Highly Cooperative Behavior of Peptide Nucleic Acid-Linked DNA-Modified Gold-Nanoparticle and Comb-Polymer Aggregates

DNA-modified gold nanoparticles (AuNPs) and comb-polymer–DNA hybrids are two classes of DNA–hybrid materials that have been used in a variety of diagnostic systems for the detection of nucleic acids, proteins, metal ions, and small molecules.^[1-10] When complementary mixtures of these materials are combined, they assemble to form aggregates linked through DNA duplexes. Upon heating, the aggregates dehybridize, and exhibit exceptionally sharp DNA dissociations, or melting, transitions, and increased melting temperatures (T_m), in comparison to unmodified DNA-duplex melting behavior.^[10-13] These sharp transitions are responsible for the high selectivity of diagnostics systems based upon the use of these structures as probes. Indeed, with them one can easily discriminate subtle changes in binding stability resulting from single-base pair mismatches.^[10,14]

The sharp melting transitions associated with DNA-linked Au–DNA and polymer–DNA aggregates have been attributed to cooperativity, which arises from polyvalent binding and charged interactions between closely spaced duplexes. The basic premise of this model is that counterions surrounding one duplex can stabilize neighboring duplexes. Therefore, when initial dissociative events take place, the remaining duplex linkages are destabilized, and the system cascades rapidly to a fully dehybridized state over a narrow temperature range. This explanation prompts us to question whether particle aggregates can be held together with peptide nucleic acids (PNAs),^[15,16] uncharged oligonucleotides, and exhibit similarly sharp melting behaviors.

Although PNA-modified AuNPs have been synthesized,^[17] these structures exhibit low aqueous solubility and colloid stability in the absence of surfactants or charged amino-acid residues. Because we have also encountered significant difficulties in preparing such structures, we have taken a different approach to studying the melting process in the context of PNA-based systems. In this strategy, we work with DNA-modified particles but use a PNA linker to effect aggregate assembly (Scheme 1). Interestingly, we have discovered that these systems also exhibit similarly sharp melting transitions, despite the uncharged state of the PNA linker. In addition, we observe a surprising reverse dependence of the melting temperature T_m of these PNA-linked aggregates on salt concentration, in comparison to that of unmodified PNA:DNA duplexes. Theoretical modeling and experiments suggest that this behavior can be attributed to the condensed ion clouds associated with closely spaced PNA:DNA duplexes within the aggregates. Finally, we show that this is a general phenomenon, which extends beyond AuNPs to another scaffold, DNA-modified comb polymers, which can also engage in cooperative binding with complementary nucleic acids (Scheme 1).

Scheme 1.

Schematic illustration of DNA-modified AuNPs or polymers linked together with complementary PNA or DNA. For our experiment, the polymer backbone is a neutral polynorbornene containing ~5 DNA strands/polymer, consequently having a much lower net charge than the 13 nm DNA-modified gold nanoparticle (~40 DNA strands/particle).^[18] (See Fig. S6 of SI for a color version of this figure.)

To create a basis for comparison, we first examine the melting behavior of unmodified PNA:DNA duplexes. In line with previous reports,^[19] PNA:DNA and DNA:DNA duplexes of the same sequence (Scheme 1) display opposite T_m dependence on salt concentration (Fig. 1). However, the variation in T_m as a function of salt concentration is more pronounced for the DNA:DNA duplexes, consistent with the higher affinity of DNA:DNA duplexes for the surrounding cation cloud.^[20] Due to the repulsive interactions between the two strands in a DNA:DNA duplex, hybridization dictates that counterions must condense along the polyanionic backbones of the individual strands, resulting in a net increase in the number of cations associated with the duplex. On the other hand, the neutral backbone in PNA does not repel the complementary DNA strand, and consequently does not require a net flux of condensed cations to stabilize the PNA:DNA duplex. In fact, cations must be released from the ion cloud normally associated with a DNA single strand as it hybridizes with a complementary PNA strand.^[19] As a result, increasing the salt concentration better stabilizes a DNA:DNA duplex, but conversely favors dissociation of a PNA:DNA duplex into single strands. However, the efflux of cations associated with PNA:DNA duplex formation is less than the influx of cations that condense when the DNA:DNA duplex is formed, leading to a weaker salt-dependence of T_m for a PNA:DNA duplex than the analogous DNA:DNA duplex (Fig. 1).^[19]

Figure 1.

