PITTSBURGH--By exploiting an HIV protein that readily traverses cell membranes, Carnegie Mellon University scientists have developed a new way to introduce a gene-like molecule called a peptide nucleic acid (PNA) directly into live mammalian cells, including human embryonic stem (ES) cells. The work, published online December 2 in Chemical Communications, http://www.rsc.org/is/journals/current/chemcomm/ccadvarts.htm, holds considerable promise in genetic engineering, diagnostics and therapeutics.
"Our results show that PNAs could be effectively delivered into mammalian cells without requiring delivery vehicles," said Danith Ly, an assistant professor of chemistry in the Mellon College of Science (MCS) at Carnegie Mellon. Ly worked with leading author and graduate student Anca Dragulescu-Andrasi on this research.
Until now, getting PNAs into living cells has been difficult. While other laboratories have developed ways to shuttle PNAs into cells, these methods remain largely ineffective and limited to small-scale experimental setups, according to Ly. "We found that our modified PNAs were not only taken up by cells, but they also were localized predominantly in the cell nucleus, a specialized compartment in the cell where messenger RNAs are made," Ly said.
Messenger RNA (mRNA), the genetic information that is translated into proteins, is the target of an emerging field called antisense therapy.
"We found that we could modify PNAs so that they bind sequence-specifically to mRNA," Ly said. By binding to specific mRNAs, these agents could dampen the production of select disease-causing proteins, he added.
First reported in the early 1990s, PNAs are small synthetic molecules in which a protein-like backbone is combined with the nucleobases found in DNA and RNA. These nucleobases enable PNA to bind to DNA and RNA in a complementary, highly specific manner. Because the cell machinery cannot recognize the unnatural backbone of PNA, it fails to break down this structure, making PNAs very stable, long-lived molecules.
To enable the PNAs to enter cells, Ly modified the PNA backbone so that it contained a short sequence of chemical groups inspired by a region of the HIV-1 virus called the Tat transduction domain, which normally regulates gene expression. The modified PNAs are called GPNAs because they contain guanidinium functional groups. Ly found that GPNAs, in addition to their superior cell uptake properties, could be designed to bind sequence-specifically to RNA, with binding affinity and selectivity rivaling that of PNA. Ly and his colleagues visualized the uptake of GPNAs into living cells by attaching them to fluorescent probes.
GPNAs could gain widespread use in genetic diagnostics, therapeutics and engineering, according to Ly. For instance, scientists could use this technology to quickly identify whether specific tissues contain a cancer-causing version of a gene and are pre-cancerous. Because they enter embryonic stem cells, GPNAs potentially could be used to control gene expression and direct what kinds of tissues these malleable cells ultimately become. By infusing GPNAs to block the translation of specific RNAs, researchers also could "down-regulate" the production of disease-related proteins. Scientists could use GPNAs to temporarily inhibit production of different regulatory proteins in cells, which could prove especially helpful in modeling diseases that involve multiple genetic mistakes occurring over time. Thus, this approach could help to tease apart the sequence of molecular events that lead to diseases such as cancer or diabetes in animal models.
Ly is currently extending his research to show that GPNAs are absorbed throughout the body in mice that receive these agents.
Source: Eurekalert & othersLast reviewed: By John M. Grohol, Psy.D. on 21 Feb 2009
Published on PsychCentral.com. All rights reserved.
Ring the bells that still can ring. Forget your perfect offering. There is a crack, a crack in everything. That's how the light gets in.
~ Leonard Cohen