the cross-binding of adenosine to theophylline aptamers. For the DNA aptamer for theophylline, cross-binding to adenosine was observed, and the affinity was 18 to 38-fold lower for adenosine based on assays using isothermal titration calorimetry and ThT fluorescence spectroscopy. The binding complexes were characterized using NMR spectroscopy, and both adenosine and theophylline showed an overall similar binding structure to the DNA theophylline aptamer, although small differences were also observed. In contrast, the RNA aptamer did not show binding to adenosine, although both aptamers have very similar relative selectivity for various methylxanthines including caffeine. After a negative selection, a few new aptamers with completely different primary sequences for theophylline were obtained and they did not show binding to adenosine. Thus, there are many ways for aptamers to bind theophylline and some can have cross-binding to adenosine. In biology, theophylline, caffeine, and adenosine can bind to the same protein receptors to regulate sleep, and their binding to the same DNA motifs may suggest an early role of nucleic acids in similar regulatory functions.

Cross-Binding of Four Adenosine/ATP Aptamers to Caffeine, Theophylline, and Other Methylxanthines.

The classical DNA aptamer for adenosine and ATP was selected twice using ATP as the target in 1995 and 2005, respectively. In 2022, this motif appeared four more times from selections using adenosine, ATP, theophylline, and caffeine as targets, suggesting that this aptamer can also bind methylxanthines. In this work, using thioflavin T fluorescence spectroscopy, this classical DNA aptamer showed Kd values for adenosine, theophylline, and caffeine of 9.5, 101, and 131 µM, respectively, and similar Kd values were obtained using isothermal titration calorimetry. Binding to the methylxanthines was also observed for the newly selected Ade1301 aptamer but not for the Ade1304 aptamer. The RNA aptamer for ATP also had no binding to the methylxanthines. Molecular dynamics simulations were performed using the classical DNA and RNA aptamers based on their NMR structures, and the simulation results were consistent with the experimental observations, explaining the selectivity profiles. This study suggests that a broader range of target analogues need to be tested for aptamers. For the detection of adenosine and ATP, the Ade1304 aptamer is a better choice due to its better selectivity.

Machine Learning Directed Aptamer Search from Conserved Primary Sequences and Secondary Structures.

Computer-aided prediction of aptamer sequences has been focused on primary sequence alignment and motif comparison. We observed that many aptamers have a conserved hairpin, yet the sequence of the hairpin can be highly variable. Taking such secondary structure information into consideration, a new algorithm combining conserved primary sequences and secondary structures is developed, which combines three scores based on sequence abundance, stability, and structure, respectively. This algorithm was used in the prediction of aptamers from the caffeine and theophylline selections. In the late rounds of the selections, when the libraries were converged, the predicted sequences matched well with the most abundant sequences. When the libraries were far from convergence and the sequences were deemed challenging for traditional analysis methods, this algorithm still predicted aptamer sequences that were experimentally verified by isothermal titration calorimetry. This algorithm paves a new way to look for patterns in aptamer selection libraries and mimics the sequence evolution process. It will help shorten the aptamer selection time and promote the biosensor and chemical biology applications of aptamers.

Simultaneous Detection of L-Lactate and D-Glucose Using DNA Aptamers in Human Blood Serum.

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.

Gold Nanoparticles Synthesized Using Various Reducing Agents and the Effect of Aging for DNA Sensing.

Gold nanoparticles (AuNPs) are one of the most commonly used reagents in colloidal science and biosensor technology. In this work, we first compared AuNPs prepared using four different reducing agents including citrate, glucose, ascorbate, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). At the same absorbance at the surface plasmon peak of 520-530 nm, citrate-AuNPs and glucose-AuNPs adsorbed more DNA and achieved higher affinity to the adsorbed DNA. In addition, citrate-AuNPs had better sensitivity than glucose-AuNPs for label-free DNA detection. Then, using citrate-AuNPs, the effect of aging was studied by incubation of the AuNPs at 22 °C (room temperature) and at 4 °C for up to 6 months. During aging, the colloidal stability and DNA adsorption efficiency gradually decreased. In addition, the DNA sensing sensitivity using a label-free method also dropped around 4-fold after 6 months. Heating at boiling temperature of the aged citrate-AuNPs could not rejuvenate the sensing performance. This study shows that while citrate-AuNPs are initially better than the other three AuNPs in their colloid properties and sensing properties, this edge in performance might gradually decrease due to constantly changing surface properties caused from the aging effect.

Protection of DNA by metal ions at 95 °C: from lower critical solution temperature (LCST) behavior to coordination-driven self-assembly.