Melting temperature of PNA:DNA and DNA:DNA duplexes, respectively, as a function of salt concentration. The duplexes are 18 base pairs consisting of two complete strands based on the complementary sequences shown in Scheme 1.

Given the important role that charge condensation plays in the hybridization behavior of DNA-linked aggregates,^[12,20,21] we were surprised to observe both enhanced melting temperatures and sharp melting profiles for AuNP–DNA aggregates formed in the presence of a charge-neutral PNA strand (Fig. 2A). Similar to DNA-linked AuNP–DNA hybrid systems, the presence of these PNA-linked aggregates was indicated by a concomitant color change from red to blue, which arises from a dampening and shifting of the AuNP surface plasmon band.^[22] The full-width at half-maximum (FWHM) of the first derivative for the melting profile is 1–2 °C, strikingly similar to the analogous DNA-linked aggregate system (FWHM = 1–2 °C, Fig. 2B). This sharp melting behavior demonstrates the potential for utilizing PNA in AuNP probe applications, where the high selectivity associated with the cooperative AuNP assemblies can be combined with the exceptional stability of PNA.

Figure 2.

Melting profiles of A) PNA-linked AuNP–DNA assemblies, B) DNA-linked AuNP–DNA assemblies, C) PNA-linked polymer–DNA assemblies, and D) DNA-linked polymer–DNA assemblies. To optimize hybridization efficiency, an acetylated N-terminus PNA strand was used, which significantly increased the amount of hybridization based on changes in the UV–vis absorbance. (See Fig. S7 of SI for a color version of this figure.)

Unlike the inverse salt-dependence observed for the T_m of unmodified PNA:DNA duplexes (Fig. 1), the PNA-linked AuNP–DNA aggregates exhibit a direct dependence of the T_m on salt concentration, similar to that for the DNA-linked system (Fig. 2A and B, respectively). One possible reason for this reversal of salt dependence is the influence of counterions on repulsive forces between the DNA-modified AuNPs. Given the large steric footprint of a DNA duplex, at most only 44%^[23] of the ~40–45^[18] surface strands on the nanoparticle can be hybridized with free DNA, leaving a significant amount of negatively charged single strands on the surface of the nanoparticles (~4 unhybridized strands/100 nm², see Supporting Information (SI)). Hence, during aggregation, these excess surface charges repel those on adjacent AuNPs, and thus require the presence of external counterions to screen the repulsive interactions. Notably, for the same concentration domain, the change in T_m as a function of salt concentration for the PNA-linked AuNPs is about five times less than that of the DNA-linked AuNP assemblies (see Fig. S1 in ESI), consistent with the lesser salt dependence observed for the unmodified PNA:DNA versus DNA:DNA duplex systems (Fig. 1).

Suspecting that the aforementioned reversal in salt concentration can be modulated by changing the scaffold of the DNA-modified material, we examined the melting behavior of PNA-linked comb polymer–DNA hybrid aggregates, which consist of a charge-neutral polymer backbone and DNA strands as side chains. For comparison to the AuNP system (8 DNA strands/100 nm²), we selected a polymer–DNA hybrid with the highest DNA density possible (5 DNA strands/100 nm², 5 DNA strands per 17 repeating units).^[10,24] As observed for PNA-linked AuNP–DNA hybrids, PNA-linked polymer–DNA hybrid aggregates also exhibit sharp melting transitions and monotonic T_m dependence on salt concentration (Fig. 2C). Remarkably, however, PNA-linked polymer–DNA aggregates can form even at 0.1 m NaCl, much below the 0.2 m NaCl limit for the assembly of PNA-linked AuNP–DNA hybrids. This observation clearly indicates that the nature of our two DNA-modified scaffolds exert significant effects on the PNA-linked aggregate behavior. Assuming a minimum hybridization efficiency of 60% for the polymer–DNA hybrids (see SI) and 44%^[23] for the AuNP–DNA hybrids, the greater number of charges attributed to unhybridized DNA strands on the AuNP–DNA hybrid (~4 vs. 2 unhybridized strands/100 nm², AuNP–and polymer–DNA hybrids, respectively) leads to particle repulsion, preventing aggregate formation at lower salt concentration, such as 0.2 m. Consistent with this hypothesis of greater repulsion in the AuNP system, the variation in T_m as a function of salt concentration for the PNA-linked AuNP–DNA aggregates (Fig. 2A) is approximately three times larger than that for the PNA-linked polymer–DNA hybrids (Fig. 2A), over the 0.2–0.5 m concentration domain (see Fig. S1 in SI). However, the direct salt-dependence of the T_m trend for both of the PNA-linked hybrid materials, in contrast to that of unmodified PNA:DNA duplexes, suggests that the aggregate presence is the primary cause for reversing the salt-dependence of “native” PNA:DNA interactions.

