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.     

Crowding Shifts the FMN Recognition Mechanism of Riboswitch Aptamer from Conformational Selection to Induced Fit

In bacteria, the binding between the riboswitch aptamer domain and ligand is regulated by environmental cues, such as low Mg²⁺ in macrophages during pathogenesis to ensure spatiotemporal expression of virulence genes. Binding was investigated between the flavin mononucleotide (FMN) riboswitch aptamer and its anionic ligand in the presence of molecular crowding agent without Mg²⁺ ion, which mimics pathogenic conditions. Structural, kinetic, and thermodynamic analyses under the crowding revealed more dynamic conformational rearrangements of the FMN riboswitch aptamer compared to dilute Mg²⁺‐containing solution. It is hypothesized that under crowding conditions FMN binds through an induced fit mechanism in contrast to the conformational selection mechanism previously demonstrated in dilute Mg²⁺solution. Since these two mechanisms involve different conformational intermediates and rate constants, these findings have practical significance in areas such as drug design and RNA engineering.     

Specific Binding of a d‐RNA G‐Quadruplex Structure with an l‐RNA Aptamer

G‐quadruplex (G4) structures are of general importance in chemistry and biology, such as in biosensing, gene regulation, and cancers. Although a large repertoire of G4‐binding tools has been developed, no aptamer has been developed to interact with G4. Moreover, the G4 selectivity of current toolkits is very limited. Herein, we report the first l ‐RNA aptamer that targets a d ‐RNA G‐quadruplex (rG4). Using TERRA rG4 as an example, our results reveal that this l ‐RNA aptamer, Ap3‐7, folds into a unique secondary structure, exhibits high G4 selectivity and effectively interferes with TERRA‐rG4–RHAU53 binding. Our approach and findings open a new door in further developing G4‐specific tools for diverse applications.     

Competitive Assay for Theophylline Based on an Abasic Site‐Containing DNA Duplex Aptamer and a Fluorescent Ligand

A fluorescence assay for theophylline, one of the common drugs for acute and chronic asthmatic conditions, has been developed based on an abasic site‐containing DNA duplex aptamer (AP aptamer) in combination with an abasic site‐binding fluorescent ligand, riboflavin. The assay is based on the competitive binding of theophylline and riboflavin at the abasic (AP) site of the AP aptamer. In the absence of theophylline, riboflavin binds to the receptor nucleotide opposite the AP site, which leads to fluorescence quenching of the riboflavin. Upon addition of theophylline, competitive binding occurs between theophylline and riboflavin, which results in an effective fluorescence restoration due to release of riboflavin from the AP site. From an examination of the optimization of the AP aptamers, the complex of riboflavin with a 23‐mer AP aptamer (5′‐TCT GCG TCC AGX GCA ACG CAC AC‐3′/5′‐GTG TGC GTT GCC CTG GAC GCA GA‐3′; X : the AP site (Spacer C3, a propylene residue)) possessing cytosine as a receptor nucleotide was found to show a selective and effective fluorescence response to theophylline; the limit of detection for theophylline was 1.1 μM . Furthermore, fluorescence detection of theophylline was successfully demonstrated with high selectivity in serum samples by using the optimized AP aptamer and riboflavin.     

Biocompatible Infinite‐Coordination‐Polymer Nanoparticle–Nucleic‐Acid Conjugates for Antisense Gene Regulation

Herein, we report the synthesis of DNA‐functionalized infinite‐coordination‐polymer (ICP) nanoparticles as biocompatible gene‐regulation agents. ICP nanoparticles were synthesized from ferric nitrate and a ditopic 3‐hydroxy‐4‐pyridinone (HOPO) ligand bearing a pendant azide. Addition of Fe^III to a solution of the ligand produced nanoparticles, which were colloidally unstable in the presence of salts. Conjugation of DNA to the Fe^III–HOPO ICP particles by copper‐free click chemistry afforded colloidally stable nucleic‐acid nanoconstructs. The DNA–ICP particles, when cross‐linked through sequence‐specific hybridization, exhibited narrow, highly cooperative melting transitions consistent with dense DNA surface loading. The ability of the DNA–ICP particles to enter cells and alter protein expression was also evaluated. Our results indicate that these novel particles carry nucleic acids into mammalian cells without the need for transfection agents and are capable of efficient gene knockdown.     

Rational Design of a Redox‐Labeled Chiral Target for an Enantioselective Aptamer‐Based Electrochemical Binding Assay

A series of redox‐labeled L ‐tyrosinamide (L ‐Tym) derivatives was prepared and the nature of the functional group and the chain length of the spacer were systematically varied in a step‐by‐step affinity optimization process of the tracer for the L ‐Tym aptamer. The choice of the labeling position on L ‐Tym proved to be crucial for the molecular recognition event, which could be monitored by cyclic voltammetry and is based on the different diffusion rates of free and bound targets in solution. From this screening approach an efficient electroactive tracer emerged. Comparable dissociation constants K_d were obtained for the unlabeled and labeled targets in direct or competitive binding assays. The enantiomeric tracer was prepared and its enantioselective recognition by the corresponding anti‐D ‐Tym aptamer was demonstrated. The access to both enantiomeric tracer molecules opens the door for the development of one‐pot determination of the enantiomeric excess when using different labels with well‐separated redox potentials for each enantiomer.     

