Robert Penchovsky

Riboswitch-based antibacterial drug discovery using high-throughput screening methods

Introduction: Bacterial riboswitches are structured RNA domains usually residing at the 5′ untranslated region of messenger RNAs that can directly bind specific metabolites. They serve as logic gates regulating gene expression. As a result, riboswitches enable mRNAs to regulate their own expression without the need for any regulatory proteins. The first riboswitches were found just 10 years ago. Over the last decade, more than dozen different riboswitch classes were identified in many bacterial species, and their number is still growing. These findings indicate that bacteria widely use RNA switches to sense changes in cell physiology and to regulate metabolic pathways. Areas covered: The authors discuss the main mechanisms for riboswitch control of gene expression in bacteria. Various riboswitch classes were found in human bacterial pathogens to control the synthesis of essential cell metabolites as discussed in this review. Some riboswitches can be used as novel targets for antibacterial drug discovery. This review presents the current and possible future high-throughput screening approaches for targeting riboswitches in the process of drug development. Expert opinion: Bacterial riboswitches of 17 different classes are discovered in 36 human bacterial pathogens that can be targeted for addressing the ever-growing need for new antibiotics. In this regard, the adaptation of various in silico, in vitro, and in vivo high-throughput screening methods for probing specific RNA switches are crucial for the success of antibacterial drug discovery process.

Engineering Integrated Digital Circuits with Allosteric Ribozymes for Scaling up Molecular Computation and Diagnostics

Here we describe molecular implementations of integrated digital circuits, including a three-input AND logic gate, a two-input multiplexer, and 1-to-2 decoder using allosteric ribozymes. Furthermore, we demonstrate a multiplexer-decoder circuit. The ribozymes are designed to seek-and-destroy specific RNAs with a certain length by a fully computerized procedure. The algorithm can accurately predict one base substitution that alters the ribozymes logic function. The ability to sense the length of RNA molecules enables single ribozymes to be used as platforms for multiple interactions. These ribozymes can work as integrated circuits with the functionality of up to five logic gates. The ribozyme design is universal since the allosteric and substrate domains can be altered to sense different RNAs. In addition, the ribozymes can specifically cleave RNA molecules with triplet-repeat expansions observed in genetic disorders such as oculopharyngeal muscular dystrophy. Therefore, the designer ribozymes can be employed for scaling up computing and diagnostic networks in the fields of molecular computing and diagnostics and RNA synthetic biology.

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.

Computational design of allosteric ribozymes as molecular biosensors.

Nucleic acids have proven to be a very suitable medium for engineering various nanostructures and devices. While synthetic DNAs are commonly used for self-assembly of nanostructures and devices in vitro, functional RNAs, such as ribozymes, are employed both in vitro and in vivo. Allosteric ribozymes have applications in molecular computing, biosensoring, high-throughput screening arrays, exogenous control of gene expression, and others. They switch on and off their catalytic function as a result of a conformational change induced by ligand binding. Designer ribozymes are engineered to respond to different effectors by in vitro selection, rational and computational design methods. Here, I present diverse computational methods for designing allosteric ribozymes with various logic functions that sense oligonucleotides or small molecules. These methods yield the desired ribozyme sequences within minutes in contrast to the in vitro selection methods, which require weeks. Methods for synthesis and biochemical testing of ribozymes are also discussed.

Designing drugs that overcome antibacterial resistance: where do we stand and what should we do?

Introduction: In recent years, infections caused by multidrug-resistant bacterial pathogens have become a huge issue to public healthcare systems. Indeed, the misuse of antibiotics has led to, over the past 30 years, the emergence of a number of resistant bacterial strains including Staphylococcus aureus, Neisseria gonorrhoeae, Escherichia coli and Mycobacterium tuberculosis. Unfortunately, efforts to produce new antibiotics have not been sufficient to cope with the emergence of these new antibiotic-resistant (AR) strains. Areas covered: There is an urgent need to invent and employ unconventional strategies for antimicrobial drug development to tackle the rising global threats imposed by the spread of antimicrobial resistance. Herein, the authors discuss these novel design strategies and provide their expert perspective on the subject. Expert opinion: To deal with the growing threat of AR, it is important to cut down the use of antibiotics to the very minimum to diminish the risk of unknown drug-resistant bacteria and increase antibacterial vaccination programs. Furthermore, it is important to develop new classes of antibiotics that can deal with multidrug-resistant bacterial pathogens.