Structure-switching signaling aptamers

Aptamers are single-stranded nucleic acids with defined tertiary structures for selective binding to target molecules. Aptamers are also able to bind a complementary DNA sequence to form a duplex structure. In this report, we describe a strategy for designing aptamer-based fluorescent reporters that function by switching structures from DNA/DNA duplex to DNA/target complex. The duplex is formed between a fluorophore-labeled DNA aptamer and a small oligonucleotide modified with a quenching moiety (denoted QDNA). When the target is absent, the aptamer binds to QDNA, bringing the fluorophore and the quencher into close proximity for maximum fluorescence quenching. When the target is introduced, the aptamer prefers to form the aptamer-target complex. The switch of the binding partners for the aptamer occurs in conjunction with the generation of a strong fluorescence signal owing to the dissociation of QDNA. Herein, we report on the preparation of several structure-switching reporters from two existing DNA aptamers. Our design strategy is easy to generalize for any aptamer without prior knowledge of its secondary or tertiary structure, and should be suited for the development of aptamer-based reporters for real-time sensing applications.     

Structure-switching allosteric deoxyribozymes

DNA aptamers and DNA enzymes (DNAzymes or deoxyribozymes) are single-stranded DNA molecules with ligand-binding and catalytic capabilities, respectively. Allosteric DNA enzymes (aptazymes) are deoxyribozymes whose activity can be regulated by the binding state of an appended aptamer domain and have many potential uses in the fields of drug discovery and diagnostics. In this report, we describe a simple, yet potentially general, DNA aptazyme rational design strategy that requires no structural characterization of the constituent deoxyribozymes and aptamers. It is based on the concept originally developed in our laboratory for the design of structure-switching signaling aptamers that change structural states from a DNA-DNA duplex to a DNA-target complex upon target binding. In our new strategy, an antisense oligonucleotide is used to regulate the enzymatic activity of a linked aptamer-deoxyribozyme by annealing with a stretch of nucleotides on each side of the aptamer-DNAzyme junction. Structural reorganization of the aptamer domain upon target binding relieves the suppressive effect of this regulatory oligonucleotide on the attached DNA enzyme. Consequently, the target-binding event triggers the catalytic action of the aptazyme. We have demonstrated this concept using two RNA-cleaving deoxyribozymes, each adjoined to a DNA aptamer that binds ATP. These allosteric DNA enzymes exhibit the same ligand-binding specificity as the parental DNA aptamer and show up to 30-fold rate enhancement in the presence of ATP. The described methodology provides a convenient approach for rationally designing catalytic DNA-based biosensors.     

Stabilizing structure-switching signaling RNA aptamers by entrapment in sol-gel derived materials for solid-phase assays

Structure-switching, fluorescence-signaling DNA and RNA aptamers have been reported as highly versatile molecular recognition elements for biosensor development. While structure-switching DNA aptamers have been utilized for solid-phase sensing, equivalent RNA aptamers have yet to be successfully utilized in solid-phase sensors due to their lack of chemical stability and susceptibility to nuclease attack. In this study, we examined entrapment into sol-gel derived organic-inorganic composite materials as a platform for immobilization of structure-switching fluorescence-signaling RNA aptamer reporters, using both the synthetic theophylline- and naturally occurring thiamine pyrophosphate-binding RNA aptamers as test cases. Structure-switching versions of both aptamers were entrapped into a series of sol-gel derived composites, ranging from highly polar silica to hydrophobic methylsilsesquioxane-based materials, and the target-binding and signaling capabilities of these immobilized aptamers were assessed relative to solution. Both immobilized aptamers demonstrated sensitivity and selectivity similar to that of free aptamers when entrapped in a composite material derived from 40% (v/v) methyltrimethoxysilane/tetramethoxysilane. Importantly, this material also conferred protection from nuclease degradation and imparted long-term chemical stability to the RNA reporter systems. Given the versatility of sol-gel entrapment for development of biosensors, microarrays, bioaffinity columns, and other devices, this entrapment method should provide a useful platform for numerous solid-phase RNA aptamer-based devices.     