Energy Landscape of Aptamer/Protein Complexes Studied by Single‐Molecule Force Spectroscopy

Aptamers are single‐stranded nucleic acid molecules selected in vitro to bind to a variety of target molecules. Aptamers bound to proteins are emerging as a new class of molecules that rival commonly used antibodies in both therapeutic and diagnostic applications. With the increasing application of aptamers as molecular probes for protein recognition, it is important to understand the molecular mechanism of aptamer–protein interaction. Recently, we developed a method of using atomic force microscopy (AFM) to study the single‐molecule rupture force of aptamer/protein complexes. In this work, we investigate further the unbinding dynamics of aptamer/protein complexes and their dissociation‐energy landscape by AFM. The dependence of single‐molecule force on the AFM loading rate was plotted for three aptamer/protein complexes and their dissociation rate constants, and other parameters characterizing their dissociation pathways were obtained. Furthermore, the single‐molecule force spectra of three aptamer/protein complexes were compared to those of the corresponding antibody/protein complexes in the same loading‐rate range. The results revealed two activation barriers and one intermediate state in the unbinding process of aptamer/protein complexes, which is different from the energy landscape of antibody/protein complexes. The results provide new information for the study of aptamer–protein interaction at the molecular level.     

Optimization of Experimental Parameters to Explore Small‐Ligand/Aptamer Interactions through Use of 1H NMR Spectroscopy and Molecular Modeling

Aptamers constitute an emerging class of molecules designed and selected to recognize any given target that ranges from small compounds to large biomolecules, and even cells. However, the underlying physicochemical principles that govern the ligand‐binding process still have to be clarified. A major issue when dealing with short oligonucleotides is their intrinsic flexibility that renders their active conformation highly sensitive to experimental conditions. To overcome this problem and determine the best experimental parameters, an approach based on the design‐of‐experiments methodology has been developed. Here, the focus is on DNA aptamers that possess high specificity and affinity for small molecules, L ‐tyrosinamide, and adenosine monophosphate. Factors such as buffer, pH value, ionic strength, Mg²⁺‐ion concentration, and ligand/aptamer ratio have been considered to find the optimal experimental conditions. It was then possible to gain new insight into the conformational features of the two ligands by using ligand‐observed NMR spectroscopic techniques and molecular mechanics.     

A Stably Protonated Adenine Nucleotide with a Highly Shifted pKa Value Stabilizes the Tertiary Structure of a GTP‐Binding RNA Aptamer

RNA tertiary structure motifs are stabilized by a wide variety of hydrogen‐bonding interactions. Protonated A and C nucleotides are normally not considered to be suitable building blocks for such motifs since their pK _a values are far from physiological pH. Here, we report the NMR solution structure of an in vitro selected GTP‐binding RNA aptamer bound to GTP with an intricate tertiary structure. It contains a novel kind of base quartet stabilized by a protonated A residue. Owing to its unique structural environment in the base quartet, the pK _a value for the protonation of this A residue in the complex is shifted by more than 5 pH units compared to the pK _a for A nucleotides in single‐stranded RNA. This is the largest pK _a shift for an A residue in structured nucleic acids reported so far, and similar in size to the largest pK _a shifts observed for amino acid side chains in proteins. Both RNA pre‐folding and ligand binding contribute to the pK _a shift.     

Pt–S Bond‐Mediated Nanoflares for High‐Fidelity Intracellular Applications by Avoiding Thiol Cleavage

The Au?S bond is the classic way to functionalize gold nanoparticles (AuNPs). However, cleavage of the bond by biothiols and other chemicals is a long‐standing problem hindering practical applications, especially in cells. Instead of replacing the thiol by a carbene or selenol for stronger adsorption, it is now shown that the Pt?S bond is much more stable, fully avoiding cleavage by biothiols. AuNPs were deposited with a thin layer of platinum, and an AuNP@Pt‐S nanoflare was constructed to detect the miRNA‐21 microRNA in living cells. This design retained the optical and cellular uptake properties of DNA‐functionalized AuNPs, while showing high‐fidelity signaling. It discriminated target cancer cells even in a mixed‐cell culture system, where the Au‐S based nanoflare was less sensitive. Compared to previous methods of changing the ligand chemistry, coating a Pt shell is more accessible, and previously developed methods for AuNPs can be directly adapted.     

