Switchable Fluorescent Light-Up Aptamers Based on Riboswitch Architectures

Fluorescent light-up RNA aptamers (FLAPs) such as Spinach or Mango can bind small fluorogens and activate their fluorescence. Here, we adopt a switching mechanism otherwise found in riboswitches and use it to engineer switchable FLAPs that can be activated or repressed by trigger oligonucleotides or small metabolites. The fluorophore binding pocket of the FLAPs comprises guanine (G) quadruplexes, whose critical nucleotides can be sequestered by corresponding anti-FLAP sequences, leading to an inactive conformation and thus preventing association with the fluorophore. We modified the FLAPs with designed toehold hairpins that carry either an anti-FLAP or an anti-anti-FLAP sequence within the loop region. The addition of an input RNA molecule triggers a toehold-mediated strand invasion process that refolds the FLAP into an active or inactive configuration. Several of our designs display close-to-zero leak signals and correspondingly high ON/OFF fluorescence ratios. We also modified purine aptamers to sequester a partial anti-FLAP or an anti-anti-FLAP sequence to control the formation of the fluorogen-binding conformation, resulting in FLAPs whose fluorescence is activated or deactivated in the presence of guanine or adenine. We demonstrate that switching modules can be easily combined to generate FLAPs whose fluorescence depends on several inputs with different types of input logic.     

Monitoring Fluorescence Response of Amphiphilic Flapping Molecules in Compressed Monolayers at the Air–Water Interface

The air–water interface, which is the boundary of two phases with a large difference in polarity, gives a distinct environment compared with bulk water or air. Since the interface provides a field for various biomolecules to work, it is important to understand the molecular behaviors at the interface. Here, polarity‐independent flapping viscosity probes (FLAP) equipped with hydrophobic/hydrophilic substituents have been synthesized and studied at the air–water interface. In situ fluorescence (FL), which is related to the internal motion and orientation, of three different FLAPs were investigated at the interface, and the internal motion of the molecule was indicated to be suppressed at the interface. In addition, the molecular response was compared with that of conventional viscosity probes (molecular rotors), which indicates the different behaviors of FLAP probably due to the distinct molecular orientation as well as molecular motion.     

Aptamers with Tunable Affinity Enable Single-Molecule Tracking and Localization of Membrane Receptors on Living Cancer Cells

Tumor cell-surface markers are usually overexpressed or mutated protein receptors for which spatiotemporal regulation differs between and within cancers. Single-molecule fluorescence imaging can profile individual markers in different cellular contexts with molecular precision. However, standard single-molecule imaging methods based on overexpressed genetically encoded tags or cumbersome probes can significantly alter the native state of receptors. We introduce a live-cell points accumulation for imaging in nanoscale topography (PAINT) method that exploits aptamers as minimally invasive affinity probes. Localization and tracking of individual receptors are based on stochastic and transient binding between aptamers and their targets. We demonstrated single-molecule imaging of a model tumor marker (EGFR) on a panel of living cancer cells. Affinity to EGFR was finely tuned by rational engineering of aptamer sequences to define receptor motion and/or native receptor density.     

Intramers as promising new tools in functional proteomics

Aptamers are valuable tools for studying numerous aspects of biological processes, opening up new experimental opportunities to analyse the function of a wide range of cellular molecules. Functional RNA molecules can be rapidly selected in vitro from complex combinatorial mixtures of different sequences. Recently, it was shown that in vitro selection processes can be automated: the first generation selection robots will soon mean aptamers for several targets can be isolated in parallel within days rather than weeks. Aptamers not only exhibit highly specific molecular recognition properties but are also able to modulate the function of their cognate targets in a highly specific manner by agonistic or antagonistic mechanisms. These properties prompted the development of novel technologies to exploit the use of aptamers to modulate distinct functions of biological targets. Recent controlled expression of aptamers inside cells demonstrated their impressive potential as rapidly generated intracellular inhibitors of biomolecules. Intracellularly applied aptamers are also called 'intramers'. Here we discuss recent developments and strategies for intramer-based technologies that have the potential to greatly facilitate characterisation of unknown protein functions in the context of their natural expression status in vivo. Thus, intramer-based technologies offer many promising applications in functional genomics, proteomics and drug discovery.     