RE-SELEX: restriction enzyme-based evolution of structure-switching aptamer biosensors

Aptamers are widely employed as recognition elements in small molecule biosensors due to their ability to recognize small molecule targets with high affinity and selectivity. Structure-switching aptamers are particularly promising for biosensing applications because target-induced conformational change can be directly linked to a functional output. However, traditional evolution methods do not select for the significant conformational change needed to create structure-switching biosensors. Modified selection methods have been described to select for structure-switching architectures, but these remain limited by the need for immobilization. Herein we describe the first homogenous, structure-switching aptamer selection that directly reports on biosensor capacity for the target. We exploit the activity of restriction enzymes to isolate aptamer candidates that undergo target-induced displacement of a short complementary strand. As an initial demonstration of the utility of this approach, we performed selection against kanamycin A. Four enriched candidate sequences were successfully characterized as structure-switching biosensors for detection of kanamycin A. Optimization of biosensor conditions afforded facile detection of kanamycin A (90 μM to 10 mM) with high selectivity over three other aminoglycosides. This research demonstrates a general method to directly select for structure-switching biosensors and can be applied to a broad range of small-molecule targets.

RE-SELEX is the first homogenous method for in vitro evolution of structure-switching DNA aptamers.     

Highly Specific Recognition of Guanosine Using Engineered Base-Excised Aptamers

Purines and their derivatives are highly important molecules in biology for nucleic acid synthesis, energy storage, and signaling. Although many DNA aptamers have been obtained for binding adenine derivatives such as adenosine, adenosine monophosphate, and adenosine triphosphate, success for the specific binding of guanosine has been limited. Instead of performing new aptamer selections, we report herein a base-excision strategy to engineer existing aptamers to bind guanosine. Both a Na⁺-binding aptamer and the classical adenosine aptamer have been manipulated as base-excising scaffolds. A total of seven guanosine aptamers were designed, of which the G16-deleted Na⁺ aptamer showed the highest bindng specificity and affinity for guanosine with an apparent dissociation constant of 0.78 mm . Single monophosphate difference in the target molecule was also recognizable. The generality of both the aptamer scaffold and excised site were systematically studied. Overall, this work provides a few guanosine binding aptamers by using a non-SELEX method. It also provides deeper insights into the engineering of aptamers for molecular recognition.     

Aptamer switch probe based on intramolecular displacement

A novel aptamer-based molecular probe design employing intramolecular signal transduction is demonstrated. The probe is composed of three elements: an aptamer, a short, partially cDNA sequence, and a PEG linker conjugating the aptamer with the short DNA strand. We have termed this aptamer probe an "aptamer switch probe", or ASP. The ASP design utilizes both a fluorophore and a quencher which are respectively modified at the two termini of the ASP. In the absence of the target molecule, the short DNA will hybridize with the aptamer, keeping the fluorophore and quencher in close proximity, thus switching off the fluorescence. However, when the ASP meets its target, the binding between the aptamer and the target molecule will disturb the intramolecular DNA hybridization, move the quencher away from the fluorophore, and, in effect, switch on the fluorescence. Both ATP and human alpha-thrombin aptamers were engineered to demonstrate this design, and both showed that fluorescence enhancement could be quantitatively mediated by the addition of various amounts of target molecules. Both of these ASPs presented excellent selectivity and prompt response toward their targets. With intrinsic advantages resulting from its intramolecular signal transduction architecture, the ASP design holds promising potential for future applications, such as biochip and in situ imaging, which require reusability, excellent stability, prompt response, and high sensitivity.     

Simple chemiluminescence aptasensors based on resonance energy transfer

In the present work, two aptamer-based probes and related sensor systems were developed with chemiluminescence signaling. The detection was based on "turning-on" chemiluminescence with switching "off" of the resonance energy transfer after the aptamer's recognition of the target molecule. In this design, a DNA/aptamer duplex linked a chemiluminescence group and a gold nanoparticle together. Only low-intensity chemiluminescence was obtained due to the highly efficient resonance energy transfer. After introducting the target molecule, structure-switching took place with turning off the energy transfer; thus, a restoration and turning on of the chemiluminescence was obtained. The two designs differed in the chemiluminescence groups, since one was a covalently linked luminol molecule, while the other was a conjugated horseradish peroxidase for the catalysis of further chemiluminescence reactions. These schemes provided simple and effective sensing toward a model analyte, adenosine.     

