Bioconjugate Chem. 1996, 7, 529−531
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Stabilization of DNA Triple-Helix Formation by Appended Cationic Peptides Ching-Hsuan Tung,† Kenneth J. Breslauer,‡ and Stanley Stein*,†,‡ Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, New Jersey 08854, and Department of Chemistry, Rutgers University, Piscataway, New Jersey 08855. Received April 1, 1996X
We have investigated the impact of appended cationic peptides on the triplex-forming and thermalstabilizing abilities of an oligonucleotide “third strand” using a homopurine-homopyrimidine 37-mer as the host duplex target. The family of appended cationic peptides studied here contains arginine, lysine, ornithine, and diaminobutyric acid, with the residues being linked through either their R-amino or side chain amino groups. On the basis of optical melting profiles, we find that peptides with different amino acid compositions, but with four positive charges in common, are able to enhance, in a similar manner, the triplex-forming capability of an oligonucleotide. We note the implications of these results for the rational design of third-strand probes.
Triplex-forming oligonucleotides (TFOs) bind to the major groove of double-stranded DNA through Hoogsteen or reverse Hoogsteen hydrogen bonding. Since binding to genomic DNA by a TFO can inhibit transcription, strategic targets for TFOs have included human diseaserelated genes such as Ki-ras (1), HER-2/neu (2), c-erbB (3), c-myc (4), HIV-1 (5), and the interferon-responsive element (6). In other applications, artificial endonucleases have been created by attaching reactive groups to TFOs (7-9), with sequence-specific cleavage at a unique site in the human genome being mediated by a TFO (10). Targeted mutations also have been achieved by linking a TFO to a photoactivatable group, such as psoralen (11). In addition, TFOs connected to solid supports have been used to achieve affinity purification of double-stranded DNA (12). To obtain greater TFO binding strengths and sequence specificities, various TFO design strategies have been employed (13, 14). These strategies include the use of modified nucleotides (ref 15 and references therein), the addition of intercalators (16-18) and/or polyamines (ref 19 and references therein) to solution, as well as the direct covalent linking of an intercalator (20, 21), a hydrophobic group (22), or a cross-linking agent (23) to the TFO. We (24) and others (25, 26) have demonstrated that attaching a polyamine to the terminus of a TFO facilitates triplex formation. In other studies, we have shown that a cationic peptide, appended to an antisense strand, will enhance duplex formation when challenged with either DNA (27) or RNA (28) single-stranded targets. In this report, we show that cationic peptides attached to oligonucleotides also favor triplex formation. We focus here on appended tetrapeptides which possess four positive charges to facilitate comparison with a previous study in which we showed that polyamines with four positive charges enhance triplex formation (24). These tetracationic appended tetrapeptides are shown in the * Corresponding author: Stanley Stein, CABM, 679 Hoes Lane, Piscataway, NJ 08854. Telephone: 908-235-5319. Fax: 908-235-4850. E-mail:
[email protected]. † Center for Advanced Biotechnology and Medicine. ‡ Rutgers University. X Abstract published in Advance ACS Abstracts, July 15, 1996.
S1043-1802(96)00040-7 CCC: $12.00
Table 1. Thermal Stabilities of the Different Cationic Peptide-TFO Conjugatesa
Xxx, structure
Tm of triplex
arginine, NH-CH[(CH2)3NHCdNH2+(NH2)]-CO lysine, NH-CH[(CH2)4NH3+]-CO ornithine, NH-CH[(CH2)3NH3+]-CO diaminobutyric acid, NH-CH[(CH2)2NH3+]-CO -lysine, NH-(CH2)4-CH(NH3+)-CO δ-ornithine, NH-(CH2)3-CH(NH3+)-CO
38 40 39 39 39 38
a
Generic TFO is shown in alignment with the duplex target.
first column of Table 1. Note that three of the four charges are associated with basic amino acids, while the fourth charge results from the R-amino group at the N-terminus of the peptide. Further note that the appended cationic peptides contain different positively charged amino acids, with the residues being connected via either their R-amino or side chain amino group. Conjugation to the oligonucleotide is achieved via the side chain of a cysteine residue. We separately synthesized with a 5′-amino group the TFO strand shown in Table 1, as well as the six amidated tetrapeptides listed. On the basis of the results of previous work (29, 30), we chose to incorporate the 5-methylated analog of cytosine into the TFO to enhance its triplex-forming capacity. After purification, the TFO and the peptide were coupled as previously described (27) using a bifunctional reagent N-[(iodoacetyl)oxy]succinimide, to yield the conjugate
(Xxx)3-NH-CH-CO(NH2) | CH2-S-CH2-CO-NH-(CH2)6oligodeoxynucleotide where Xxx is a basic amino acid. The TFO-peptide conjugate then was mixed in a 1:1 molar ratio with a solution of the target 37-mer duplex shown at the top of Table 1. Triplex formation was assessed by melting profiles at 260 nm in 10 mM sodium phosphate and 100 mM sodium chloride (pH 6.5) based on well-established characteristic melting behaviors defined in more exten© 1996 American Chemical Society
530 Bioconjugate Chem., Vol. 7, No. 5, 1996
Tung et al. Table 2. Thermal Stabilities of Various Lengths of Peptide and TFOa
Figure 1. UV melting of the triarginine tetrapeptide-oligonucleotide conjugates. Curves from top to bottom are 21-mer, 18-mer, and 15-mer TFO.
