Research Profiles: An insight into the Ly Lab

Danith Ly is an assistant professor in the department of chemistry. His lab focuses on research and development at the interface of chemistry and biology, with emphasis on the development of chemical tools and the application of genomics and proteomics technologies to better understand the foundations of biological problems.

A molecule at the center of his research is peptide nucleic acid (PNA), a synthetic analogue of DNA and RNA. PNA expresses a unique ability to form sequence-specific hybrids with complementary DNA and RNA strands in accordance with the Watson-Crick base pairing rules. The resulting hybrid exhibits both thermal stability and enzymatic stability towards proteases and nucleases. PNA’s greatest use would be in its ability to silence the expression of a targeted gene at the mRNA (and possibly at the DNA) level. There are currently obstacles to overcome before PNA can be used as an effective tool for therapeutics, diagnostics, and basic research. Undergraduates in the Ly lab are currently working on a variety of projects involving PNA with the goal of creating an efficient molecular tool to regulate gene expression.

A major obstacle to the success of PNA involves its ability to permeate the cell membrane and be effectively taken up by cells. PNA, due to its molecular structure, has both hydrophobic and hydrophilic properties. While the hydrophobic elements are able to pass through the lipid bilayer, the hydrophilic part gets lodged in the lipid, eventually causing lysis, or destruction, of the cell.

“We have devised a scheme in order to increase the cellular permeability of PNA without inhibiting its ability to freely diffuse through the cell and hybridize with RNA or DNA,” said Brian Belardi, a sophomore chemistry major who is currently working on a project to tackle the issue of cellular uptake. “This [modified PNA] involves the use of a disulfide ‘linker’ molecule, which contains a modified positively charged guanidinium group. The positively charged PNA will increase the cellular uptake, and then ... the reducing environment of the cell will reduce the disulfide bond, cleaving the positively charged group. Thus, this will hopefully allow PNA to diffuse freely through the cell without any electrostatic interference.”

“What makes Professor Ly’s research so nice is that he takes groups of peptides that are important, and attaches them to the backbone of PNA. This is a novel approach to nucleic acid analog,” said Bruce Armitage, a professor in the chemistry department who is also working with PNA. Currently, Armitage is collaborating with Ly to develop PNAs that can be used as anti-cancer agents. The specially designed PNAs will block the expression of three specific proteins involved in cancer.

A derivative of PNA, known as GPNA, has already been developed that can traverse the cell membrane and be used to regulate gene expression. Its synthesis required the incorporation of the guanidinium functional groups into the PNA backbone. Currently, Andy Hsieh, a senior biology major, is working on utilizing this molecule to better understand gene function in model organisms. “I am trying to apply the GPNA in our lab to a model organism, specifically vertebrates. A zebra fish is externally fertilized and translucent, making it easy to manipulate and observe under a microscope. We can identify gene function by knocking down a specific gene using GPNA. By incubating the GPNA with zebra fish embryos, we wish to uncover many genes that are essential in the developmental process in vertebrates,” said Hsieh.

One of Ly’s major projects and the underlying theme of the lab involves the development of a molecular tool involving PNA to better understand the relationship between human embryonic stem cells (HESC) and tumorigenesis.

Ly’s interest in HESC lies at the heart of a recently emerged hypothesis, which suggests that stem cells may be the origins of (certain) cancer.

The hypothesis states that cancer cells which develop into tumors are a direct result of the malfunctioning of stem cells. Stem cells, during their relatively short existence, are regulated by several pathways—one of which is the canonical Wnt signaling pathway. When turned on, this pathway tells ESC to continue dividing without regard to reparations. The lack of cell maintenance could lead to accumulation of damaged cells that would manifest into genetic diseases later in life, such as cancer.

The Wnt signaling pathway is turned off at a later stage of development. But if it doesn’t get turned off due to mutations, this can lead to cancerous cells which form tumors. This hypothesis is supported by the fact that the Wnt pathway is turned on in both stem cells and cancerous cells, although it is turned off in normal adult cells. Also, tumors are made up of a heterogenous mixture of cells. This is a strong
indication that tumor cells can differentiate into other cell types. This goes against the standard belief that cancerous cells that form tumors are caused by the uncontrolled reproduction of a single cell, which would result in a homogenous mixture.

This new discovery would make sense of why cancer drugs are often not effective on tumors.“Most cancer therapies exert their anti-tumorigenic effect by activating the cell cycle checkpoints. However, the cell cycle checkpoints are not fully developed in these primitive ES cells. What works with somatic cells may not work with ES cells,” Ly said.

Ly hopes to one day be able to use PNA as a molecular tool to address this hypothesis. Specially designed PNA molecules could be used to short circuit the Wnt pathway and other related pathways in cancer cells by turning off expression of certain genes and analyzing their effects on cancer cell production. “PNA is one of the tools that we’re developing to address the cellular origins of cancer,” said Ly.

The PNA molecule must be further modified before it could be used to study this hypothesis. A current problem that exists is in the penetration of ESC. They are significantly more difficult to penetrate than regular cells due to the unique cell membrane (the composition of which is not fully understood) and a specialized pump system which actively removes foreign material from cells. Ly is trying to engineer these molecules so that they can penetrate the cellular membrane of ES cells.

PNA technology competes with current technology being co-developed to silence genes, known as RNA interference (RNAi). The advantage of PNA is that it does not need any chemical enhancers to stimulate its activity inside a cell. “RNAi relies on intracellular enzymes, whereas PNA works independently,” said Armitage.

PNA is already being used by researchers at the University of Pittsburgh Medical Center to downregulate epidermal growth factor receptor (EGFR) in patients with head or neck tumors. This gene was found to play an important role in tumor aggressiveness, and PNA-regulated silencing of the gene has resulted in tumor shrinkage.

The use of PNA as a molecular tool to better understand both gene function and the relationship between tumor and embryonic stem cells holds great promise.