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The impact of nucleic acid secondary structure on PNA hybridization Bruce A. Armitage Hybridization of oligonucleotides and their analogues to complementary DNA or RNA sequences is complicated by the presence of secondary and tertiary structure in the target. In particular, folding of the target nucleic acid imposes substantial thermodynamic penalties to hybridization. Slower kinetics for hybridization can also be observed, relative to an unstructured target. The development of high affinity oligonucleotide analogues such as peptide nucleic acid (PNA) can compensate for the thermodynamic and kinetic barriers to hybridization. Examples of structured targets successfully hybridized by PNA oligomers include DNA duplexes, DNA hairpins, DNA quadruplexes and an RNA hairpin embedded within a mRNA.

Bruce A. Armitage Dept of Chemistry Carnegie Mellon University 4400 Fifth Avenue Pittsburgh PA 15213-3890, USA e-mail: [email protected]

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▼ Fifty years have passed since Watson and Crick proposed the double-helical model for the 3D structure of DNA [1]. The biological implications of the model were famously understated in the paper, but cannot be overemphasized, because the molecular basis of genetics and reproduction follow from the complementary pairing of the two DNA strands. Less obvious at the time was the revolution that would be launched in bioorganic chemistry due to the elegantly simple strategy of hydrogen bond-mediated molecular recognition of specific nucleic acid sequences. Using the rules for nucleobase pairing, that is guanine with cytosine and adenine with thymine, synthetic oligomers can now be designed for diverse applications including antisense therapy, clinical diagnostics, microarray assays and nucleic acid purification. In each case, the oligomer hybridizes to its complementary sequence on the DNA or RNA target, forming a double-helical complex in the same fashion as natural DNA. It seems trivial to design an effective hybridization agent: given the sequence of nucleobases on the target strand, one simply synthesizes the complementary sequence

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based on the Watson-Crick rules for base pairing. However, there are several obstacles to effective hybridization, the most significant being the presence of secondary (and perhaps tertiary) structure in the target. When duplex DNA is the target, the secondary structure is the double helix and the functional groups required for recognition are already involved in hydrogen bonding to the complementary strand. When mRNA is the target, the lack of a complementary strand allows the RNA to fold into a complex 3D structure stabilized by a combination of hydrogen bonding and other non-covalent interactions. In both cases, the structure inherent in the target must be disrupted in order for the hybridization agent to gain access to its target sequence. Thus, structured targets incur a thermodynamic penalty for hybridization that is not present for unstructured targets, leading to a less favorable free energy of reaction (Fig. 1, ∆GA versus ∆GB). For in vitro assays and applications, the structure can be destabilized by decreasing ionic strength or raising temperature, although this strategy for recovering the free energy of hybridization requires that the resulting hybrid not be similarly destabilized by the change in environmental conditions. No such luxury is available for in vivo applications, where the temperature, ionic strength or pH cannot be varied substantially without deleterious effect on the organism. To overcome the energetic penalty for hybridization to structured targets, an alternative strategy is to increase the stability of the hybrid duplex by using higher affinity oligomers. This review will focus on one such analogue, peptide nucleic acid (PNA). PNA was first reported in 1991 by Nielsen, Buchardt, Egholm and Berg [2–4] and consists of the natural nucleobases conjugated to a polyamide

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Figure 1. Comparison of ∆G for hybridization to unstructured (A) and structured (B) targets. Higher affinity probes increase the driving force for the reaction (∆GC).

backbone (Fig. 2). The bases extend from the backbone at approximately the same distances as in natural DNA or RNA. This allows a PNA oligomer to hybridize to its complementary DNA or RNA sequence in a sequence-specific, cooperative fashion, because formation of one base pair facilitates formation of adjacent pairs [5]. Moreover, as the PNA backbone is uncharged, hybridization does not suffer from the electrostatic repulsions incurred when a DNA oligomer is used. This effectively lowers the free energy of the hybrid and recovers some of the energy penalty due to structure in the target (Fig. 1, ∆GB versus ∆GC). The effect of target folding on the kinetics of PNA hybridization is more difficult to predict. In some cases, where the target sequence is base-paired to another region of the nucleic acid, then one can reasonably expect the kinetics to be slower than when the target sequence is fully accessible. However, because so little is known about hybridization kinetics for folded targets, general statements should best be avoided and each system studied carefully. The few cases where kinetics have been analyzed will be described below. The remainder of the review will focus on hybridization of PNA to four different structured targets: (1) duplex DNA, (2) DNA hairpins, (3) quadruplex DNA and (4) RNA hairpins.

Duplex DNA targets Hybridization to duplex DNA holds great appeal for regulation of gene expression, although the presence of a tightly and thermodynamic barrier to binding by WatsonCrick pairing. PNA was originally designed to bind duplex

DNA in the major groove through Hoogsteen hydrogen bonding, analogous to triplex-forming oligonucleotides. However, the original paper on PNA demonstrated that a T10-PNA oligomer bound to a DNA restriction fragment at an A10 sequence through strand invasion, meaning that the complementary T10 DNA strand was locally displaced to allow the PNA to bind the A10 target through WatsonCrick pairing [2]. Further experiments revealed that a second PNA strand was bound by Hoogsteen pairing, giving an exceptionally stable PNA2-DNA triplex (Fig. 3) [6,7]. This is an impressive example of how high affinity can be used to overcome the secondary structure inherent in duplex DNA to access the sequence information, and has led to an interesting set of applications including both enhanced [8–10] and repressed [11–15] transcription, rare enzyme cutting [16–20], DNA purification [21] and plasmid functionalization [22–25]. Strand invasion was originally thought to be restricted to homopyrimidine PNA sequences because these permit Hoogsteen pairing of the second PNA strand. However, homopurine PNAs have been reported to successfully invade duplex DNA targets [26]. In addition, cationic peptides attached to the PNA termini have been found to permit strand invasion at a variety of mixed-sequence DNA targets, although this was most favorable for super-coiled DNA targets [27,28]. Evidently, the additional stabilization as a result of electrostatic attraction between the peptide and the DNA could couple into uncoiling of the DNA to permit strand invasion without Hoogsteen stabilization of the PNA-DNA hybrid. The kinetics of strand invasion are slow under most circumstances and, in the original experiments, required relatively low ionic strength conditions. This is because the DNA duplex must be opened in order for the Watson-Crick PNA to bind to its complement and low ionic strength