Quenching of fluorophore-labeled DNA oligonucleotides by divalent metal ions: implications for selection, design, and applications of signaling aptamers and signaling deoxyribozymes

Recent years have seen a dramatic increase in the use of fluorescence-signaling DNA aptamers and deoxyribozymes as novel biosensing moieties. Many of these functional single-stranded DNA molecules are either engineered to function in the presence of divalent metal ion cofactors or designed as sensors for specific divalent metal ions. However, many divalent metal ions are potent fluorescence quenchers. In this study, we first set out to examine the factors that contribute to quenching of DNA-bound fluorophores by commonly used divalent metal ions, with the goal of establishing general principles that can guide future exploitation of fluorescence-signaling DNA aptamers and deoxyribozymes as biosensing probes. We then extended these studies to examine the effect of specific metals on the signaling performance of both a structure-switching signaling DNA aptamer and an RNA-cleaving and fluorescence-signaling deoxyribozyme. These studies showed extensive quenching was obtained when using divalent transition metal ions owing to direct DNA-metal ion interactions, leading to combined static and dynamic quenching. The extent of quenching was dependent on the type of metal ion and the concentration of supporting monovalent cations in the buffer, with quenching increasing with the number of unpaired electrons in the metal ion and decreasing with the concentration of monovalent ions. The extent of quenching was independent of the fluorophore, indicating that quenching cannot be alleviated simply by changing the nature of the fluorescent probe. Our results also show that the DNA sequence and the local secondary structure in the region of the fluorescent tag can dramatically influence the degree of quenching by divalent transition metal ions. In particular, the extent of quenching is predominantly determined by the fluorophore location with respect to guanine-rich and duplex regions within the strand sequence. Examination of the effect of both the type and concentration of metal ions on the performance of a fluorescence-signaling aptamer and a signaling deoxyribozyme confirms that judicious choice of divalent transition metal ions is important in maximizing signals obtained from such systems.     

Kinetic capillary electrophoresis-based affinity screening of aptamer clones

DNA aptamers are single stranded DNA (ssDNA) molecules artificially selected from random-sequence DNA libraries for their specific binding to a certain target. DNA aptamers have a number of advantages over antibodies and promise to replace them in both diagnostic and therapeutic applications. The development of DNA aptamers involves three major stages: library enrichment, obtaining individual DNA clones, and the affinity screening of the clones. The purpose of the screening is to obtain the nucleotide sequences of aptamers and the binding parameters of their interaction with the target. Highly efficient approaches have been recently developed for the first two stages, while the third stage remained the rate-limiting one. Here, we introduce a new method for affinity screening of individual DNA aptamer clones. The proposed method amalgamates: (i) aptamer amplification by asymmetric PCR (PCR with a primer ratio different from unity), (ii) analysis of aptamer-target interaction, combining in-capillary mixing of reactants by transverse diffusion of laminar flow profiles (TDLFP) and affinity analysis using kinetic capillary electrophoresis (KCE), and (iii) sequencing of only aptamers with satisfying binding parameters. For the first time we showed that aptamer clones can be directly used in TDLFP/KCE-based affinity analysis without an additional purification step after asymmetric PCR amplification. We also demonstrated that mathematical modeling of TDLFP-based mixing allows for the determination of K_d values for the in-capillary reaction of an aptamer and a target and that the obtained K_d values can be used for the accurate affinity ranking of aptamers. The proposed method does not require the knowledge of aptamer sequences before screening, avoids lengthy (3-5 h) purification steps of aptamer clones, and minimizes reagent consumption to nanoliters.     

Nonequilibrium capillary electrophoresis of equilibrium mixtures: a universal tool for development of aptamers

Aptamers are DNA (or RNA) ligands selected from large libraries of random DNA sequences and capable of binding different classes of targets with high affinity and selectivity. Both the chances for the aptamer to be selected and the quality of the selected aptamer are largely dependent on the method of selection. Here we introduce selection of aptamers by nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM). The new method has a number of advantages over conventional approaches. First, NECEEM-based selection has exceptionally high efficiency, which allows aptamer development with fewer rounds of selection. Second, NECEEM can be equally used for selecting aptamers and finding their binding parameters. Finally, due to its comprehensive kinetic capabilities, the new method can potentially facilitate selection of aptamers with predefined K(d), k(off), and k(on) of the aptamer-target interaction. In this proof-of-principle work, we describe the theoretical bases of the method and demonstrate its application to a one-step selection of DNA aptamers with nanomolar affinity for protein farnesyltransferase.     

