Theophylline Aptamer Research Articles

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.

Trans-acting aptazyme for conditional gene knockdown in eukaryotic cells

Trans-acting hammerhead ribozyme inherits the advantages of being the smallest and best-characterized RNA-cleaving ribozyme, offering high modularity and the ability to cleave any desired sequence without the aid of any protein, as long as the target sequence contains a cleavage site. However, achieving precise control over the trans-acting hammerhead ribozyme would enable safer and more accurate regulation of gene expression. Herein, we described an intracellular selection of hammerhead aptazyme that contains a theophylline aptamer on stem II based on toxin protein IbsC. Based on the intracellular selection, we obtained three new cis-acting hammerhead aptazymes. Moreover, the corresponding trans-acting aptazymes could be efficiently induced by theophylline to knock down different targeted genes in eukaryotic cells. Notably, the best one, T195, exhibited a ligand-dependent and dose-dependent response to theophylline, and the cleavage efficiency could be enhanced by incorporating multiplex aptazymes.

Kinetic competition between theophylline sensitive ribozyme cleavage and termination in living cells by 3D orbital tracking-fluorescent cross-correlation spectroscopy.

Synthetic RNA-based regulatory tools such as riboswitches are promising candidates for the realization of complex engineered genetic circuits. However, previous efforts to implement such tools in synthetic biology have yielded inconsistent results. Often the quantification of performance is measured using protein expression, even though these devices act at the RNA level. To overcome this, we use in vivo RNA labeling of Hemoglobin B in Human Epithelial Kidney T-Rex Cells to determine two objectives. First, we will determine whether a theophylline-dependent hammerhead ribozyme riboswitch alters gene expression by inducing early termination of transcription or via post-transcriptional mRNA cleavage. The second objective will use a self-cleaving hammerhead ribozyme to separate transcription elongation from termination. This will allow for the determination of transcription termination kinetics. Quantification for both objectives will be completed using 3D Orbital Tracking-Fluorescent Cross-Correlation Spectroscopy (3DOT-FCCS). To determine the levels of post-transcription mRNA cleavage, we will use Raster Image Correlation Spectroscopy (RICS) to measure mRNA stability via mRNA lifetime. Preliminary data suggests that the theophylline aptamer stabilizes the ribozyme, correlating to an increased RNA dwell time and termination. However, when the aptamer is absent, there is a decrease. This indicates early termination and premature transcript release from the DNA template are occurring with the ribozyme. In the future, we will examine the transcribing order of Intron 1 and Intron 2, splicing behaviors, post-termination splicing using RICS, and RNA lifetime.

Discovery of small molecules that target a tertiary-structured RNA.

There is growing interest in therapeutic intervention that targets disease-relevant RNAs using small molecules. While there have been some successes in RNA-targeted small-molecule discovery, a deeper understanding of structure-activity relationships in pursuing these targets has remained elusive. One of the best-studied tertiary-structured RNAs is the theophylline aptamer, which binds theophylline with high affinity and selectivity. Although not a drug target, this aptamer has had many applications, especially pertaining to genetic control circuits. Heretofore, no compound has been shown to bind the theophylline aptamer with greater affinity than theophylline itself. However, by carrying out a high-throughput screen of low-molecular-weight compounds, several unique hits were identified that are chemically distinct from theophylline and bind with up to 340-fold greater affinity. Multiple atomic-resolution X-ray crystal structures were determined to investigate the binding mode of theophylline and four of the best hits. These structures reveal both the rigidity of the theophylline aptamer binding pocket and the opportunity for other ligands to bind more tightly in this pocket by forming additional hydrogen-bonding interactions. These results give encouragement that the same approaches to drug discovery that have been applied so successfully to proteins can also be applied to RNAs.

Theophylline-induced synergic activation of guide RNA to control CRISPR/Cas9 function.

Ligand-induced activation of CRISPR/Cas9 function is achieved based on a synergic approach through the integration of the theophylline aptamer into protein-unrecognized regions of guide RNA. This design of allosteric regulation opens a new window towards the broad involvement of RNA aptamers for conditional control of CRISPR/Cas9 function.

