A. General research interest

Our group works at the interface between chemistry and biology. Our overall research interest is to examine unusual functions of nucleic acids and to be creative about them. The molecules that we are interested in studying include artificial or natural single-stranded DNA or RNA molecules with often surprising properties.  Our study on DNA or RNA has several distinct features. First, we examine DNA or RNA not for its well-recognized role as the genetic material to store and transmit genetic information for living organisms, but rather for its less-known utility as a simple polymer to carry out catalytic and binding functions. Second, we study DNA or RNA not in their rigid double-stranded form but in their flexible single-stranded configuration. Third, usually there is no natural source to fetch the DNA or RNA molecules for our study, but rather we create our own molecules using a technique called "In vitro selection". In vitro selection is a simple yet powerful combinatorial approach that allows simultaneous screening of up to 1016 different DNA or RNA molecules in a single mixture for rare sequences with unusual functions.

 

It has been well demonstrated that nucleic acids, in addition to their roles in the storage and transmission of genetic information, can also act as enzymes (ribozymes and DNAzymes) and receptors (aptamers and riboswitches). My group is interested both in the study of basic functions of these molecules (basic science focus of the lab) and in the exploration of these molecules as novel molecular tools for therapeutics, biomolecular detection, drug discovery and nanotechnology (applied science focus of the lab).

 

B. Basic science projects

DNAzymes. DNAzymes refer to single-stranded DNA molecules with catalytic function. They can be made through simple test-tube evolution experiments and hold great potential for many biomedical applications. Mother Nature does not use DNAzymes for cellular functions; however, we can create them by in vitro selection. My lab has been studying three types of DNAzymes: RNase DNAzymes, signaling DNAzymes and kinase DNAzymes. RNase DNAzymes are capable of cleaving an RNA sequence at a specific junction, and thus, they hold therapeutic potential because they can be used to degrade specific mRNA molecules in cells. Signaling DNAzymes link the catalytic event to the generation of a fluorescent signal through the cleavage of an RNA unit that is embedded in a DNA substrate but surrounded by two nucleotides separately modified with a fluorophore and a quencher; signaling DNAzymes can be used to design real-time enzymatic sensors for protein and small molecule detection. Kinase DNAzymes transfer the g-phosphate from an NTP (such as ATP or GTP) to the 5'-hydroxyl of DNA; they can be developed into sensors for NTPs and thus provide useful tools to monitor the activity of a plethora of NTP-utilizing enzymes such as kinases, ATPases and GTPases. The long-term goal of our DNAzyme research is to fully understand the catalytic limitations of DNA and to generate important knowledge that can benefit the search for better DNAzymes and their suitable applications. Within this context, we actively pursue the following thee aims: creation of new DNAzymes that can catalyze important chemical and biological reactions, investigation of structural and mechanistic properties of some of the DNAzymes already created by us, engineering suitable DNAzymes for biomedical applications ranging from cancer therapeutics to disease diagnostic tools (see application projects below).

 

Aptamers. Aptamers are single-stranded DNA or RNA molecules that are capable of specific molecular recognition (binding) of a target molecule (such as a protein or a metabolite). You can think an aptamer as a "key" and a target as a "lock". Mother Nature does use aptamers as an integral part of riboswitch (see discussion below); however, most time we create aptamers in house by in vitro selection. To date, many labs in the world have created a large array of aptamers for biological targets spanning amino acids, nucleotides, carbohydrates, proteins, and even whole cells. Since aptamers can specifically recognize the targets for which they are created for, they can be used as highly specific "molecular detectives" (biosensors). My lab has been interested in developing novel approaches for the design of fluorescent and colorimetric aptamer biosensors. Advantages of fluorescent sensors include abundant choices of chromogenic species for aptamer modification, minimal health risks in the handling and use of such sensors, and the availability of instrumentation capable of detecting fluorescence at high sensitivity. The most obvious benefit that colorimetric assays offer is the elimination of sophisticated instruments because human eyes can see color changes directly, at least for qualitative analysis. Quantitative analysis can also be achieved easily through the use of simple spectrophotometers. Specific applications of aptamers in my lab will be further discussed in application projects below.

