Novel binding and efficient cellular uptake of guanidine-based peptide nucleic acids (GPNA)

Article abstract of DOI:10.1021/ja029665m

Incorporation of a guanidine functional group into the PNA backbone facilitates cellular uptake of PNA into mammalian cells with efficiency comparable to that of the TAT transduction domain. The modified PNA recognizes and binds to the complementary DNA s

Full text of DOI:10.1021/ja029665m

Published on Web 05/17/2003
Novel Binding and Efficient Cellular Uptake of Guanidine-Based Peptide
Nucleic Acids (GPNA)
Peng Zhou,† Miaomiao Wang,† Lei Du, Gregory W. Fisher,‡ Alan Waggoner,‡ and Danith H. Ly†*
Department of Chemistry and Center for Light Microscope Imaging and Biotechnology,
Carnegie Mellon UniVersity, 4400 Fifth AVenue, Pittsburgh, PennsylVania 15253
Received December 10, 2002; E-mail: dly@andrew.cmu.edu
Scheme 1
Peptide nucleic acid (PNA) is a synthetic analogue of DNA and
RNA in which the natural sugar-phosphate backbone has been
replaced by achiral N-(2-aminoethyl) glycine units (Scheme 1).1
PNA can form sequence-specific hybrids with complementary DNA
and RNA strands in accordance with the Watson-Crick base pairing
rules. The resulting hybrid duplexes exhibit exceptional thermal
stability, while the unnatural backbone renders PNA stable to both
nucleases and proteases. Since its initial report, PNA has spawned
considerable interest in its development as therapeutics, diagnostics,
and molecular tools for basic research.2
However, the promise of PNA in in vivo applications has
dissipated with the discovery that PNA is not taken up readily by
mammalian cells. Previous studies involving PNA have been
performed in vitro,3 with the exception of direct microinjection and,4
more recently, through the use of carrier peptides.5 This inherent
limitation has prevented PNA from finding widespread applications
in therapeutics as well as in basic research involving live cells and
intact organisms. The growing awareness of these unfulfilled
promises of PNA has prompted us to investigate a novel strategy
for delivery of PNA into mammalian cells.
Our approach was inspired by the remarkable uptake properties
to be highly soluble in water compared to the unmodified PNA,
which has a strong tendency to aggregate in solution.
of the human HIV-1 Tat transduction domainswhich comprises
relatively short basic sequence (GRKKRRQRRR).6 Fusion of this
To determine whether GPNA was capable of recognizing and
domain to other proteins (regardless of size)7 and synthetic
binding to a complementary DNA sequence (A10), we measured
the thermal stability of a GPNA-DNA mixture at 1:1 and 2:1 ratios
utilizing UV spectroscopy. Our results showed that the mixture
yielded a well-defined melting curve at both ratios with the Tm value
of 78.0 °C (4.8 °C higher than that of the unmodified PNA2-DNA
triplex) (Figure S1, Supporting Information). Hybridization of
GPNA to DNA containing internal T-C and T-T mismatches
molecules (including nanoparticles)8 facilitated effective uptake of
these complexes into mammalian cells. Intraperitoneal (IP) injection
of these fused proteins into mice resulted in systemic uptake by
cells in every tissue, including those in the brain regions.9 More
intriguingly, these fused proteins remained fully active in vivo.10
Wender and colleagues have recently shown that TAT transduction
domain could be replaced entirely with a homoarginine peptoid
without compromising uptake efficiency.11 On the basis of these
lowered the Tm by ∼10 °C, while a T-G mismatch destabilized
the duplex by ∼5 °C (Figure S2, Supporting Information). These
findings, we hypothesized that incorporation of the arginine side
mismatched ∆Tm are comparable to those observed with unmodified
chain (guanidinium functional group) into the PNA backbone
PNA. On the other hand, GPNA containing a sequence identical
(GPNA, Scheme 1) would effectively facilitate PNA uptake into
to that of DNA did not yield defined melting transition. Taken
mammalian cells, not only in cultures but also in intact mammalian
together, these results suggest that, despite the positive charged side
organisms.
chains, GPNA retains high level of sequence specificity normally
exhibited by PNA.
By starting with arginine instead of glycine in the synthesis of
PNA backbone, we introduced the guanidinium functional group
The binding properties of GPNA were further characterized by
into the PNA backbone (Scheme S1, Supporting Information).12
circular dichroism (CD). Figure 1 shows the CD spectra of single-
stranded DNA and GPNA, GPNA-DNA duplex, and PNA2-DNA