While polyvalent metal ions and heating can both degrade nucleic acids, we herein report that a combination of them leads to stabilization. After incubating 4 mM various metal ions and DNA oligonucleotides at 95 °C for 3 h at pH 6 or 8, metal ions were divided into four groups based on gel electrophoresis results. Mg2+ can stabilize DNA at pH 6 without forming stable nanoparticles at room temperature. Co2+, Cu2+, Cd2+, Mn2+ and Zn2+ all protected the DNA and formed nanoparticles, whereas the nanoparticles formed with Fe2+ and Ni2+ were so stable that they remained even in the presence of EDTA. At pH 8, Ce3+ and Pb2+ showed degraded DNA bands. For Mg2+, better protection was achieved with higher metal and DNA concentrations. By monitoring temperature-programmed fluorescence change, a sudden drop in fluorescence intensity attributable to the lower critical solution temperature (LCST) transition of DNA was found to be around 80 °C for Mg2+, while this transition temperature decreased with increasing Mn2+ concentration. The unexpected thermal stability of DNA enabled by metal ions is useful for extending the application of DNA at high temperatures, forming coordination-driven nanomaterials, and it might offer insights into the origin of life on the early Earth.

A DNA Aptamer for Theophylline with Ultrahigh Selectivity Reminiscent of the Classic RNA Aptamer.

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.

Adsorption of DNA Oligonucleotides by Self-Assembled Metalloporphyrin Nanomaterials.

Porphyrin assemblies have controllable morphology, high biocompatibility, and good optical properties and were widely used in biomedical diagnosis and treatment. With the development of DNA biotechnology, combining DNA with porphyrin assemblies can broaden the biological applications of porphyrins. Porphyrin assemblies can serve as nanocarriers for DNA, although the fundamental interactions between them are not well understood. In this work, zinc meso-tetra(4-pyridyl)porphyrin (ZnTPyP) assemblies were prepared in the presence of various surfactants and at different pH values, yielding a variety of aggregation forms. Among them, the hexagonal stacking form exposes more pyridine substituents, and the hydrogen bonding force between the substituents and the DNA bases allows the DNA to be quickly adsorbed on the surface of the assemblies. The effects of DNA sequence and length were systematically tested. In particular, the adsorption of duplex DNA was less efficient compared to the adsorption of single-stranded DNA. This fundamental study is useful for the further combination of DNA and porphyrin assemblies to prepare new functional hybrid nanomaterials.

Homogeneous assays for aptamer-based ethanolamine sensing: no indication of target binding.

Ethanolamine is an important analyte for environmental chemistry and biological sciences. A few DNA aptamers were previously reported for binding ethanolamine with a dissociation constant (Kd) as low as 9.6 nM. However, most of the previous binding assays and sensing work used either immobilized ethanolamine or immobilized aptamers. In this work, we studied three previously reported DNA sequences, two of which were supposed to bind ethanolamine while the other could not bind. Isothermal titration calorimetry revealed no binding for any of these sequences. In addition, due to their guanine-rich sequences, thioflavin T was used as a probe. Little fluorescence change was observed with up to 1 µM ethanolamine. Responses within the millimolar range of ethanolamine were attributed to the general fluorescence quenching effect of ethanolamine instead of aptamer binding. Finally, after studying the adsorption of ethanolamine to gold nanoparticles (AuNPs), we confirmed the feasibility of using AuNPs as a probe when the concentration of ethanolamine was below 0.1 mM. However, no indication of specific aptamer binding was observed by comparing the three DNA sequences for their color changing trends. This work articulates the importance of careful homogeneous binding assays using free target molecules.

Sensing Metal Ions with Phosphorothioate-Modified DNAzymes.

Phosphorothioate (PS) modification refers to replacing one of the nonbridging oxygen atoms in nucleic acids with sulfur. PS modifications can be easily introduced during solid-phase DNA synthesis. It has been extensively used in ribozyme and DNAzyme research to achieve a bioinorganic understanding of metal binding, bioanalytical applications of metal detection, and chemical biology of DNA modification. It allows for the access of new chemistry, not available to natural DNA. Since each PS modification is accompanied by the production of a chiral phosphorus center, a key technical challenge is to separate the two diastereomers called Rp and Sp. In this chapter, we describe our methods of HPLC-based separation followed by ligation to generate a long and fluorescently modified DNAzyme substrate. Subsequently, the use of the modified substrate for activity assay to understand metal binding and for metal ion detection is also described.

Selection of Aptamers for Sensing Caffeine and Discrimination of Its Three Single Demethylated Analogues.

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.

Probing Metal-Dependent Phosphate Binding for the Catalysis of the 17E DNAzyme.

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.

In vitro selection and application of lanthanide-dependent DNAzymes.