A selective adenosine sensor derived from a triplex DNA aptamer

The aim of this study is to develop a selective adenosine aptamer sensor using a rational approach. Unlike traditional RNA aptamers developed from SELEX, duplex DNA containing an abasic site can function as a general scaffold to rationally design aptamers for small aromatic molecules. We discovered that abasic site-containing triplex DNA can also function as an aptamer and provide better affinity than duplex DNA aptamers. A novel adenosine aptamer sensor was designed using such a triplex. The aptamer is modified with furano-dU in the binding site to sense the binding. The sensor bound adenosine has a dissociation constant of 400 nM, more than tenfold stronger than the adenosine aptamer developed from SELEX. The binding quenched furano-dU fluorescence by 40%. It was also demonstrated in this study that this sensor is selective for adenosine over uridine, cytidine, guanosine, ATP, and AMP. The detection limit of this sensor is about 50 nM. The sensor can be used to quantify adenosine concentrations between 50 nM and 2 μM.     

Label free,highly sensitive and selective recognition of small molecule using gold surface confined aptamers

A highly sensitive and selective small molecule detection platform has been developed using aptamers immobilized on electrode surface. In our system, two aptamers were used – one of which is for ATP recognition and the other is for signal produce. We designed two probes L1 (containing ATP aptamer and a part of hemin aptamer) and L2 (containing the complementary strand of ATP aptamer and the rest of hemin aptamer) and immobilized L1 on electrode surface. L2 was used to hybridize to L1 and form L1–L2 duplex which brought the two parts of hemin aptamer into close proximity. Then hemin can be captured by this duplex and detected by electrochemical methods. When we introduced ATP into the system, the ATP binding destroyed the duplex and L2 diffused into the solution. As a result, hemin cannot be captured to bring electrochemical signal.     

Principal factors that determine the extension of detection range in molecular beacon aptamer/conjugated polyelectrolyte bioassays

A strategy to extend the detection range of weakly-binding targets is reported that takes advantage of fluorescence resonance energy transfer (FRET)-based bioassays based on molecular beacon aptamers (MBAs) and cationic conjugated polyelectrolytes (CPEs). In comparison to other aptamer-target pairs, the aptamer-based adenosine triphosphate (ATP) detection assays are limited by the relatively weak binding between the two partners. In response, a series of MBAs were designed that have different stem stabilities while keeping the constant ATP-specific aptamer sequence in the loop part. The MBAs are labeled with a fluorophore and a quencher at both termini. In the absence of ATP, the hairpin MBAs can be opened by CPEs via a combination of electrostatic and hydrophobic interactions, showing a FRET-sensitized fluorophore signal. In the presence of ATP, the aptamer forms a G-quadruplex and the FRET signal decreases due to tighter contact between the fluorophore and quencher in the ATP/MBA/CPE triplex structure. The FRET-sensitized signal is inversely proportional to [ATP]. The extension of the detection range is determined by the competition between opening of the ATP/MBA G-quadruplex by CPEs and the composite influence by ATP/aptamer binding and the stem interactions. With increasing stem stability, the weak binding of ATP and its aptamer is successfully compensated to show the resistance to disruption by CPEs, resulting in a substantially broadened detection range (from millimolar up to nanomolar concentrations) and a remarkably improved limit of detection. From a general perspective, this strategy has the potential to be extended to other chemical- and biological-assays with low target binding affinity.     

