Kyung-Ae Yang

Recognition and sensing of low-epitope targets via ternary complexes with oligonucleotides and synthetic receptors.

Oligonucleotide-based receptors or aptamers can interact with small molecules, but the ability to achieve high-affinity and selectivity of these interactions depends strongly on functional groups or epitopes displayed by the binding targets. Some classes of targets are particularly challenging: for example, monosaccharides have scarce functionalities and no aptamers have been reported to recognize, let alone distinguish from each other, glucose and other hexoses. Here we report aptamers that differentiate low-epitope targets such as glucose, fructose, or galactose by forming ternary complexes with high-epitope organic receptors for monosaccharides. In a follow-up example, we expand this method to isolate high-affinity oligonucleotides against aromatic amino acids complexed in situ with a non-specific organometallic receptor. The method is general and enables broad clinical use of aptamers for detection of small molecules in mix-and-measure assays, as demonstrated by monitoring postprandial waves of phenylalanine in human subjects.

Optimizing cross-reactivity with evolutionary search for sensors.

We report a straightforward evolutionary procedure to build an optimal sensor array from a pool of DNA sequences oriented toward three-way junctions. The individual sensors were mined from this pool under separate selection pressures to interact with four steroids, while allowing cross-reactivity, in a manner designed to achieve perfect classification of individual steroids. The resulting sensor array had three sensors and displayed discriminatory capacity between steroid classes over full ranges of concentrations. We propose that similar protocols can be used whenever we have two or more classes of samples, with individual classes being defined through gross differences in ratios of dominant families of responsive components.

The use of gold nanoparticle aggregation for DNA computing and logic-based biomolecular detection.

The use of DNA molecules as a physical computational material has attracted much interest, especially in the area of DNA computing. DNAs are also useful for logical control and analysis of biological systems if efficient visualization methods are available. Here we present a quick and simple visualization technique that displays the results of the DNA computing process based on a colorimetric change induced by gold nanoparticle aggregation, and we apply it to the logic-based detection of biomolecules. Our results demonstrate its effectiveness in both DNA-based logical computation and logic-based biomolecular detection.

In vitro selection and amplification protocols for isolation of aptameric sensors for small molecules

We recently optimized a procedure that directly yields aptameric sensors for small molecules in so-called structure-switching format. The protocol has a high success rate, short time, and is sufficiently simple to be readily implemented in a non-specialist laboratory. We provide a stepwise guide to this selection protocol.

In vitro molecular pattern classification via DNA-based weighted-sum operation

Recent progress in molecular computation suggests the possibility of pattern classification in vitro. Weighted sum is a primitive operation required by many pattern classification problems. Here we present a DNA-based molecular computation method for implementing the weighted-sum operation and its use for molecular pattern classification in a test tube. The weights of the classifier are encoded as the mixing ratios of the differentially labeled probe DNA molecules, which are competitively hybridized with the input-encoding target molecules to compute the decision boundary of classification. The computation result is detected by fluorescence signals. We experimentally verify the underlying weight encoding scheme and demonstrate successful discrimination of two-group labels of synthetic DNA mixture patterns. The method can be used for direct computation on biomolecular data in a liquid state.

EvoOligo: Oligonucleotide Probe Design With Multiobjective Evolutionary Algorithms

Probe design is one of the most important tasks in successful deoxyribonucleic acid microarray experiments. We propose a multiobjective evolutionary optimization method for oligonucleotide probe design based on the multiobjective nature of the probe design problem. The proposed multiobjective evolutionary approach has several distinguished features, compared with previous methods. First, the evolutionary approach can find better probe sets than existing simple filtering methods with fixed threshold values. Second, the multiobjective approach can easily incorporate the users custom criteria or change the existing criteria. Third, our approach tries to optimize the combination of probes for the given set of genes, in contrast to other tools that independently search each gene for qualifying probes. Lastly, the multiobjective optimization method provides various sets of probe combinations, among which the user can choose, depending on the target application. The proposed method is implemented as a platform called EvoOligo and is available for service on the Web. We test the performance of EvoOligo by designing probe sets for 19 types of Human Papillomavirus and 52 genes in the Arabidopsis Calmodulin multigene family. The design results from EvoOligo are proven to be superior to those from well-known existing probe design tools, such as OligoArray and OligoWiz.

