Bioconjugate Chem. 2005, 16, 501−503
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Enhanced Oligonucleotide Binding to Self-Assembled Nanofibers Mustafa O. Guler,† Jonathan K. Pokorski,† Daniel H. Appella,† and Samuel I. Stupp*,†,‡,§ Department of Chemistry, Department of Materials Science and Engineering, and Feinberg School of Medicine, Northwestern University, 2220 Campus Drive, Evanston, Illinois 60208 . Received February 24, 2005; Revised Manuscript Received March 23, 2005
A peptide nucleic acid/peptide amphiphile conjugate (PNA-PA) that self-assembles into fiber-shaped nanostructures was designed to bind oligonucleotides with high affinity and specificity. Oligonucleotide binding to PNA-PA nanofibers was studied using fluorescence polarization, and thermal stability was examined by UV-vis measurement of duplex melting temperatures. The self-assembled PNAPA DNA system was observed to bind more strongly than the corresponding DNA-DNA duplex. We also observed single base specificity with a 16 °C in thermal stability. As expected from the previous PNA studies, PNA-PA RNA binding is also stronger than the corresponding RNA-RNA duplex.
Recent developments in molecular biology demonstrate the importance of RNA isolation and detection experiments such as Northern analysis (1, 2), nuclease protection assays (3), RT-PCR (reverse transcription-polymerase chain reaction) (4), RNA mapping (5), and in vitro translation (6). Among the different methodologies used to separate and purify RNA sequences for genetic research, isolation of mRNA containing polyadenylic (poly A) regions plays an important role in detection and quantification of low-copy mRNAs (7). Therefore, developing isolation tools for poly A-containing mRNAs using thymine-functionalized high molecular weight polymers (e.g. cellulose, polystyrene beads) has been an active area of research (8, 9). Hydrogels derived from self-assembled molecules that specifically bind oligonucleotides are interesting targets for new biomaterials (10). In this study, we exploit the nanoscale structure of self-assembled nanofiber networks as an approach to bind poly A sequences. We have designed and synthesized a novel peptide nucleic acid/peptide amphiphile conjugate (PNA-PA). These molecules contain a peptide nucleic acid segment followed by a peptide sequence terminated by an alkyl segment. The PNA-PA molecules self-assemble upon increasing pH to above pH 7 to form a self-supporting gel consisting of a network of nanofibers. This behavior is similar to that of peptide amphiphiles (PAs) reported previously by our laboratory which self-assemble after pH changes or electrolyte neutralize or shield molecular charges (11-16). The PNA-PA was designed to contain a KGGGAAAK peptide sequence since nanofiber formation is promoted by intermolecular β-sheets among peptide amphiphiles (17). Branching at one of the lysine residues was used to provide an attachment point for the PNA recognition unit on the self-assembled nanofibers and also to increase water solubility (see Figure 1). PNA was chosen over DNA for several reasons (18-23). First, the uncharged PNA backbone results in thermally stron* To whom correspondence should be addressed. E-mail:
[email protected]. † Department of Chemistry. ‡ Department of Materials Science and Engineering. § Feinberg School of Medicine.
ger PNA-DNA and PNA-RNA duplexes and triplexes than DNA-DNA and RNA-RNA counterparts, especially at very low ionic strength. Second, PNA synthesis is compatible with solid-phase peptide synthesis (SPPS) used for PAs. The PNA-PA was synthesized by SPPS. Branching of the peptide headgroup was achieved by using orthogonal protecting group chemistry for the amines at the R and positions of a lysine residue. A poly-thymine PNA heptamer was then built on the PA. The PNA-PA was found to be soluble in water at pH 4. Solutions with concentrations over 1 wt % were observed to form selfsupporting gel by increasing the pH above 7. Gel formation was found to be reversible when pH was reduced to 4. Transmission electron microscopy (TEM) and circular dichroism (CD) spectroscopy were used to characterize the supramolecular structure of the PNA-PA. TEM micrographs of the PNA-PA revealed formation of uniform nanofibers with a diameter of 8 ( 1 nm and lengths ranging from 100 nm to several micrometers, as shown in Figure 2 and also in Supporting Information. Circular dichroism spectra of the self-assembled PNAPA revealed a β-sheet signal at 215 nm (nπ* transition) resulting from hydrogen bonding between the peptide segments (see Supporting Information) (24). As expected, the PNA segment did not show any CD signal given its achiral structure. The thermal stability of duplexes formed by nucleotide oligomers and PNA-PA nanofibers was determined by their melting temperature (Tm) using UV to detect their thermal denaturation (25). Compared to previous work with PNAs, the PNA-PA nanofibers formed stronger complexes with both polyadenylic DNA and RNA heptamers (26, 27). This resulted in a ca. 25 °C increase in Tm over the corresponding polythymine PNA heptamer (Table 1). Furthermore, the PNA-PA retained sequence specificity when tested with 5′-AAATAAA-3′ single strand DNA (ssDNA). This complex melted at 55.5 °C, which is ca. 16 °C lower than the melting temperature of the fully matched sequence when bound to PNA-PA nanofibers. Oligonucleotide binding to the PNA-PA was also tested with fluorescence polarization with 5′-AAAAAAA3′-fluorescein ssDNA. The fluorescence polarization of
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Figure 1. Structure of peptide nucleic acid/peptide amphiphile conjugate (PNA-PA). The thymine nucleic acid base was attached through an acetyl linker to an N-(2-aminoethyl)glycine PNA derivative, shown in red.