sive studies that used a range of additional techniques (26-30). Simply stated, under appropriate conditions, triplex formation results in the appearance within a melting profile of a second transition at a temperature below the transition of the target duplex. Each sample for melting was prepared by taking a solution containing all three strands at 1 µM and heating to 90 °C for 15 min, followed by slow cooling to room temperature. The resulting solution was then kept at 4 °C overnight to facilitate proper annealing, followed by heating to 100 °C at a rate of 0.5 °C/min. Melting temperatures were determined from the first derivative of the resulting absorbance versus temperature profiles with an accuracy of (0.5 °C. Inspection of the melting profiles shown in Figure 1 reveals the peptide-conjugated TFOs to exhibit two transitions, the first corresponding to the triplex-toduplex conversion and the second reflecting the duplexto-single strand transition at 67 °C. Similar biphasic melting behavior was observed for all other TFO-peptide conjugates studied. The assignment of the second transition to duplex disruption was based on the melting profile of the “free” 37-mer duplex in the absence of the TFO, while the triplex nature of the first transition was confirmed by a negative circular dichroism band at 217 nm (24). Significantly, the “control” unappended third strand does not form a thermally stable triplex under these conditions. Thus, the appended oligopeptide induces the formation of a triplex that otherwise would not form under the conditions of this study. We previously observed a similar influence of appended polyamines (24). Inspection of the Tm data listed in Table 1 reveals that, for TFO-peptide conjugates with the same oligonucleotide chain length and peptide charge, we observe little difference between the thermal stabilities of the triplexes induced by the various peptide conjugates, with the Tm values clustering between 38 and 40 °C, compared with 42 °C for the corresponding spermine conjugate (24). Thus, the topology/amino acid composition of the appended cationic tetrapeptide is less critical to the triplexforming capacity, as judged by the thermal stability of the resulting complex, than is the net charge of the peptide portion of the TFO, which is +4 for all the conjugates in Table 1. To assess the influence of oligonucleotide chain length on TFO capacity, we synthesized a family of conjugates, with a constant peptide component (arginine) and with nucleic acid chain lengths of 15, 18, and 21 nucleotides. The Tm data in Table 2 reveal the triplex-forming capacities of these three conjugates to be nearly independent of chain length. This observation demonstrates
TFO
Tm of triplex
Arg3-21-mer Arg3-18-mer Arg3-15-mer δ-Orn3-21-mer δ-Orn8-21-mer
38 38 37 39 40
a The structures of the TFOs are shown in alignment with the duplex target.
that, under the conditions employed here, an oligonucleotide as short as a 15-mer is sufficient for triplex formation when a cationic peptide is attached, even though triplex formation is not observed for the unconjugated 15-mer. Furthermore, the near constancy of the triplex Tm with increasing chain length of the appended TFO suggests that the system is approaching polymerlike behavior in that the thermal stability has become independent of chain length. We also have assessed preliminarily the influence of peptide chain length on triplex thermal stability by studying two conjugates with constant nucleic acid lengths (21-mers), but varied peptide lengths. As shown by the data in Table 2, we find that in going from three to eight δ-ornithine residues in the peptide little, if any, change in Tm is observed. Thus, for this residue and its linkage to a 21-mer oligonucleotide, the peptide chain length does not impact on the triplex-inducing capacity or the thermal stability of the resulting triplex. Additional studies are required to assess the generality of this observation. In conclusion, attaching a cationic peptide to the terminus of an oligonucleotide can enhance its capacity to form a triplex with a target duplex. From the limited series of conjugates evaluated in this work, it appears that the nature and length of the cationic peptide is less important than is the charge that the appended peptide imparts to the TFO. We also have shown that it is possible to use shorter oligonucleotides as the triplexforming strand, if they are appended to cationic peptides. It should be noted that appended cationic peptides may impart additional desirable properties to TFOs, such as enhanced cellular uptake (31) and nuclear localization (32). Recently, free basic oligopeptides have been used to stabilize triplex formation with sequence dependent activity being observed (33). Clearly, future studies on TFOs with varying charge levels which are challenged by a broader range of duplex targets will be needed to define more completely the role of charge on the triplexforming properties, including sequence specificities. ACKNOWLEDGMENT
The assistance of Walter A. Dickerhof in running the melting profiles is appreciated. LITERATURE CITED (1) Mayfield, C., Squibb, M., and Miller, D. (1994) Inhibition of nuclear protein binding to the human Ki-ras promoter by triplex-forming oligonucleotides. Biochemistry 33, 33583363.
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