 

Riboswitches. Riboswitches are found in natural systems and they are RNA sequences found mostly in the 5¢-untranslated regions (5¢-UTR) of many mRNAs in bacteria and other organisms. Many riboswitches have been discovered for many important cellular metabolites such as adenine, lysine, and thiamine pyrophosphate. A given riboswitch performs two linked functions: sensing the concentration of a metabolite and exerting a control over the expression of a gene or genes. These functions are carried out via the actions of two sequence elements: an aptamer domain to act as a binding site for a specific metabolite, and an adjacent control element to act as an on/off switch for downstream genes. The discovery of riboswitches also points to a brand new direction for creating RNA-based sensors as tools for molecular biology research. My lab is particularly interested in applying riboswitches as intracellular biosensors. We engineer such biosensors by placing a reporter gene (such as green fluorescent protein) under the control of a riboswitch. With these sensors in hand, we can apply them as tools to study cell physiology and gene functions. For example, we can create a cell-based sensor for amino acid lysine and use it to study relevant genes for their roles in the biosynthesis of lysine. The importance of lysine biosynthetic pathway in prokaryotes and the lack of an orthologous pathway in humans make the relevant genes attractive targets for the development of antibiotics. For this purpose, we are also interested in applying such lysine biosensor as means to conduct high throughput screening (HTS) to search for small-molecule inhibitors of this pathway. These inhibitors may eventually be developed into a new generation of antibiotics to fight bacterial infection.

 

Small RNAs. Small RNAs (sRNAs) encoded in the intergenic regions of bacterial genomes are important players in the regulation of critical cellular processes, including stress response and pathogenesis. For example, sRNAs ranging from 40 to 500 nucleotides in size have been demonstrated to control the expression of target mRNAs by regulating the stability or the translation initiation of the RNA transcript. sRNAs have also been shown to interact with and modulate the activity of associated proteins. My lab is very interested in genomewide screens aimed at isolating novel sRNAs in E. coli and other organisms. We have already isolated several novel sRNAs from E. coli and are actively studying their cellular roles. For example, we have recently discovered a small RNA known as RygC and its function in cells is speculated to be the regulation of the production of a toxic protein called IbsC. Presently, the biochemical properties and precise physiological role of the IbsC toxin remain elusive. Many questions pertaining to its regulation, activation, and mechanism of action are not yet answered. Therefore, my lab aims to provide the answers to questions such as: why does a bacterial cell produce a protein toxic to itself? Under what conditions does the protein toxin express? How does RygC regulate the expression of IbsC?  Findings from such studies will provide a comprehensive picture of the fate and function of sRNAs and related protein toxins, which in turn will significantly enhance our knowledge about bacterial physiology and pathogenesis.

 

C. Applied science projects

DNAzymes as therapeutics. Apoptosis (programmed cell death) is essential for both normal development and tissue homeostasis; abnormal regulation of apoptosis has been attributed to many human diseases, such as cancer, autoimmunity, and neurodegenerative disorders. Several anti-apoptotic members of the Bcl-2 protein family are known to dictate the apoptosis or survival of cancer cells; cancer cells can escape from chemo- or radiotherapy-induced apoptosis by overproducing these proteins. Therefore, these so-called apoptotic blocks are attractive candidates as therapeutic targets. RNA-cleaving DNAzymes (RNase DNAzymes) can perform sequence-specific cleavage of RNA, and therefore, they can be very useful enzymatic agents to degrade specific cellular RNA molecules in cells and have considerable therapeutic potential. We are working on creating and studying RNase DNAzymes that target the mRNAs of important anti-apoptotic proteins of the Bcl-2 family, with a long-term goal of developing RNase DNAzymes as anti-cancer therapeutics.