Identifying conformational changes of aptamer binding to theophylline: A combined biolayer interferometry, surface-enhanced Raman spectroscopy, and molecular dynamics study

Theophylline is a potent bronchodilator for the treatment of asthma, bronchitis, and emphysema. Its narrow therapeutic window (20–100 μM) demands that the blood concentration of theophylline be monitored carefully, which can be achieved by aptamer capture. Thus, an understanding of what occurs when aptamers bind to theophylline is critical for identifying a high-affinity and high-specificity aptamer, which improve the sensitivity and selectivity of theophylline detection. Consequently, there is an urgent need to develop a simple, convenient, and nondestructive method to monitor conformational changes during the binding process. Here, we report the determination of the affinity of a selected aptamer and theophylline via biolayer interferometry (BLI) experiments. Additionally, using surface-enhanced Raman spectroscopy (SERS), the conformational changes on theophylline–aptamer binding were identified from differences in the SER spectra. Finally, molecular dynamics (MD) simulations were used to identify the specific conformational changes of the aptamer during the binding process. Such a combined BLI-SERS-MD method provides an in-depth understanding of the theophylline–aptamer binding processes and a comprehensive explanation for conformational changes, which helps to select, design, and modify an aptamer with high affinity and specificity. It can also be used as a scheme for the study of other aptamer–ligand interactions, which can be applied to the detection, sensing, clinical diagnosis, and treatment of diseases.

A New Method for Converting Aptamers to Riboswitches Using the Theophylline Riboswitch

Fluorescence Activated Cell Sorting, or FACS, is a method that was used to convert the theophylline aptamer into a riboswitch. This method could theoretically be used to convert other discovered aptamers into riboswitches, however it is a costly method and is only available to those with access to these high‐tech, expensive machines. The theophylline riboswitch was previously discovered by implementing the theophylline aptamer, with random sequences linking it to the expression platform, into a specifically‐designed plasmid, and then using a FACS machine to sort the cells.We can structure a new system that would select only the correct sequence, containing the theophylline riboswitch out of a pool, without the use of a FACS machine. To do so, we place the pool of sequences containing the aptamer, linked to the Shine Dalgarno by eight randomized nucleotides, into a newly designed plasmid, placNEO, and transform the plasmid into bacteria cells. Then, the use of replica plating along with screening will select the cells that only contain the plasmid with the correct riboswitch sequence. By doing so, we confirm that this system is efficient in converting aptamers into riboswitches without the need for a FACS machine.After an aptamer has been successfully converted into its riboswitch, the system of ratiometric fluorescence will allow for testing of the riboswitch’s function. This is done by designing a plasmid, pTRFlac, that contains genes for red and green fluorescence proteins, mCherry and GFP respectively, on either side of the inserted riboswitch. A PCR product encoding for mCherry, the riboswitch, and GFP will be inserted downstream of the lactose operon in pUC18. Ratios of the fluorescence intensities of the two florescent proteins will provide the ability to measure the riboswitch’s function through fluorescence readings.Support or Funding InformationMonmouth University Summer Research Program ICFNJ

The Theophylline Aptamer: 25 Years as an Important Tool in Cellular Engineering Research.

The theophylline aptamer was isolated from an oligonucleotide library in 1994. Since that time, the aptamer has found wide utility, particularly in synthetic biology, cellular engineering, and diagnostic applications. The primary application of the theophylline aptamer is in the construction and characterization of synthetic riboswitches for regulation of gene expression. These riboswitches have been used to control cellular motility, regulate carbon metabolism, construct logic gates, screen for mutant enzymes, and control apoptosis. Other applications of the theophylline aptamer in cellular engineering include regulation of RNA interference and genome editing through CRISPR systems. Here we describe the uses of the theophylline aptamer for cellular engineering over the past 25 years. In so doing, we also highlight important synthetic biology applications to control gene expression in a ligand-dependent manner.

Construction and application of theophylline-activated RNA switches in the regulation of expression of recombinant proteins in Bacillus subtilis