Supramolecular Fluorescence Resonance Energy Transfer in Nucleobase‐Modified Fluorogenic RNA Aptamers

RNA aptamers form compact tertiary structures and bind their ligands in specific binding sites. Fluorescence‐based strategies reveal information on structure and dynamics of RNA aptamers. Herein, we report the incorporation of the universal emissive nucleobase analog 4‐cyanoindole into the fluorogenic RNA aptamer Chili, and its application as a donor for supramolecular FRET to the bound ligands DMHBI⁺ or DMHBO⁺. The photophysical properties of the new nucleobase–ligand‐FRET pair revealed structural restraints for the overall RNA aptamer organization and identified nucleotide positions suitable for FRET‐based readout of ligand binding. This strategy is generally suitable for binding‐site mapping and may also be applied for responsive aptamer devices.     

Multifunctional G‐Quadruplex Aptamers and Their Application to Protein Detection

Two significant G‐quadruplex aptamers named AGRO100 and T30695 are identified as multifunctional aptamers that can bind the protein ligands nucleolin or HIV‐1 integrase and hemin. Besides their strong binding to target proteins, both AGRO100 and T30695 exhibit high hemin‐binding affinities comparable to that of the known aptamer (termed PS2M) selected by the in vitro evolution process. Most importantly, their corresponding hemin–DNA complexes reveal excellent peroxidase‐like activities, higher than that of the reported hemin–PS2M DNAzyme. This enables these multifunctional aptamers to be applied to the sensitive detection of proteins, which is demonstrated by applying AGRO100 to the chemiluminescence detection of nucleolin expressed at the surface of HeLa cells. Based on the specific AGRO100–nucleolin interaction, the surface‐expressed nucleolin of HeLa cells is labeled in situ with the hemin–AGRO100 DNAzyme, and then determined in the luminol–H₂O₂ system. Through this approach, the sensitive detection of total nucleolin expressed at the surface of about 6000 HeLa cells is accomplished. Our results suggest that exploiting new functions of existing aptamers will help to extend their potential applications in the biochemical field.     

Filterable Agents for Hyperpolarization of Water,Metabolites, and Proteins

Hyperpolarization is generated by dissolution dynamic nuclear polarization (d‐DNP) using a polymer‐based polarizing agent dubbed FLAP (filterable labeled agents for polarization). It consists of a thermo‐responsive poly(N‐isopropylacrylamide), also known as pNiPAM‐COOH, labeled with nitroxide radicals. The polymer powder is impregnated with an arbitrary solution of interest and frozen as is. Dissolution is followed by a simple filtration, leading to hyperpolarized solutions free from any contaminants. We demonstrated the use of FLAP to hyperpolarize partially deuterated water up to P(¹H)=6 % with a long relaxation T₁ >36 s characteristic of high purity. Water hyperpolarization can be transferred to drugs, metabolites, or proteins that are waiting in an NMR spectrometer, either by exchange of labile protons or through intermolecular Overhauser effects. We also show that FLAPs are suitable polarizing agents for ¹³C‐labeled metabolites such as pyruvate, acetate, and alanine.     

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.     

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.     

Selection and Characterization of Vimentin-Binding Aptamer Motifs for Ovarian Cancer

The application of aptamers in biomedicine is emerging as an essential technology in the field of cancer research. As small single-stranded DNA or RNA ligands with high specificity and low immunogenicity for their targets, aptamers provide many advantages in cancer therapeutics over protein-based molecules, such as antibodies. Vimentin is an intermediate filament protein that is overexpressed in endothelial cells of cancerous tissue. High expression levels of vimentin have been associated with increased capacity for migration and invasion of the tumor cells. We have selected and identified thioated aptamers with high specificity for vimentin using human ovarian cancer tissues. Tentative binding motifs were chosen for two vimentin aptamers based on predicted secondary structures. Each of these shorter, tentative binding motifs was synthesized, purified, and characterized via cell binding assays. Two vimentin binding motifs with high fidelity binding were selected and further characterized via cell and tissue binding assays, as well as flow cytometric analysis. The equilibrium binding constants of these small thioated aptamer constructs were also determined. Future applications for the vimentin binding aptamer motifs include conjugation of the aptamers to synthetic dyes for use in targeted imaging and therapy, and ultimately more detailed and precise monitoring of treatment response and tumor progression in ovarian pathology.     

FRET in a Synthetic Flavin‐ and Bilin‐binding Protein

The last decade has seen development and application of a large number of novel fluorescence‐based techniques that have revolutionized fluorescence microscopy in life sciences. Preferred tags for such applications are genetically encoded fluorescent proteins (FP), mostly derivatives of the green fluorescent protein (GFP). Combinations of FPs with wavelength‐separated absorption/fluorescence properties serve as excellent tools for molecular interaction studies, for example, protein–protein complexes or enzyme–substrate interactions, based on the FRET phenomenon (Förster resonance energy transfer). However, alternatives are requested for experimental conditions where FP proteins or FP couples are not or less efficiently applicable. We here report as a “proof of principle” a specially designed, non‐naturally occurring protein (LG1) carrying a combination of a flavin‐binding LOV‐ and a photochromic bilin‐binding GAF domain and demonstrate a FRET process between both chromophores.     