L-Argininamide biosensor based on S1 nuclease hydrolysis signal amplification

A sensitive method is presented for the detection of L-argininamide. It is based on the amplification of the hydrolysis of S1 nuclease of single-stranded regions of an aptamer-target complex. The S1 nuclease, which is sequence-independent, is used to “recycle” target molecules, thus leading to strongly enhanced chemiluminescence (CL). L-Argininamide was chosen as model analyte. The DNA aptamer and its complementary DNA were labeled with the CL reagent N-(4-aminobutyl)-N-ethylisoluminol (ABEI). The DNA complementary to the aptamer was labeled with ABEI and immobilized on magnetic beads (MBs) coated with gold. The aptamer was also labeled with ABEI and self-assembled on the MBs. A duplex was formed due to hybridization between the DNA aptamer and the DNA complementary to the aptamer. In the presence of the target L-argininamide, a stem-loop aptamer structure is formed which subsequently denatures the duplex. This switch from a duplex structure to a stem-loop structure causes the formation of single-stranded regions both in the target-aptamer and in the single-stranded DNA on the MBs. The nuclease hydrolyzes the single-stranded regions and single-stranded DNA. Ultimately, L-argininamide is released which then interacts with another aptamer on the MB, thereby leading to one more L-argininamide. This autocatalytic cycle can generate substantial quantities of ABEI which then can be sensitively determined by the diperiodatonickelate-isoniazide reaction system. L-argininamide can be detected in the concentration range from 3.0?×?10^?4 to 3.0?×?10^?7 M, and the limit of detection is 1.0?×?10^?7 M.

Dynamic,Bioresponsive Hydrogels via Changes in DNA Aptamer Conformation

DNA aptamers are integrated into synthetic hydrogel networks with the aim of creating hydrogels that undergo volume changes when exposed to target molecules. Specifically, single‐stranded DNA aptamers in cDNA‐bound, extended state are incorporated into hydrogel networks as cross‐links, so that the nanoscale conformational change of DNA aptamers upon binding to target molecules will induce macroscopic volume decreases of hydrogels. Hydrogels incorporating adenosine triphosphate (ATP)–binding aptamers undergo controllable volume decreases of up to 40.3 ± 4.6% when exposed to ATP, depending on the concentration of DNA aptamers incorporated in the hydrogel network, temperature, and target molecule concentration. Importantly, this approach can be generalized to aptamer sequences with distinct binding targets, as demonstrated here that hydrogels incorporating an insulin‐binding aptamer undergo volume changes in response to soluble insulin. This work provides an example of bioinspired hydrogels that undergo macroscopic volume changes that stem from conformational shifts in resident DNA‐based cross‐links.     

Surface plasmon resonance spectroscopy study of interfacial binding of thrombin to antithrombin DNA aptamers

We have applied surface plasmon resonance (SPR) spectroscopy, in combination with one-step direct binding, competition, and sandwiched assay schemes, to study thrombin binding to its DNA aptamers, with the aim to further the understanding of their interfacial binding characteristics. Using a 15-mer aptamer that binds thrombin primarily at the fibrinogen-recognition exosite as a model, we have demonstrated that introducing a DNA spacer in the aptamer enhances thrombin-binding capacity and stability, as similarly reported for hydrocarbon linkers. The bindings are aptamer surface coverage and salt concentration dependent. When free aptamers or DNA sequences complementary to the immobilized aptamer are applied after the formation of thrombin/aptamer complexes, bound thrombin is displaced to a certain extent, depending on the stability of the complexes formed under different conditions. When the 29-mer aptamer (specific to thrombin's heparin-binding exosite) is immobilized on the surface, its affinity to thrombin appears to be lower than the immobilized 15-mer aptamer, although the 29-mer aptamer is known to have a higher affinity in the solution phase. These findings underline the importance of aptamers' ability to fold into intermolecular structures and their accessibility for target capture. Using a sandwiched assay scheme followed by an additional signaling step involving biotin-streptavidin chemistry, we have observed the simultaneous binding of the 15- and 29-mer aptamers to thrombin protein at different exosites and have found that one aptamer depletes thrombin's affinity to the other when they bind together. We believe that these findings are invaluable for developing DNA aptamer-based biochips and biosensors.     