To further explain the reversal in salt dependence for the PNA:DNA duplexes in an aggregate, and its relation to sharp melting, we must take into account the molecular-level interactions between the neighboring strands in the aggregate. As previously discussed, the salt dependence of unmodified PNA:DNA duplexes is relatively weak compared to that of DNA:DNA duplexes (Fig. 1), which can be understood in terms of the counterion condensation effects. In our earlier work, we demonstrated that the effect of salt on the melting behavior of DNA:DNA aggregates (either polymers–DNA^[21] or AuNP–DNA^[12]) fits well within the framework of the condensed-counterion, cooperative mechanism.^[20] To see if this same mechanism might apply to the aggregates involving PNA:DNA duplexes, we have used molecular dynamics simulations to perform an analysis of the ion distributions around a PNA:DNA duplex pair (two duplexes) and a PNA:DNA-duplex cluster (four duplexes, see Fig. S5 in SI). The simulations demonstrate that the local (condensed) net-charge fraction increases when the interduplex distance for the pair or cluster drops below 5 nm, virtually identical to the distance calculated for the DNA:DNA pairs and clusters.^[20] Significantly, these data also indicate that more condensed counterions associate with the close-packed PNA:DNA aggregates than with the isolated PNA:DNA duplex. Because of the presence of more condensed counterions in the PNA:DNA aggregates, we propose that within a critical separation distance, the PNA:DNA pair or cluster associates more condensed counterions than single-stranded DNA, leading to a release of condensed counterions when the close-packed PNA:DNA aggregates melt (similar to the behavior of DNA:DNA duplexes). For the close-packed PNA:DNA aggregates, the presence of more condensed cations on the PNA and DNA strands when hybridized rather than dissociated results in a T_m that increases with increasing salt concentration, in contrast to the reverse behavior that occurs with isolated PNA:DNA duplexes.

Notably, sharp melting in DNA-based aggregates has been modeled as a phase transition from gel to sol states,^[25,26] which ignores nearby DNA:DNA interactions and the condensed ion clouds between neighboring duplexes.^[27,28] In two recent papers, we have demonstrated how it is possible to “embed” the cooperative model within the overall framework of the phase-transition model, such that both are utilized to determine the overall melting curve.^[25,26] The relative importance of these two models depends on two factors: the size of the aggregates (more linked particles lead to more important phase-transition effects) and the number of cooperatively interacting duplexes (as determined by duplex density). In particular, the effect of salt would be significant in the counterion condensation model, but not on phase-transition properties. Even if the phase-transition behavior is considered to be dominant, cooperativity still influences the T_m^[26] and its dependence on added salt.^[25] In particular, if the cooperative model suggests that the T_m increases with added salt, then this salt-dependent behavior will be observed even when the overall sharpness is determined by the phase-transition mechanism. On the other hand, if the PNA:DNA duplexes do not approach one another to within the critical reversion distance (5 nm), the melting transition might still be sharp due to the phase-transition mechanism, but the salt effect would be reversed and similar to that of the isolated PNA:DNA duplexes. We conclude from this and the results in Figure 2 that cooperativity originating from neighboring duplex interactions is important in both the PNA-linked gold-nanoparticle and polymer aggregates. Significantly, the ramifications of these results are that PNA hybrid materials must allow for neighboring PNA:DNA duplex interactions to exhibit the high level of cooperativity observed in our systems.

In conclusion, we have discovered that PNA-linked AuNP–DNA and polymer–DNA hybrid aggregates display sharp melting transitions when compared with unmodified PNA:DNA duplexes. Significantly, we have uncovered an unusual and general salt dependence of the melting temperature of PNA:DNA duplexes within a hybrid aggregate, which is reverse of the trend observed in unmodified PNA:DNA duplexes. Experimental observations supported by molecular simulations lend credence to duplex cooperativity and ion condensation playing significant roles in AuNP– and polymer–DNA hybrid-aggregate melting properties. It is the presence of closely spaced duplexes inside the aggregates that leads to the positive salt-dependent behavior, which in turn results in cooperative interactions between neighboring duplexes. This work represents a significant step in extending the cooperativity manifested in the sharp melting transitions of DNA-linked hybrids to uncharged biopolymers such as PNA, and provides further insight into the role that charge plays in cooperativity.