Bacillus subtilis can be widely used as an important microorganism for metabolic engineering and recombinant proteins expression in industrial biotechnology and synthetic biology. However, it is difficult to make accurate regulation of exogenous gene by biological tools in B. subtilis, which limits the application of B. subtilis in synthetic biology. The purpose of this study is to develop regulatory tools for precise control of gene expression by using non-coding RNAs, by which the activation of heterologous gene could be achieved without the auxiliary protein factors. We constructed the synthetic riboswitch E and aptazyme AZ using the theophylline aptamer. Six different native promoters from B. subtilis were functionally adapted with the E and AZ to fabricate an array of novel regulatory elements activated by theophylline. Then, we determined the performance of these elements using green fluorescence protein as reporter, and then further verified using red fluorescence protein and pullulanase as cargo proteins. Results showed that the same kind of RNA elements with different promoters showed different levels of efficiency. Promoter PsigW and E combination (sigWE) had the highest induction rate in B. subtilis. Compared with the control group, it can produce the induction rate of 16.8. Promoter PrpoB and AZ combination (rpoBAZ) showed the highest induction rate of 6.2. SigWE mediated mCherry induction rate was 9.2, and P43E mediated pullulanase induction rate was 32.8, in which enzyme activity reached 81 U/mL. This study confirmed that GFP, mCherry and pullulan can all be regulated by riboswitch and aptazyme, but there were differences between different combinations of promoters with RNA regulators.

Second-harmonic generation-based methods to detect and characterize ligand-induced RNA conformational changes.

Second-harmonic generation (SHG) is a biophysical tool that senses ligand-induced conformational changes in biomolecules. The Biodesy Delta™ has been developed as a high-throughput screening platform to monitor conformational changes in proteins and oligonucleotides by SHG to support drug discovery efforts. This work will outline (1) an overview of this technology, (2) detailed protocols for optimizing screening-ready SHG assays on RNA targets, (3) practical considerations for developing robust and informative SHG measurements, and (4) a case study that demonstrates the application of these recommendations on an RNA target. The previously published theophylline aptamer SHG assay [1] was further optimized to maximize the assay window between the positive control (theophylline) and the negative control (caffeine). Optimization of this assay provides practical considerations for building a robust SHG assay on an RNA target, including testing for specific tethering of the conjugate to the surface as well as testing tool compound response stability, reversibility, and concentration-dependence/affinity. A more robust, better-performing theophylline aptamer SHG assay was achieved that would be more appropriate for conducting a screen.

Next-level riboswitch development-implementation of Capture-SELEX facilitates identification of a new synthetic riboswitch.

The development of synthetic riboswitches has always been a challenge. Although a number of interesting proof-of-concept studies have been published, almost all of these were performed with the theophylline aptamer. There is no shortage of small molecule-binding aptamers; however, only a small fraction of them are suitable for RNA engineering since a classical SELEX protocol selects only for high-affinity binding but not for conformational switching. We now implemented RNA Capture-SELEX in our riboswitch developmental pipeline to integrate the required selection for high-affinity binding with the equally necessary RNA conformational switching. Thus, we successfully developed a new paromomycin-binding synthetic riboswitch. It binds paromomycin with a KD of 20 nM and can discriminate between closely related molecules both in vitro and in vivo. A detailed structure–function analysis confirmed the predicted secondary structure and identified nucleotides involved in ligand binding. The riboswitch was further engineered in combination with the neomycin riboswitch for the assembly of an orthogonal Boolean NOR logic gate. In sum, our work not only broadens the spectrum of existing RNA regulators, but also signifies a breakthrough in riboswitch development, as the effort required for the design of sensor domains for RNA-based devices will in many cases be much reduced.

In silico design of ligand triggered RNA switches.

This contribution sketches a work flow to design an RNA switch that is able to adapt two structural conformations in a ligand-dependent way. A well characterized RNA aptamer, i.e., knowing its Kd and adaptive structural features, is an essential ingredient of the described design process. We exemplify the principles using the well-known theophylline aptamer throughout this work. The aptamer in its ligand-binding competent structure represents one structural conformation of the switch while an alternative fold that disrupts the binding-competent structure forms the other conformation. To keep it simple we do not incorporate any regulatory mechanism to control transcription or translation. We elucidate a commonly used design process by explicitly dissecting and explaining the necessary steps in detail. We developed a novel objective function which specifies the mechanistics of this simple, ligand-triggered riboswitch and describe an extensive in silico analysis pipeline to evaluate important kinetic properties of the designed sequences. This protocol and the developed software can be easily extended or adapted to fit novel design scenarios and thus can serve as a template for future needs.

Molecular simulations and Markov state modeling reveal the structural diversity and dynamics of a theophylline-binding RNA aptamer in its unbound state.