Near‐Infrared‐Light‐Mediated Imaging of Latent Fingerprints based on Molecular Recognition

Photoluminescence is one of the most sensitive techniques for fingerprint detection, but it also suffers from background fluorescence and selectivity at the expense of generality. The method described herein integrates the advantages of near‐infrared‐light‐mediated imaging and molecular recognition. In principle, upconversion nanoparticles (UCNPs) functionalized with a lysozyme‐binding aptamer were used to detect fingerprints through recognizing lysozyme in the fingerprint ridges. UCNPs possess the ability to suppress background fluorescence and make it possible for fingerprint imaging on problematic surfaces. Lysozyme, a universal compound in fingerprints, was chosen as the target, thus simultaneously meeting the selectivity and generality criteria in photoluminescence approaches. Fingerprints on different surfaces and from different people were detected successfully. This strategy was used to detect fingerprints with cocaine powder by using UCNPs functionalized with a cocaine‐binding aptamer.     

Aptamer-Mediated Reversible Transactivation of Gene Expression by Light

The investigation and manipulation of cellular processes with subcellular resolution requires non-invasive tools with spatiotemporal precision and reversibility. Building on the interaction of the photoreceptor PAL with an RNA aptamer, we describe a variation of the CRISPR/dCAS9 system for light-controlled activation of gene expression. This platform significantly reduces the coding space required for genetic manipulation and provides a strong on-switch with almost no residual activity in the dark. It adds to the current set of modular building blocks for synthetic biological circuit design and is broadly applicable.     

A DNA‐Mediated Chemically Induced Dimerization (D‐CID) Nanodevice for Nongenetic Receptor Engineering To Control Cell Behavior

Small‐molecule regulation is a powerful switching tool to manipulate cell signal transduction for a desired function; however, most available methods usually require genetic engineering to endow cells with responsiveness to user‐defined small molecules. Herein, we demonstrate a nongenetic approach for small‐molecule‐controlled receptor activation and consequent cell behavior manipulation that is based on DNA‐mediated chemically induced dimerization (D‐CID). D‐CID uses a programmable chemical‐responsive DNA nanodevice to trigger DNA strand displacement and induce the activation of c‐Met, a tyrosine kinase receptor cognate for hepatocyte growth factor, through dimerization. Through the use of various functional nucleic acids, including aptamers and DNAzymes, as recognition modules, the versatility of D‐CID in inducing c‐Met signaling upon addition of various small‐molecular or ionic cues, including ATP, histidine, and Zn²⁺, is demonstrated. Moreover, owing its multi‐input properties, D‐CID can be used to manipulate the behaviors of multiple cell populations simultaneously in a selective and programmable fashion.     

Arrest of Rolling Circle Amplification by Protein‐Binding DNA Aptamers

Certain DNA polymerases, such as ?29 DNA polymerase, can isothermally copy the sequence of a circular template round by round in a process known as rolling circle amplification (RCA), which results in super‐long single‐stranded (ss) DNA molecules made of tandem repeats. The power of RCA reflects the high processivity and the strand‐displacement ability of these polymerases. In this work, the ability of ?29DNAP to carry out RCA over circular templates containing a protein‐binding DNA aptamer sequence was investigated. It was found that protein–aptamer interactions can prevent this DNA polymerase from reading through the aptameric domain. This finding indicates that protein‐binding DNA aptamers can form highly stable complexes with their targets in solution. This novel observation was exploited by translating RCA arrest into a simple and convenient colorimetric assay for the detection of specific protein targets, which continues to showcase the versatility of aptamers as molecular recognition elements for biosensing applications.     

Regulation of Protein Activity and Cellular Functions Mediated by Molecularly Evolved Nucleic Acids

Regulation of protein activity is essential for revealing the molecular mechanisms of biological processes. DNA and RNA achieve many uniquely efficient functions, such as genetic expression and regulation. The chemical capability to synthesize artificial nucleotides can expand the chemical space of nucleic acid libraries and further increase the functional diversity of nucleic acids. Herein, a versatile method has been developed for modular expansion of the chemical space of nucleic acid libraries, thus enabling the generation of aptamers able to regulate protein activity. Specifically, an aptamer that targets integrin alpha3 was identified and this aptamer can inhibit cell adhesion and migration. Overall, this chemical‐design‐assisted in vitro selection approach enables the generation of functional nucleic acids for elucidating the molecular basis of biological activities and uncovering a novel basis for the rational design of new protein‐inhibitor pharmaceuticals.     

Complex Formation of the Tetracycline‐Binding Aptamer Investigated by Specific Cross‐Relaxation under DNP

While dynamic nuclear polarization (DNP) under magic‐angle spinning (MAS) is generally a powerful method capable of greatly enhancing the sensitivity of solid‐state NMR spectroscopy, hyperpolarization also gives rise to peculiar spin dynamics. Here, we elucidate how specific cross‐relaxation enhancement by active motions under DNP (SCREAM‐DNP) can be utilized to selectively obtain MAS‐NMR spectra of an RNA aptamer in a tightly bound complex with a methyl‐bearing ligand (tetracycline) due to the effective CH₃‐reorientation at an optimized sample temperature of approximately 160 K. SCREAM‐DNP can spectrally isolate the complex from non‐bound species in an RNA mixture. This selectivity allows for a competition assay between the aptamer and a mutant with compromised binding affinity. Variations in molecular structure and methyl dynamics, as observed by SCREAM‐DNP, between free tetracycline and RNA‐bound tetracycline are discussed.