Highly sensitive and selective detection of lanthanide ions is a major analytical challenge. In recent years, the use of DNA for this purpose has been pursued. For such highly charged cations, it is difficult to select their aptamers due to strong nonspecific binding. On the other hand, the use of catalytic DNA or DNAzymes has an advantage to overcome this problem, especially DNAzymes with RNA-cleaving activity. In this chapter, a few such DNAzymes are introduced and methods for in vitro selection of lanthanide-dependent RNA-cleaving DNAzymes are described in detail, including the selection protocols, the DNA sequences used, the characterization of selected DNAzymes and their conversion into biosensors. All of the experiments use only fluorophore-labeled DNA, and radioisotope labeling is completely avoided. The resulting DNAzymes can distinguish lanthanides from non-lanthanide metals, tell the difference between light and heavy lanthanides, and can be used together to discriminate individual lanthanides.

In vitro Selection of Chemically Modified DNAzymes.

DNAzymes are in vitro selected DNA oligonucleotides with catalytic activities. RNA cleavage is one of the most extensively studied DNAzyme reactions. To expand the chemical functionality of DNA, various chemical modifications have been made during and after selection. In this review, we summarize examples of RNA-cleaving DNAzymes and focus on those modifications introduced during in vitro selection. By incorporating various modified nucleotides via polymerase chain reaction (PCR) or primer extension, a few DNAzymes were obtained that can be specifically activated by metal ions such as Zn2+ and Hg2+. In addition, some modifications were introduced to mimic RNase A that can cleave RNA substrates in the absence of divalent metal ions. In addition, single modifications at the fixed regions of DNA libraries, especially at the cleavage junctions, have been tested, and examples of DNAzymes with phosphorothioate and histidine-glycine modified tertiary amine were successfully obtained specific for Cu2+, Cd2+, Zn2+, and Ni2+. Labeling fluorophore/quencher pair right next to the cleavage junction was also used to obtain signaling DNAzymes for detecting various metal ions and cells. Furthermore, we reviewed work on the cleavage of 2'-5' linked RNA and L-RNA substrates. Finally, applications of these modified DNAzymes as biosensors, RNases, and biochemical probes are briefly described with a few future research opportunities outlined at the end.

Sensing Adenosine and ATP by Aptamers and Gold Nanoparticles: Opposite Trends of Color Change from Domination of Target Adsorption Instead of Aptamer Binding.

The 27 mer DNA aptamer for adenosine and adenosine 5'-triphosphate (ATP) is a popular model system for designing biosensors. Various strategies have been reported for label-free colorimetric detection using gold nanoparticles (AuNPs). It is generally accepted that free aptamers can protect AuNPs against salt-induced aggregation, whereas target-bound aptamers cannot. However, these studies only considered the aptamer binding to its target, and the adsorption of the aptamer on AuNPs, but none considered the adsorption of target molecules by AuNPs. We herein report that the adsorption of adenosine destabilized citrate-capped AuNPs with an apparent Kd of just 7.7 µM adenosine, whereas that of ATP stabilized the AuNPs because of the negative charges from the triphosphate group. The adsorbed ATP inhibited the adsorption of DNA. Using the aptamer and a nonbinding mutant, ATP and guanosine 5'-triphosphate (GTP) had the same colorimetric response, and so did adenosine and guanosine, regardless of the DNA sequence, indicating that the color change mainly reflected the adsorption of the nucleosides and nucleotides instead of aptamer binding. The related literature examples using this aptamer were classified into three types and individually analyzed, where the reported color changes can all be explained by the adsorption of target analytes.

Dissecting the Effect of Salt for More Sensitive Label-Free Colorimetric Detection of DNA Using Gold Nanoparticles.

Taking advantage of the protection effect of single-stranded DNA oligonucleotides, gold nanoparticles (AuNPs) remain dispersed and retain a red color with the addition of a low concentration of salt, while AuNPs would aggregate in the presence of double-stranded DNA. This difference has been used to design label-free colorimetric sensors for DNA detection. NaCl is the most commonly used salt to induce the aggregation of AuNPs. In this work, we aimed to test if other salts can provide even better sensor performance and to understand the effects of the cations and anions in salts. We first studied the effect of anions, including halides (NaF, NaCl, NaBr, and NaI), and other common salts (NaNO3, NaClO4, Na2SO4, Na2S2O3, sodium phosphate, and sodium citrate). Among them, weakly adsorbing ones such as F-, citrate, and phosphate appeared to yield better sensitivity than Cl-. Anions can directly adsorb on the AuNPs and affect DNA adsorption. We then tested cations, and only group 1A metals (LiCl, NaCl, KCl, RbCl, and CsCl) can signal DNA adsorption, while divalent metals (MgCl2, CaCl2, MnCl2, and NiCl2) barely showed the effect of DNA. CsCl only works for strongly adsorbing DNA, such as A15, but not weakly adsorbing T15. Overall, NaF is a better salt than NaCl by having a 2.3-fold higher sensitivity, which was confirmed in a DNA sensing assay. This work has identified a better salt yielding higher sensitivity, and sensing work relying on the change of the aggregation state of AuNPs can benefit from this study.