RNA aptamers are oligonucleotides that bind with high specificity and affinity to target ligands. In the absence of bound ligand, secondary structures of RNA aptamers are generally stable, but single-stranded and loop regions, including ligand binding sites, lack defined structures and exist as ensembles of conformations. For example, the well-characterized theophylline-binding aptamer forms a highly stable binding site when bound to theophylline, but the binding site is unstable and disordered when theophylline is absent. Experimental methods have not revealed at atomic resolution the conformations that the theophylline aptamer explores in its unbound state. Consequently, in the present study we applied 21 microseconds of molecular dynamics simulations to structurally characterize the ensemble of conformations that the aptamer adopts in the absence of theophylline. Moreover, we apply Markov state modeling to predict the kinetics of transitions between unbound conformational states. Our simulation results agree with experimental observations that the theophylline binding site is found in many distinct binding-incompetent states and show that these states lack a binding pocket that can accommodate theophylline. The binding-incompetent states interconvert with binding-competent states through structural rearrangement of the binding site on the nanosecond to microsecond timescale. Moreover, we have simulated the complete theophylline binding pathway. Our binding simulations supplement prior experimental observations of slow theophylline binding kinetics by showing that the binding site must undergo a large conformational rearrangement after the aptamer and theophylline form an initial complex, most notably, a major rearrangement of the C27 base from a buried to solvent-exposed orientation. Theophylline appears to bind by a combination of conformational selection and induced fit mechanisms. Finally, our modeling indicates that when Mg2+ ions are present the population of binding-competent aptamer states increases more than twofold. This population change, rather than direct interactions between Mg2+ and theophylline, accounts for altered theophylline binding kinetics.

Inducible Bcl-2 gene RNA interference mediated by aptamer-integrated HDV ribozyme switch.

The regulation of RNA interference (RNAi) could be a powerful method for the study of temporal and dose dependent effects of gene expression. In this study, we designed the hepatitis delta virus (HDV) ribozyme with an embedded theophylline aptamer as the sensor domain and the pri-miRNA of endogenous gene Bcl-2 as the effector domain to engineer an RNAi-regulatory device in MCF-7 cells. The system allowed us to control gene expression by adding theophylline into the culture media in a dose dependent fashion. This is the pioneering application of ribozyme switches to activate RNAi for modulating endogenous genes in mammalian cells. The platform sets the stage for investigations of other endogenous genes that regulate various biological functions such as differentiation, cell division or cell death, and provides a promising interface with other universal RNAi-based decision-making circuits that operate in mammalian cells. It can be used to study more genes associated with cancer and screen for potential drug targets for gene therapy.

Regulation of MAP4K4 gene expression by RNA interference through an engineered theophylline-dependent hepatitis delta virus ribozyme switch.

Riboswitches are functional non-coding RNA regulatory components that play an important role in the regulation of gene expression in diverse organisms. In particular, using riboswitches to modulate RNA interference (RNAi) enables temporal and spatial control of gene expression in mammalian cells. Herein, a ribozyme gene switch to activate RNAi was fabricated for the artificial regulation of versatile gene silencing through the interaction of an RNA aptamer with small molecules. The device comprised an allosteric HDV ribozyme with an embedded theophylline aptamer and a primary miRNA (pri-miRNA) to silence the MAP4K4 gene in hepatic (HepG2) cells, aiming to achieve dose-dependent control of the activation of RNAi, and then the regulation of the MAP4K4 gene by theophylline. Finally, we demonstrated the feasibility and applicability of utilizing HDV ribozyme switches to activate RNAi for regulating an endogenous gene in mammalian cells.

Multi-objective optimization for RNA design with multiple target secondary structures.

BackgroundRNAs are attractive molecules as the biological parts for synthetic biology. In particular, the ability of conformational changes, which can be encoded in designer RNAs, enables us to create multistable molecular switches that function in biological circuits. Although various algorithms for designing such RNA switches have been proposed, the previous algorithms optimize the RNA sequences against the weighted sum of objective functions, where empirical weights among objective functions are used. In addition, an RNA design algorithm for multiple pseudoknot targets is currently not available.ResultsWe developed a novel computational tool for automatically designing RNA sequences which fold into multiple target secondary structures. Our algorithm designs RNA sequences based on multi-objective genetic algorithm, by which we can explore the RNA sequences having good objective function values without empirical weight parameters among the objective functions. Our algorithm has great flexibility by virtue of this weight-free nature. We benchmarked our multi-target RNA design algorithm with the datasets of two, three, and four target structures and found that our algorithm shows better or comparable design performances compared with the previous algorithms, RNAdesign and Frnakenstein. In addition to the benchmarks with pseudoknot-free datasets, we benchmarked MODENA with two-target pseudoknot datasets and found that MODENA can design the RNAs which have the target pseudoknotted secondary structures whose free energies are close to the lowest free energy. Moreover, we applied our algorithm to a ribozyme-based ON-switch which takes a ribozyme-inactive secondary structure when the theophylline aptamer structure is assumed.ConclusionsCurrently, MODENA is the only RNA design software which can be applied to multiple pseudoknot targets. Successful design results for the multiple targets and an RNA device indicate usefulness of our multi-objective RNA design algorithm.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-015-0706-x) contains supplementary material, which is available to authorized users.

