Yaroslav Chushak

In silico selection of RNA aptamers

In vitro selection of RNA aptamers that bind to a specific ligand usually begins with a random pool of RNA sequences. We propose a computational approach for designing a starting pool of RNA sequences for the selection of RNA aptamers for specific analyte binding. Our approach consists of three steps: (i) selection of RNA sequences based on their secondary structure, (ii) generating a library of three-dimensional (3D) structures of RNA molecules and (iii) high-throughput virtual screening of this library to select aptamers with binding affinity to a desired small molecule. We developed a set of criteria that allows one to select a sequence with potential binding affinity from a pool of random sequences and developed a protocol for RNA 3D structure prediction. As verification, we tested the performance of in silico selection on a set of six known aptamer–ligand complexes. The structures of the native sequences for the ligands in the testing set were among the top 5% of the selected structures. The proposed approach reduces the RNA sequences search space by four to five orders of magnitude—significantly accelerating the experimental screening and selection of high-affinity aptamers.

Development of a 2,4-Dinitrotoluene-Responsive Synthetic Riboswitch in E. coli Cells

Riboswitches are RNA sequences that regulate expression of associated downstream genes in response to the presence or absence of specific small molecules. A novel riboswitch that activates protein translation in E. coli cells in response to 2,4-dinitrotoluene (DNT) has been engineered. A plasmid library was constructed by incorporation of 30 degenerate bases between a previously described trinitrotoluene aptamer and the ribosome binding site. Screening was performed by placing the riboswitch library upstream of the Tobacco Etch Virus (TEV) protease coding sequence in one plasmid; a second plasmid encoded a FRET-based construct linked with a peptide containing the TEV protease cleavage site. Addition of DNT to bacterial culture activated the riboswitch, initiating translation of TEV protease. In turn, the protease cleaved the linker in the FRET-based fusion protein, causing a change in fluorescence. This new riboswitch exhibited a 10-fold increase in fluorescence in the presence of 0.5 mM DNT compared to the system without target.

Detection of orexin A neuropeptide in biological fluids using a zinc oxide field effect transistor.

Biomarkers which are indicative of acute physiological and emotional states are studied in a number of different areas in cognitive neuroscience. Currently, many cognitive studies are conducted based on programmed tasks followed by timed biofluid sampling, central laboratory processing, and followed by data analysis. In this work, we present a sensor platform capable of rapid biomarker detection specific for detecting neuropeptide orexin A, found in blood and saliva and known as an indicator of fatigue and cognitive performance. A peptide recognition element that selectively binds to orexin A was designed, characterized, and functionalized onto a zinc oxide field effect transistor to enable rapid detection. The detection limit using the sensor platform was sub-picomolar in water, and picomolar to nanomolar levels in saliva and serum. The transistor and recognition element sensor platform can be easily expanded, allowing for multiple biomarkers to be detected simultaneously, lending itself to complex biomarker analysis applicable to rapid feedback for neuroscience research and physiological monitoring.

Single-Round Patterned DNA Library Microarray Aptamer Lead Identification

A method for identifying an aptamer in a single round was developed using custom DNA microarrays containing computationally derived patterned libraries incorporating no information on the sequences of previously reported thrombin binding aptamers. The DNA library was specifically designed to increase the probability of binding by enhancing structural complexity in a sequence-space confined environment, much like generating lead compounds in a combinatorial drug screening library. The sequence demonstrating the highest fluorescence intensity upon target addition was confirmed to bind the target molecule thrombin with specificity by surface plasmon resonance, and a novel imino proton NMR/2D NOESY combination was used to screen the structure for G-quartet formation. We propose that the lack of G-quartet structure in microarray-derived aptamers may highlight differences in binding mechanisms between surface-immobilized and solution based strategies. This proof-of-principle study highlights the use of a computational driven methodology to create a DNA library rather than a SELEX based approach. This work is beneficial to the biosensor field where aptamers selected by solution based evolution have proven challenging to retain binding function when immobilized on a surface.

Computational design of RNA libraries for in vitro selection of aptamers.

Selection of aptamers that bind a specific ligand usually begins with a random library of RNA sequences, and many aptamers selected from such random pools have a simple stem-loop structure. We present here a computational approach for designing a starting library of RNA sequences with increased formation of complex structural motifs and enhanced affinity to a desired target molecule. Our approach consists of two steps: (1) generation of RNA sequences based on customized patterning of nucleotides with increased probability of forming a base pair and (2) a high-throughput virtual screening of the generated library to select aptamers with binding affinity to a small-molecule target. We developed a set of criteria that allows one to select a sequence with potential binding affinity from a pool of random sequences and designed a protocol for RNA 3D structure prediction. The proposed approach significantly reduces the RNA sequence search space, thus accelerating the experimental screening and selection of high-affinity aptamers.

