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.