 

DNAzyme as protein biosensors. Currently the major classes of sensors for events in live cells are chemical dyes and fluorescence proteins. Both have significant limitations and there is a great need to develop both in-cell sensors and those that can be used in ‘mix and read' assays. We are interested in developing novel protein sensors using signaling DNAzymes discussed above. Signaling DNAzyme sensors will perform three linked functions: ligand binding, catalysis and fluorescence generation; we believe they can be used as platform probes for engineering real-time fluorescent sensors for the detection of a specific protein of interest. For example, we are actively conducting a project aimed to create signaling DNAzymes for detecting some apoptotic factors in cancer cells and studying biological roles of proteins that regulate apoptosis, which is an important step in the understanding of molecular mechanism of cancer and can lead to the discovery of novel therapeutic targets for such a deadly disease. Certain apoptotic factors have also been implicated as biomarkers for diseases such as cancer; therefore, a convenient method to detect these protein molecules may eventually be developed into a diagnostic test.

 

Aptamers and DNAzymes as biosensors for food-borne pathogens. Testing for food-borne pathogens is essential to the public health. Highlighted by the recent Listeria outbreak at Maple Leaf Foods, there is a significant need to develop efficient, easy-to-use and cheap methods for food pathogen detection because existing methods are time consuming and labor-intensive processes, requiring highly skilled personnel and expensive reagents. We are developing biosensing aptamers and DNAzymes that are capable of performing speedy detection of food-borne pathogens such as Salmonella, Listeria and E. coli O157. For example, we are working on creating signaling DNAzyme based bacterial sensors. As discussed above, signaling DNAzymes are engineered to perform three linked functions: target recognition, enzymatic catalysis and fluorescence generation. The unmatched advantage of signaling DNAzyme technology is the development of a simple, ‘mix-and-read' type of assay that is faster to perform and easier to use. The second advantage is the fact that signaling DNAzymes are a catalytic system and have a multiple turnover ability, resulting in faster reporting time. The third advantage is straightforward automation of such an assay for high-throughput sample analysis because manual DNA extraction or amplification is not required. In addition, the fact that the reagents are DNA provides for several practical benefits: signaling DNAzymes can be produced at a low cost, with high batch-to-batch consistency, and long shelf-life.

 

Bioactive papers.  It remains a significant challenge to develop simple, rapid, and inexpensive bioassays that can be applied for detection of biological agents in health, food and water. Currently, most of such analyses are performed in modern laboratories equipped with expensive instruments and staffed with highly qualified personnel. Although a few applications utilize simple test strips, generally speaking, most bioassays are not suited for use by average users. Thus, the development of easy-to-use biosensors is of tremendous interest in both research community and many industrial sectors. Such devices are deemed even more useful in the developing world where the access to sophisticated testing facilities is extremely limited. My lab is a member of a nationwide network called "Sentinel Bioactive Paper Network" and the goal of the network is to develop easy-to-use paper strips for medical diagnosis, food-borne pathogen detection, and environmental monitoring. We are actively developing paper strips that can detect toxic metals, organic pollutants, disease markers, or even whole cells (like food-borne pathogens or cancer cells). For example, we have recently successfully created a bioactive paper that can sensitively detect DNA.   

 

DNA nanotechnology.  My lab is also interested in designing nanomaterials and nanomachines that contain DNAzymes or aptamers. Sensors made from nanomaterials (such as gold nanoparticles) modified with DNAzymes and DNA aptamers have the potential to achieve high sensitivity, low sample volume requirement, and capabilities for real time detection and high-throughput analysis-the major characteristics of an ideal biosensing technology. For example, we are very interested in developing DNA-modified gold nanoparticle (AuNP) as colorimetric biosensors. The ability to use AuNP as a colorimetric reporter relies on the fact that the dispersed AuNP solution is red whereas the aggregated AuNP solution is purple or blue. We have also established several forms of DNA-modified AuNP based assays that can be used to detect small molecules such as ATP and proteins such as enzymes. We have also found out that DNA-modified AuNPs can be printed on paper (like ink) and resultant bioactive papers can be used to perform the specific detection of a target of interest (including small molecules and proteins). These findings have given us the confidence that we can develop AuNP technologies that can be eventually commercialized for the detection of a variety of targets that are important to our health and lives.