Microarrays as Model Biosensor Platforms to Investigate the Structure and Affinity of Aptamers

Immobilization of nucleic acid aptamer recognition elements selected free in solution onto the surface of biosensor platforms has proven challenging. This study investigated the binding of multiple aptamer/target pairs immobilized on a commercially available microarray as a model system mimicking biosensor applications. The results indicate a minimum distance (linker length) from the surface and thymine nucleobase linker provides reproducible binding across varying conditions. An indirect labeling method, where the target was labeled with a biotin followed by a brief Cy3-streptavidin incubation, provided a higher signal-to-noise ratio and over two orders of magnitude improvement in limit of detection, compared to direct Cy3-protein labeling. We also showed that the affinities of the aptamer/target interaction can change between direct and indirect labeling and conditions to optimize for the highest fluorescence intensity will increase the sensitivity of the assay but will not change the overall affinity. Additionally, some sequences which did not initially bind demonstrated binding when conditions were optimized. These results, in combination with studies demonstrating enhanced binding in nonselection buffers, provided insights into the structure and affinity of aptamers critical for biosensor applications and allowed for generalizations in starting conditions for researchers wishing to investigate aptamers on a microarray surface.

Identifying Potential Protein Targets for Toluene Using a Molecular Similarity Search, in Silico Docking and in Vitro Validation

The toxicity of chemicals greatly depends on their interaction with macromolecular targets. The main goal of this study was to develop an approach for predicting protein targets for chemical toxins using a molecular similarity search of toxin–target information collected in the Toxin and Toxin-Target Database. The developed method was used to identify new targets for toluene which could predict potential cellular toxicity and to validate the approach with in vitro laboratory studies. We obtained 124 potential targets for toluene from a molecular similarity search. Results were further analysed using in silico molecular docking methods. The binding of toluene to two proteins, hemoglobin and serum albumin, was validated by the measurement of binding using microscale thermophoresis. The measured binding constant between toluene and hemoglobin was 1.9 μM, while albumin demonstrated toluene-induced aggregation. These results demonstrate the applicability of an exploratory in silico toxicity tool, based on a molecular similarity search and protein–ligand docking, for identification of potential targets for chemical toxins.

Riboswitch-based sensor in low optical background

Riboswitches are a type of natural genetic control element that use untranslated sequence in the RNA to recognize and bind to small molecules that regulate expression of that gene. Creation of synthetic riboswitches to novel ligands depends on the ability to screen for analyte binding sensitivity and specificity. In our work, we have coupled a synthetic riboswitch to an optical reporter assay based on fluorescence resonance energy transfer (FRET) between two genetically-coded fluorescent proteins. Specifically, a theophylline-sensitive riboswitch was placed upstream of the Tobacco Etch Virus (TEV) protease coding sequence, and a FRET-based construct, BFP-eGFP or eGFP-REACh, was linked by a peptide encoding the recognition sequence for TEV protease. Cells expressing the riboswitch showed a marked optical difference in fluorescence emission in the presence of theophylline. However, the BFP-eGFP FRET pair posses significant optical background that reduces the sensitivity of a FRET-based assay. To improve the optical assay, we designed a nonfluorescent yellow fluorescent protein (YFP) mutant called REACh (for Resonance Energy-Accepting Chromoprotein) as the FRET acceptor for eGFP. The advantage of using an eGFP-REACh pair is the elimination of acceptor fluorescence which leads to an improved detection of FRET via better signal-to-noise ratio. The EGFP-REACh fusion protein was constructed with the TEV protease cleavage site; thus upon TEV translation, cleavage occurs diminishing REACh quenching and increasing eGFP emission resulting in a 4.5-fold improvement in assay sensitivity.

FRET-Based Optical Assay for Selection of Artificial Riboswitches

Artificial riboswitches are engineered to regulate gene expression in response to a variety of non-endogenous small molecules and, therefore, can be useful tools to reprogram cellular behavior for different applications. A new synthetic riboswitch can be created by linking an in vitro-selected aptamer with a randomized expression platform followed by in vivo selection and screening. Here, we describe an in vivo selection and screening technique to discover artificial riboswitches in E. coli cells that is based on TEV protease-FRET substrate reporter system.