An evolutionary Monte Carlo algorithm for predicting DNA hybridization

Many DNA-based technologies, such as DNA computing, DNA nanoassembly and DNA biochips, rely on DNA hybridization reactions. Previous hybridization models have focused on macroscopic reactions between two DNA strands at the sequence level. Here, we propose a novel population-based Monte Carlo algorithm that simulates a microscopic model of reacting DNA molecules. The algorithm uses two essential thermodynamic quantities of DNA molecules: the binding energy of bound DNA strands and the entropy of unbound strands. Using this evolutionary Monte Carlo method, we obtain a minimum free energy configuration in the equilibrium state. We applied this method to a logical reasoning problem and compared the simulation results with the experimental results of the wet-lab DNA experiments performed subsequently. Our simulation predicted the experimental results quantitatively.

A microchip for nucleic acid isolation and enrichment

This paper presents a microchip that effectively isolates and enriches target-binding single-stranded DNA (ssDNA) in a randomized DNA mixture using solid-phase extraction (SPE) and electrophoresis. The microchip consists of isolation and enrichment microchambers that are connected by a microchannel partially filled with agarose gel. Single-stranded DNA oligomers in the randomized mixture are captured via specific binding onto human immunoglobulin E (IgE) that is immobilized on microbeads in the isolation chamber. Following elution, the oligomers are separated from impurities via electrophoretic transport through the gel-filled microchannel to the enrichment chamber. Experimental results show that this approach can enhance the sensitivity of ssDNA detection methods in dilute and complex biological samples.

Integrated Microfluidic Isolation of Aptamers Using Electrophoretic Oligonucleotide Manipulation

We present a microfluidic approach to integrated isolation of DNA aptamers via systematic evolution of ligands by exponential enrichment (SELEX). The approach employs a microbead-based protocol for the processes of affinity selection and amplification of target-binding oligonucleotides, and an electrophoretic DNA manipulation scheme for the coupling of these processes, which are required to occur in different buffers. This achieves the full microfluidic integration of SELEX, thereby enabling highly efficient isolation of aptamers in drastically reduced times and with minimized consumption of biological material. The approach as such also offers broad target applicability by allowing selection of aptamers with respect to targets that are either surface-immobilized or solution-borne, potentially allowing aptamers to be developed as readily available affinity reagents for a wide range of targets. We demonstrate the utility of this approach on two different procedures, respectively for isolating aptamers against a surface-immobilized protein (immunoglobulin E) and a solution-phase small molecule (bisboronic acid in the presence of glucose). In both cases aptamer candidates were isolated in three rounds of SELEX within a total process time of approximately 10 hours.

Integrated Microfluidic Selex Using Free Solution Electrokinetics

Systematic evolution of ligands by exponential enrichment (SELEX) offers a powerful method to isolate affinity oligonucleotides known as aptamers, which can then be used in a wide range of applications from drug delivery to biosensing. However, conventional SELEX methods rely on labor intensive and time consuming benchtop operations. A simplified microfluidic approach is presented which allows integration of the affinity selection and amplification stages of SELEX for the isolation of target-binding oligonucleotides by combining bead-based biochemical reactions with free solution electrokinetic oligonucleotide transfer. Free solution electrokinetics allows coupling of affinity selection and amplification for closed loop oligonucleotide enrichment without the need for offline processes, flow handling components or gel components, while bead based selection and amplification allow efficient manipulation of reagents and reaction products thereby realizing on-chip loop closure and integration of the entire SELEX process. Thus the approach is capable of multi-round enrichment of oligonucleotides using simple transfer processes while maintaining a high level of device integration, as demonstrated by the isolation of an aptamer pool against a protein target (IgA) with significantly higher binding affinity than the starting library in approximately 4 hours of processing time.