triplex. The DNA duplex is characterized by a right-handed Cotton
effects,13 in contrast to the left-handed helix formed by PNA2-
DNA triplex as the result of the L-lysine residue at the C-terminus.
GPNA by itself did not show CD signal, indicating the absence of
secondary structures normally observed with unmodified PNA.
Thymine was initially chosen for this study because it presented
the fewest synthetic challenges; however, it can be extended to other
natural and unnatural nucleobases. The GPNA (Tg)10 (PNA oligomer
containing 10 thymines with arginine side chain installed at the
R-L-position) was constructed from Boc-Arg(Tos)-T-OH monomers
using Boc-protected solid-phase synthesis protocol, and the final
oligomer was purified by HPLC and characterized by MALDI-
However, in the presence of complimentary DNA, GPNA induced
TOF (m/z found 3673.2 and calcd 3675.0). The GPNA was found
a left-handed split CD signal characteristic of a left-handed helix
(Figure 1). A Job plot was further utilized to probe the binding
stoichiometry between GPNA and DNA. It has been well estab-
† Department of Chemistry.
‡ Center for Light Microscope Imaging and Biotechnology.
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J. AM. CHEM. SOC. 2003, 125, 6878-6879
10.1021/ja029665m CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
fluorescence intensity was strongest in the nucleus (Figure S3,
Supporting Information). Likewise, a parallel experiment utilizing
DAPI (localizes specifically to the nucleus) as a second staining
agent showed that the images acquired from the DAPI and FITC
channels overlapped, again confirming that GPNA localized to the
nucleus (Figure S4, Supporting Information). PNA containing
alternating PNA units and arginine amino acids (NH2-Arg-T-Arg-
G-Arg-T-Arg-A-Arg-C-Arg-G-Arg-T-Arg-C-Arg-A) (Scheme S2,
Supporting Information) also showed similar uptake properties.
These results are in agreement with earlier reports for TAT and
â-TAT transduction domain,14 which indicated that the inter-
distance between each arginine side chain along the backbone has
little influence on uptake efficiency.
Although the mechanism of TAT transduction has not been fully
substantiated, several groups have demonstrated that endocytosis
does not play a significant role in this process since the rate of
uptake is independent of temperature.15 Likewise, we found that
there was no difference in the uptake properties of GPNA at 37
and 4 °C, suggesting that the uptake mechanism of GPNA is neither
endocytosis-driven nor receptor mediated (perhaps through mem-
brane flipping).
In summary, we have demonstrated that PNA containing arginine
side chains exhibited remarkable uptake properties while maintain-
ing Watson-Crick recognition with complementary DNA strands.
These findings provide tantalizing evidence suggesting that PNA
could be selectively modified to facilitate uptake into mammalian
cells. The ability to effectively introduce PNA into mammalian cells
will be critically important for in vivo applications.
Figure 1. CD spectra of single-stranded DNA and GPNA, GPNA-DNA
duplex, and PNA2-DNA triplex. The hybridization complexes were formed
by heating to 90 °C for 5min, followed by gradual cooling to room
temperature. GPNA-DNA complex contained 2 µM of DNA and GPNA
strand each, while that of PNA2-DNA triplex contained 4 µM of PNA
and 2 µΜ of DNA. Buffer contained 20 mM NaCl and 10 mM NaPi (pH
7.0). Inset: Job plots of GPNA-DNA and PNA2-DNA complexes. The
overall concentration was 2 µM, and the ratios between PNA and GPNA
with DNA were varied. CD intensity was measured at 254 nm.
Acknowledgment. We thank Professor Bruce Armitage for
critical review of the manuscript and the Carnegie Mellon University
for financial support.
Supporting Information Available: Synthetic scheme, UV melting
curve, 3D-rendering, superimposed images of DAPI and fluorescein-
labeled G-PNA, and schematic drawing of PNA oligomer containing
alternating PNA units and arginines. This material is available free of
charge via the Internet at http://pubs.acs.org.
Figure 2. Fluorescence microscope images of HCT116 cells following
incubation with unmodified PNA (A: Fl-T10-Lys), TAT domain (B: Fl-
Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg), and GPNA (C: Fl-
(Tg)10), respectively. Cells were incubated with each respective oligomer
at 37 °C for 10 min at 1 µM, followed by a thorough wash (5×) with PBS
and fixing with 4% paraformaldehyde. Cells were then imaged with confocal
fluorescence microscope.
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