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"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. |