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.     

Controlling protein activity with ligand-regulated RNA aptamers

Controlling the activity of a protein is necessary for defining its function in vivo. RNA aptamers are capable of inhibiting proteins with high affinity and specificity, but this effect is not readily reversible. We describe a general method for discovering aptamers that bind and inhibit their target protein, but addition of a specific small molecule disrupts the protein-RNA complex. A SELEX protocol was used to raise RNA aptamers to the DNA repair enzyme, formamidopyrimidine glycosylase (Fpg), and neomycin was employed in each round to dissociate Fpg-bound RNAs. We identified an RNA molecule able to completely inhibit Fpg at 100 nM concentration. Importantly, Fpg activity is recovered by the addition of neomycin. We envision these ligand-regulated aptamers (LIRAs) as valuable tools in the study of biological phenomena in which the timing of molecular events is critical.     

Antibody Activation using DNA‐Based Logic Gates

Oligonucleotide‐based molecular circuits offer the exciting possibility to introduce autonomous signal processing in biomedicine, synthetic biology, and molecular diagnostics. Here we introduce bivalent peptide–DNA conjugates as generic, noncovalent, and easily applicable molecular locks that allow the control of antibody activity using toehold‐mediated strand displacement reactions. Employing yeast as a cellular model system, reversible control of antibody targeting is demonstrated with low nM concentrations of peptide–DNA locks and oligonucleotide displacer strands. Introduction of two different toehold strands on the peptide–DNA lock allowed signal integration of two different inputs, yielding logic OR‐ and AND‐gates. The range of molecular inputs could be further extended to protein‐based triggers by using protein‐binding aptamers.     

Expanding molecular modeling and design tools to non‐natural sidechains

Protein–protein interactions encode the wiring diagram of cellular signaling pathways and their deregulations underlie a variety of diseases, such as cancer. Inhibiting protein–protein interactions with peptide derivatives is a promising way to develop new biological and therapeutic tools. Here, we develop a general framework to computationally handle hundreds of non‐natural amino acid sidechains and predict the effect of inserting them into peptides or proteins. We first generate all structural files (pdb and mol2), as well as parameters and topologies for standard molecular mechanics software (CHARMM and Gromacs). Accurate predictions of rotamer probabilities are provided using a novel combined knowledge and physics based strategy. Non‐natural sidechains are useful to increase peptide ligand binding affinity. Our results obtained on non‐natural mutants of a BCL9 peptide targeting beta‐catenin show very good correlation between predicted and experimental binding free‐energies, indicating that such predictions can be used to design new inhibitors. Data generated in this work, as well as PyMOL and UCSF Chimera plug‐ins for user‐friendly visualization of non‐natural sidechains, are all available at http://www.swisssidechain.ch . Our results enable researchers to rapidly and efficiently work with hundreds of non‐natural sidechains. © 2012 Wiley Periodicals, Inc.     

Kinetically Tunable Photostability of Fluorogen‐Activating Peptide–Fluorogen Complexes

Ease of genetic encoding, labeling specificity, and high photostability are the most sought after qualities in a fluorophore for biological detection. Furthermore, many applications can gain from the fluorogenic nature of fluoromodules and the ability to turn on the same fluoromodules multiple times. Fluorogen‐activating peptides (FAPs) bind noncovalently to their cognate fluorogens and exhibit enhanced photostability. Herein, the photostabilities of malachite green (MG)‐binding and thiazole‐orange‐binding FAPs are compared under limiting‐ and excess‐fluorogen conditions to establish distinct mechanisms for photostability that correspond to the dissociation rate of the FAP–fluorogen complex. FAPs with slow dissociation show evidence of dye encapsulation and protection from photo or environmental degradation and single‐step bleaching at the single molecule level, whereas those with rapid dissociation show repeated cycles of binding and enhanced photostability by exchange of bleached fluorogen with a new dye. A combination of generalizable selection pressure based on bleaching, flow cytometry, and site‐specific amino acid mutagenesis is used to obtain a modified FAP with enhanced photostability, due to rapid dissociation of the MG fluorogen. These studies shed light on the basic mechanisms by which noncovalent association can effect photostable labeling, and demonstrate novel reagents for photostable and intermittent labeling of biological targets.     

