Our on-going work in this area is focused on the design and testing of synthetic reagents for delivery of molecular cargos into the cytoplasmic and nuclear compartments of cells. In addition to being able to deliver these exogenous molecules into live cells and intact organisms for therapeutic and diagnostic applications, we are interested in gaining a better understanding of the mechanisms by which they traverse the extracellular and nuclear membranes of mammalian cells, normal and diseased states, in attempts to further improve the efficiency and selectivity of cellular delivery. One particular class of molecules that we are currently honing is peptide nucleic acid (PNA), as a molecular platform for regulating gene expression.
Our chemical synthesis program is focused on function, rather than on methodology development. These targeted molecules, which span over a wide range of chemical space, are designed for specific purposes—using a combination of solution and solid-phase methodologies. Examples of these targeted molecules include fluorescent dyes for labeling, probing and monitoring protein and nucleic acid localizations and dynamics; natural products for modulating protein-protein and protein-nucleic acid interactions; natural biopolymers (DNA and RNA, proteins, and carbohydrates) and their synthetic derivatives for a variety of applications in biology and medicine, including gene regulation, molecular therapies, and cancer vaccine development.
One aspect of our work is focused on the design of modified peptide nucleic acid (PNA) for recognition of double-stranded B-form DNA (B-DNA), based on the simple rules of Watson-Crick base-pairing. Previously we have shown that PNA, when preorganized into a right-handed helix by installing an appropriate stereogenic center at the γ-backbone position, can invade any sequence of double-stranded B-DNA. However, with the recurrent design, this binding mode is limited to relatively low ionic strengths—due mainly to the lack of binding free energy. Current work is focused on the design of modified nucleobases with improved hydrogen-bonding and base-stacking capabilities and on the development of 'Janus' nucleobases that are capable of forming Watson-Crick hydrogen-bonding interactions with both strands of the DNA double helix, with the overall goal of being able to target any sequence of double-stranded B-DNA under physiological conditions based on the well-established rule of Watson-Crick base-pairing.
Many genetic diseases occur as the result of single-base mutations of key, protein-encoding genes. In principle, it should be possible to treat (or perhaps cure) such genetic diseases by correcting the mutations—i.e. changing the mutated base-pairs to the original sets. Earnest attempts have been made to try to tackle this problem; however, there appears to be no solution insight. Our work in this area explores the possibility of using especially designed Janus-based γ-peptide nucleic acids (J-γPNAs) to invade genomic DNA, the endogenous repair enzymes to excise the mutant base-pairs, and the innate recombination process to correct the mutations.
Antisense and antigene are promising technologies for regulating gene expression because of the simplicity and specificity of recognition and the generality in sequence design; however, the major obstacles are cellular delivery, nonspecific binding and cytotoxicity. While some progress has been made on the cellular delivery front, the other two issues remain a bottle-neck for molecular therapies. Work in our group is focused on the design of nucleic acid platforms with specific chemical modifications and secondary structures, in attempts to modify the hybridization kinetics and thermodynamics in such a way that they are more favorable toward the perfectly-matched than the mismatched targets, as compared to the standard, linear motif.
Work in this area is focused on the development of novel nucleic acid reagents, relatively small in size (3-5nt in length), that preferentially recognize and bind secondary structures of RNA over the linear counterparts. Such molecules are valuable as molecular tools for basic research in biology, as well as remedies for treating genetic and infectious diseases with rapidly evolving targets such as cancer, bacterial and viral infections. The strategy, based on the concept of 'pot-hole fixing', utilizes 16 different combinations of Janus nucleobases to repair the mismatched hydrogen-bonding interactions within RNA molecules.
This aspect of our work is focused on the development of molecular recognition codes for programming molecular self-assembly. Specifically, we are working toward developing relatively short (3-5nt in length) and orthogonal nucleic acid recognition codes for organizing and assembling materials in aqueous solution, as well as in organic solvents. The work involves syntheses of conformationally-constrained, β-peptide and related backbones and modified nucleobases, evaluation of recognition properties, and exploration of their utility in biomedical and tissue engineering and in molecular electronics.
The notion of using peptides and peptidomimetics as drug candidates for treating genetic and infectious diseases is appealing because of the ease and flexibility of synthesis, and the large sequence space that can be generated from a limited number of chemical building blocks; however, the main drawbacks are conformational flexibility and enzymatic stability. Because of their small size and high degree of conformational freedom, they neither binding with high affinity nor selectivity; and moreover, they are easily degraded by proteases. Inspired by nature, in particular the synthesis of 'cyclotides', our group is currently working on the design of template-based strategy for construction of cyclic and knotted-structure peptides as possible antibiotics and other drug candidates.
To date more than 20 genetic diseases have been associated with unstable repeat expansion, including fragile X syndrome, dystrophia myotonica 1 and 2, Huntingtin disease and Machado-Joseph disease, just to name a few. These unstable repeats cause either loss or altered protein functions, or in some cases altered RNA functions; and presently there are no effective treatments for such genetic diseases. Our effort in this area is focused on the development of Janus-based γ-peptide nucleic acids (J-γPNAs) for targeting unstable repeated regions within genomic DNA and RNA. Our current emphasis is on MD1, a type of muscular dystrophy and mental retardation caused by gain of RNA function. The goal is to develop a relatively short nucleic acid reagent (preferentially 3nt in length) that can invade the CUG repeat and thereby prevents the MBNL1 splicing factor from binding to it. In doing so will free MBNL1, allowing it to execute its function in the cytoplasm and thereby reverting the phenotype.