Detection of oncoprotein platelet-derived growth factor using a fluorescent signaling complex of an aptamer and TOTO

There have recently been advances in the application of aptamers, a new class of nucleic acids that bind specifically with target proteins, as protein recognition probes for biomedical study. The development of a signaling aptamer with the capability of simple and rapid real-time detection of disease-related proteins has attracted increasing interest. We have recently reported a new protein-detection strategy using a signaling aptamer based on a DNA molecular light-switching complex, [Ru(phen)₂(dppz)]²⁺. In this work we have used the commercially available DNA-intercalating dye, TOTO, to replace [Ru(phen)₂(dppz)]²⁺ for detection of oncoprotein platelet-derived growth factor BB (PDGF-BB), a potential cancer marker. Taking advantage of the high affinity of the aptamer to PDGF-BB and the sensitive fluorescence change of the aptamer–TOTO signaling complex on protein binding, PDGF-BB was detected in physiological buffer with high selectivity and sensitivity. The detection limit was 0.1 nmol L⁻¹, which was better than that of other reported aptamer-based methods for PDGF-BB, including that using [Ru(phen)₂(dppz)]²⁺. The method is very simple with no need for covalent labeling of the aptamer or probe synthesis. It facilitates wide application of the signaling mechanism to the analysis and study of cancer markers and other proteins.      

A Colorimetric Biosensing Platform with Aptamers,Rolling Circle Amplification and Urease-Mediated Litmus Test

Here we report on an ultra-sensitive colorimetric sensing platform that takes advantage of both the strong amplification power of rolling circle amplification (RCA) and the high efficiency of a simple urease-mediated litmus test. The presence of a target triggers the RCA reaction, and urease-labelled DNA can hybridize to the biotinylated RCA products and be immobilized onto streptavidin-coated magnetic beads. The urease-laden beads are then used to hydrolyze urea, leading to an increase in pH that can be detected by a simple litmus test. We show this sensing platform can be easily integrated with aptamers for sensing diverse targets via the detection of human thrombin and platelet-derived growth factor (PDGF) utilizing structure-switching aptamers as well as SARS-CoV-2 in human saliva using a spike-binding trimeric DNA aptamer. Furthermore, we demonstrate that this colorimetric sensing platform can be integrated into a simple paper-based device for sensing applications.     

Bioresponsive controlled release using mesoporous silica nanoparticles capped with aptamer-based molecular gate

This communication describes the design of a novel and general bioresponsive controlled-release mesoporous silica (MS) nanoparticles system based on aptamer-target interactions. In this system, the pores of MS were capped with Au nanoparticles modified with aptamer (ATP aptamer in this case). By a competitive displacement reaction, the Au nanoparticles were uncapped in the presence of ATP molecule, and the cargo was released. Our results demonstrated that the aptamer-target interaction may be a promising route for the design of custom-made controlled-release nanodevices specifically governed by target biomolecules. Since aptamers have been obtained for a broad range of targets, including several cancer biomarkers, we believe that this aptamer-based controlled-release system should have an equally broad spectrum of applications.     

Cell-specific internalization study of an aptamer from whole cell selection

Nucleic acid aptamers have been shown many unique applications as excellent probes in molecular recognition. However, few examples are reported which show that aptamers can be internalized inside living cells for aptamer functional studies and for targeted intracellular delivery. This is mainly due to the limited number of aptamers available for cell-specific recognition, and the lack of research on their extra- and intracellular functions. One of the major difficulties in aptamers' in vivo application is that most of aptamers, unlike small molecules, cannot be directly taken up by cells without external assistance. In this work, we have studied a newly developed and cell-specific DNA aptamer, sgc8. This aptamer has been selected through a novel cell selection process (cell-SELEX), in which whole intact cells are used as targets while another related cell line is used as a negative control. The cell-SELEX enables generation of multiple aptamers for molecular recognition of the target cells and has significant advantages in discovering cell surface binding molecules for the selected aptamers. We have studied the cellular internalization of one of the selected aptamers. Our results show that sgc8 is internalized efficiently and specifically to the lymphoblastic leukemia cells. The internalized sgc8 aptamers are located inside the endosome. Comparison studies are done with the antibody for the binding protein of sgc8, PTK7 (Human protein tyrosine kinase-7) on cell surface. We also studied the internalization kinetics of both the aptamer and the antibody for the same protein on the living cell surface. We have further evaluated the effects of sgc8 on cell viability, and no cytotoxicity is observed. This study indicates that sgc8 is a promising agent for cell-type specific intracellular delivery.