High-throughput binding characterization of RNA aptamer selections using a microplate-based multiplex microcolumn device

We describe a versatile 96-well microplate-based device that utilizes affinity microcolumn chromatography to complement downstream plate-based processing in aptamer selections. This device is reconfigurable and is able to operate in serial and/or parallel mode with up to 96 microcolumns. We demonstrate the utility of this device by simultaneously performing characterizations of target binding using five RNA aptamers and a random library. This was accomplished through 96 total selection tests. Three sets of selections tested the effects of target concentration on aptamer binding compared to the random RNA library using aptamers to the proteins green fluorescent protein (GFP), human heat shock factor 1 (hHSF1), and negative elongation factor E (NELF-E). For all three targets, we found significant effects consistent with steric hindrance with optimum enrichments at predictable target concentrations. In a fourth selection set, we tested the partitioning efficiency and binding specificity of our three proteins’ aptamers, as well as two suspected background binding sequences, to eight targets running serially. The targets included an empty microcolumn, three affinity resins, three specific proteins, and a non-specific protein control. The aptamers showed significant enrichments only on their intended targets. Specifically, the hHSF1 and NELF-E aptamers enriched over 200-fold on their protein targets, and the GFP aptamer enriched 750-fold. By utilizing our device’s plate-based format with other complementary plate-based systems for all downstream biochemical processes and analysis, high-throughput selections, characterizations, and optimization were performed to significantly reduce the time and cost for completing large-scale aptamer selections.

Native Top‐Down Mass Spectrometry of TAR RNA in Complexes with a Wild‐Type tat Peptide for Binding Site Mapping

Ribonucleic acids (RNA) frequently associate with proteins in many biological processes to form more or less stable complex structures. The characterization of RNA–protein complex structures and binding interfaces by nuclear magnetic resonance (NMR) spectroscopy, X‐ray crystallography, or strategies based on chemical crosslinking, however, can be quite challenging. Herein, we have explored the use of an alternative method, native top‐down mass spectrometry (MS), for probing of complex stoichiometry and protein binding sites at the single‐residue level of RNA. Our data show that the electrostatic interactions between HIV‐1 TAR RNA and a peptide comprising the arginine‐rich binding region of tat protein are sufficiently strong in the gas phase to survive phosphodiester backbone cleavage of RNA by collisionally activated dissociation (CAD), thus allowing its use for probing tat binding sites in TAR RNA by top‐down MS. Moreover, the MS data reveal time‐dependent 1:2 and 1:1 stoichiometries of the TAR–tat complexes and suggest structural rearrangements of TAR RNA induced by binding of tat peptide.     

Modified capillary electrophoresis based measurement of the binding between DNA aptamers and an unknown concentration target

The recognition of targets such as biomacromolecules, viruses and cells by their aptamers is crucial in aptamer-based biosensor platforms and research into protein function. However, it is difficult to evaluate the binding constant of aptamers and their targets that are hard to purify and quantify, especially when the targets are undefined. Therefore, we aimed to develop a modified capillary electrophoresis based method to determine the dissociation constant of aptamers whose targets are hard to quantify. A protein target, human thrombin, and one of its aptamers were used to validate our modified method. We demonstrated that the result calculated by our method, only depending on the aptamer’s concentrations, was consistent with the classical method, which depended on the concentrations of both the aptamers and the targets. Furthermore, a series of DNA aptamers binding with avian influenza virus H9N2 were confirmed by a four-round selection of capillary electrophoresis–systematic evolution of ligands by exponential enrichment, and we identified the binding constant of these aptamers by directly using the whole virus as the target with the modified method. In conclusion, our modified method was validated to study the interaction between the aptamer and its target, and it may also advance the evaluation of other receptor–ligand interactions.     

Recent Progress in Aptamer‐Based Functional Probes for Bioanalysis and Biomedicine

Nucleic acid aptamers are short synthetic DNA or RNA sequences that can bind to a wide range of targets with high affinity and specificity. In recent years, aptamers have attracted increasing research interest due to their unique features of high binding affinity and specificity, small size, excellent chemical stability, easy chemical synthesis, facile modification, and minimal immunogenicity. These properties make aptamers ideal recognition ligands for bioanalysis, disease diagnosis, and cancer therapy. This review highlights the recent progress in aptamer selection and the latest applications of aptamer‐based functional probes in the fields of bioanalysis and biomedicine.