mRNA separation and purification, as well as potential sensor applications that require strong and specific binding to oligonucleotides. Longer peptide nucleic acid sequences could be of interest for RNA interference applications; however, further study will be necessary to determine if these sequences can be incorporated without distrupting peptide amphiphile self-assembly. Small interfering RNAs (siRNAs) incorporate into protein complexes that are critical in mRNA destruction and translation regulation (29). The self-assembling nanostructures described here can be potentially useful in this context, however the siRNA sequence presented on the nanostructures must be released in cytoplasm and be able to complex to proteins without affecting their functions. Further studies are underway to explore other PNAPA sequences and their behavior under physiological conditions. ACKNOWLEDGMENT Figure 2. TEM micrograph of negatively stained PNA-PA nanofibers. Table 1. Tm Data for PNA-PA/Oligonucleotide Complexesa Tm (°C)
PNA2DNA
PNA2RNA
mismatch
∆Tm (°C)
PNA-PA PNAb
71.4 44.4b
73.8 48.3b
55.5
-15.9
a Conditions for T m measurement: 10 mM TAPS, 0.1 mM EDTA, and 150 mM NaCl, pH 8.5, UV measured at 260 nm from 90 °C to 25 °C, in 1 °C increments. Strand concentrations were 5.0 µM in PNA and 2.5 µM in the complementary oligonucleotide. ∆Tm represents the difference in melting temperature between the complementary DNA and the DNA with the indicated mismatch. Mismatch represents 5′-AAATAAA-3′ ssDNA. b Data adopted from ref (26).
fluorescein-tagged polyadenylic ssDNA heptamer was measured to be 0.4 ( 2 milli-Polarization (mP), with an increase to 33 ( 2 mP observed in the presence of selfassembled PNA-PA nanofibers. We attribute this increase to reduced rotational freedom of the tagged DNA sequence, indicative of binding to the self-assembled nanofibers. Fluorescence polarization of fluorescein alone in the presence of the PNA-PA was measured as 0.1 ( 2 mP, indicating an absence of nonspecific binding of the dye to the PNA-PA nanofibers. We have reported a peptide nucleic acid/peptide amphiphile conjugate that binds to oligonucleotides with high affinity and specificity after self-assembly into nanostructures. Because the nanostructures are easily endocytosed by cells (28), this type of hybrid material could be attractive in biomedical research for applications such as RNA interference studies and gene silencing,
This work was funded by the US Department of Energy (DoE) under award No. DE-FG02-00ER54810, NSF Nanoscale Science and Engineering Center (NSEC) (EEC-0118025), and J.K.P. was supported by Northwestern University’s NIH Biotechnology Training Grant (T32GM008449). We thank the Electron Probe Instrumentation Center for use of its Hitachi H-8100 transmission electron microscope and the Keck Biophysics Facility for use of its Jasco J-715 CD spectrometer and Beacon 2000 fluorescence polarization system at Northwestern University. We also thank Mukti Rao and Liang-Shi Li for TEM and James Hulvat for his helpful discussions. Supporting Information Available: Experimental protocols, circular dichroism spectrum, and additional TEM images. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Alwine, J. C., Kemp, D. J., and Stark, G. R. (1977) Method for Detection of Specific RNAs in Agarose Gels by Transfer to Diazobenzyloxymethyl-Paper and Hybridization with DNA Probes. Proc. Natl. Acad. Sci. U.S.A. 74, 5350-5354. (2) Ahlquist, P. (2002) RNA-dependent RNA polymerases, viruses, and RNA silencing. Science 296, 1270-1273. (3) Calzone, F. J., Britten, R. J., and Davidson, E. H. (1987) Mapping of Gene Transcripts by Nuclease Protection Assays and Cdna Primer Extension. Methods Enzymol. 152, 611632. (4) Erlich, H. A., Gelfand, D., and Sninsky, J. J. (1991) Recent Advances in the Polymerase Chain-Reaction. Science 252, 1643-1651. (5) Paterson, B. M., Roberts, B. E., and Kuff, E. L. (1977) Structural Gene Identification and Mapping by DNA-Mes-
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