RNA Scaffold: Designed to Co-localize Enzymes.

Self-assembling RNA scaffold is designed to co-localize enzymes in engineered biological pathways through interactions between scaffold's protein docking domains and their affinity protein-enzyme fusions, in vivo. Here we introduced a noncoding RNA structure theophylline aptamer that respond to theophylline ligand in order to modulate RNA scaffold and regulate multistep enzymatic activities. We described the specifically designed RNA scaffold increased the fluorescent intensity in a splitting GFP assay by 2.25-fold, and also observed a 1.43-fold increase in multi-enzymatic efficiency in IAA synthesis pathway.

Computational Design and Biosensor Applications of Small Molecule-Sensing Allosteric Ribozymes

Here I describe accurate and time-efficient computational methods for designing small molecule-sensing allosteric ribozymes that serve as logic gates with NOT or YES Boolean logic functions. Theophylline-sensing ribozymes are engineered to have a high cleavage rate of 1.3 min(-1) under physiologically relevant conditions. They are highly specific to theophylline and do not respond to caffeine, which differs in a single methyl group. These ribozymes are designed by fusing a theophylline aptamer with an extended version of the hammerhead ribozyme by modeling secondary structures. Purine-sensing ribozymes are designed by fusing the minimal version of the hammerhead ribozyme with bacterial guanine or adenine aptamers by modeling 3D interactions. I have developed high-throughput compatible arrays based on purine RNA sensors that can be used for antibacterial drug discovery. The ribozymes can be employed as molecular sensors in various applications, including exogenous control of gene expression, high-throughput screening arrays, and molecular computing.

De novo design of a synthetic riboswitch that regulates transcription termination

Riboswitches are regulatory RNA elements typically located in the 5′-untranslated region of certain mRNAs and control gene expression at the level of transcription or translation. These elements consist of a sensor and an adjacent actuator domain. The sensor usually is an aptamer that specifically interacts with a ligand. The actuator contains an intrinsic terminator or a ribosomal binding site for transcriptional or translational regulation, respectively. Ligand binding leads to structural rearrangements of the riboswitch and to presentation or masking of these regulatory elements. Based on this modular organization, riboswitches are an ideal target for constructing synthetic regulatory systems for gene expression. Although riboswitches for translational control have been designed successfully, attempts to construct synthetic elements regulating transcription have failed so far. Here, we present an in silico pipeline for the rational design of synthetic riboswitches that regulate gene expression at the transcriptional level. Using the well-characterized theophylline aptamer as sensor, we designed the actuator part as RNA sequences that can fold into functional intrinsic terminator structures. In the biochemical characterization, several of the designed constructs show ligand-dependent control of gene expression in Escherichia coli, demonstrating that it is possible to engineer riboswitches not only for translational but also for transcriptional regulation.

Rational construction of eukaryotic OFF-riboswitches that downregulate internal ribosome entry site-mediated translation in response to their ligands

A strategy for rationally constructing a novel type of eukaryotic OFF-riboswitch, which ligand-dependently turns off translation mediated by an internal ribosome entry site (IRES), has been established. The theophylline-dependent IRES-based OFF-riboswitch obtained through the proposed strategy functioned well in wheat germ extract, independently from the downstream gene, indicating that it can regulate any gene. Despite the fact that it has one theophylline aptamer, its switching efficiency was as high as that of a previously reported theophylline-dependent OFF-riboswitch that was constructed by inserting three continuous theophylline aptamers into a 5′ untranslated region in mRNA to downregulate the normal 5′-terminus-mediated translation. In addition, because the riboswitch part that was optimized in the theophylline-dependent IRES-based OFF-riboswitch, except for the aptamer domain, can be used as-is for other aptamer-ligand pairs, an arbitrary ligand-dependent IRES-based OFF-riboswitch is easy to construct with the corresponding well-minimized aptamer.