Graphene oxide as a cartridge enable on-line assembly of photosensitizer for 1O2-based electrochemical aptasensing.

An ultrasensitive 1O2-based electrochemical aptasensor by on-line assembly of photosensitizers using graphene oxide (GO) as a cartridge is reported. In the presence of target protein lysozyme, the interaction of lysozyme with aptamer led to the dissociation of dsDNA and release of the aptamer-lysozyme complex to solution, with DNA-c retaining on the electrode; then, the photosensitizer phloxine B (PB) was assembled on the electrode since GO can simultaneously adsorb DNA-c and PB molecules. Upon irradiation by a green LED, 1O2 was generated by photocatalysis of PB molecules and then cleaved the DNA-c, leading to remarkably decreased impedance signals that linearly respond with the logarithm of lysozyme concentration. Benefitting from the efficient photosensitization ability of PB and the high PB-loading capacity of GO, the developed sensor allowed determination of 0.001 to 100 nM lysozyme with a limit of detection of about 0.14 pM. The relative standard deviation (RSD) for five independent electrodes with 1 nM lysozyme was 3.1%, indicating satisfactory reproducibility. The sensor also showed excellent selectivity toward lysozyme in the presence of interfering substances and was applied to the determination of lysozyme in urine samples with recoveries ranging from 91 to 101%. The on-line assembly of photosensitizer technique opens a new way for amplified electrosensing of biomolecules. Graphical abstract An on-line assembly of photosensitizers and DNA on electrode was developed using graphene oxide a cartridge and the photocatalytic electrosensor can be used for label-free detection of lysozyme as low as 1 pM.

Sensitivity of a classic DNAzyme for Pb2+ modulated by cations, anions and buffers.

GR5 is the first reported DNAzyme, which has RNA cleavage activity in the presence of Pb2+. Due to its excellent selectivity, GR5 has been a popular DNAzyme for developing biosensors for Pb2+. The activity of DNAzymes is often affected by pH and salt, which may in turn affect the sensitivity of related sensors. Although pH can be readily controlled by using a buffer, the effect of salt is more complex. To have a systematic understanding, we herein measured the cleavage activity of GR5 in various concentrations of Na+ and Mg2+. Both metals inhibited the DNAzyme with Pb2+, and the inhibition constants were 1.8 mM Mg2+ and 33.4 mM Na+. For anions, F- inhibited GR5 more strongly than Br-, while Cl- was the least inhibiting anion, which was consistent with the solubility of their lead salts. The reaction can work similarly in many Good's buffers, while phosphate buffer should be avoided. Finally, GR5 is an optimal sequence based on the truncation and elongation studies. This study reveals important solution conditions that should be considered when designing and testing related biosensors for metal detection.

Target Self-Enhanced Selectivity in Metal-Specific DNAzymes.

Highly selective recognition of metal ions by rational ligand design is challenging, and simple metal binding by biological ligands is often obscured by nonspecific interactions. In this work, binding-triggered catalysis is used and metal selectivity is greatly increased by increasing the number of metal ions involved, as exemplified in a series of in vitro selected RNA-cleaving DNAzymes. The cleavage junction is modified with a glycyl-histidine-functionalized tertiary amine moiety to provide multiple potential metal coordination sites. DNAzymes that bind 1, 2, and 3 Zn2+ ions, increased their selectivity for Zn2+ over Co2+ ions from approximately 20-, 1000-, to 5000-fold, respectively. This study offers important insights into metal recognition by combining rational ligand design and combinatorial selection, and it provides a set of new DNAzymes with excellent selectivity for Zn2+ ions.

The Two Classic Pb2+ -Selective DNAzymes Are Related: Rational Evolution for Understanding Metal Selectivity.

In 1994, the first DNAzyme named GR5 was reported, which specifically requires Pb2+ for its RNA cleavage activity. Three years later, the 8-17 DNAzyme was isolated. The 8-17 DNAzyme and the related 17E DNAzyme are also most active with Pb2+ , although other divalent metals can work as well. GR5 and 17E have the same substrate sequence, and their catalytic loops in the enzyme strands also have a few similar and conserved nucleotides. Considering these, we hypothesized that 17E might be a special form of GR5. To test this hypothesis, we performed systematic rational evolution experiments to gradually mutate GR5 toward 17E. By using the activity ratio in the presence of Pb2+ and Mg2+ for defining these two DNAzymes, the critical nucleotide was identified to be T12 in 17E for metal specificity. In addition, G9 in GR5 is a position not found in most 17E or 8-17 DNAzymes, and G9 needs to be added to rescue GR5 activity if T12 becomes a cytosine. This study highlights the links between these two classic and widely used DNAzymes, and offers new insight into the sequence-activity relationship related to metal selectivity.