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model for DNA structure. However, there is growing evidence that a variD P ety of non-duplex structures, includD ing triplex [36], quadruplex [37] and P Strand hairpin forms [38], are relevant in vivo. DNA invasion When one considers the difficulty of D DNA distinguishing two similar sequences P + of duplex DNA in comparison with D distinguishing between a duplex and PNA hairpin secondary structure, the appeal D Double of non-duplex forms for protein recogP duplex nition becomes evident. Thus, regions P invasion D of the genome which are important for gene expression and replication, such Drug Discovery Today as promoters, replication origins and Figure 3. Hybridization of PNA to duplex DNA. Strand invasion occurs in 2:1 or 1:1 telomeres, contain sequences that are (PNA:DNA) ratios depending on the sequence. Double duplex invasion occurs when two suspected to fold into non-duplex ‘pseudocomplementary’ PNAs are used. PNA and DNA strands are labeled P and D, respectively. structures, even in the presence of a complementary strand. Although much remains to be learned about the underlying thermodynamics controlling formation of nondestabilizes DNA-DNA duplexes [29,30]. Significant imduplex structures, initial efforts to hybridize synthetic provements in strand invasion kinetics have been made by oligomers to them have been promising. covalently attaching two homopyrimidine PNAs together An early report from Ørum and coworkers described to form a bisPNA [31,32] or by adding cationic peptides to the use of PNA to target a DNA hairpin consisting of a 4 the N-terminus of the PNA [33]. nucleotide loop and a 15 base pair stem [21]. The PNA was Nielsen and coworkers devised an alternative approach complementary to the 15 nucleotides comprising the for recognizing mixed-sequence duplex DNA targets, 3′-side of the stem. When mixed at low ionic strength, the known as the ‘double duplex invasion’ strategy, where two PNA effectively hybridized to the hairpin. However, at PNA strands simultaneously target both DNA strands higher ionic strength, hybridization was suppressed. (Fig. 3) [34,35]. The two PNAs are necessarily compleThese results are reminiscent of the strand invasion rementary to one another and so will ‘quench’ each other quirements described above, wherein PNAs were unable through hybridization because PNA-PNA duplexes are to target duplex DNA except at low ionic strength. In the more stable than DNA-DNA duplexes, even without the case of the hairpin target, it is unclear whether hybridizathermodynamic penalty for strand invasion. To avoid this s tion failed at high ionic strength for thermodynamic or problem, thiouracil ( U) was substituted for thymine, and kinetic reasons. diaminopurine (D) was substituted for adenine. Whereas s A more recent publication reported the use of PNA U and D form stable base pairs with A and T, respectively, probes to target members of the GA3-family of DNA hairthe sU-D pair is less stable than an A-T pair for steric reasons. Thus the ‘pseudocomplementary’ PNA-PNA duplex is pins (Fig. 4) [39]. Hairpins of this class are stabilized by a sufficiently destabilized to allow double duplex invasion unique secondary structure for the GA3 loop that leads to into DNA. This strategy necessarily requires that there be surprisingly high melting temperatures. Sequences consufficient A-T pairs in the target sequence to allow the taining this motif are common in replication origins and s U-D discrimination to be exploited, so highly G-C rich promoters, suggesting a biological role for the hairpin structure [40], similar to the recent discovery of a guanine sequences still cannot be targeted in this fashion. quadruplex structure involved in the regulation of c-Myc Nevertheless, this is an important advance in the sequenceoncogene expression [37]. Three different PNA probes specific recognition of duplex DNA. were synthesized and targeted to the hairpins. In each case, stable duplexes were formed at room temperature, Hairpin DNA targets even though the melting temperatures of the hybrids Non-duplex secondary structures for DNA would not, at were as much as 70°C below that of the hairpin. This is first glance, appear to be interesting targets for hybridization because the ∆G for formation of the PNA-DNA duplex was probes. Such is the lasting effect of the Watson-Crick

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Figure 4. Non-duplex nucleic acid secondary structures. Top: Hairpin; Bottom: G-quadruplex. Bottom right: hydrogen-bonded guanine tetrad stabilized by coordinated potassium ion.

more favorable by between 3 and 6 kcal mol−1, allowing hybridization to occur. However, in comparison with a reference target that was not able to fold into a stable secondary structure, a 2.1 kcal mol−1 penalty was incurred for unfolding the hairpin in order for the PNA to hybridize. Under identical conditions, the corresponding DNA probe was unable to hybridize to the hairpin demonstrating the importance of high affinity to overcome stable secondary structures. Kinetic experiments also revealed that PNA hybridized to the structured target five-times more slowly and with an activation energy that was nearly 50% greater than for the unstructured target. In addition, although longer PNAs formed more stable hybrids with the DNA hairpin target, they did so at slower rates. This was attributed to the ability of the PNA to adopt its own secondary structure and illustrates the need for careful probe design to maximize hybridization efficiency. The PNA probes targeted the DNA hairpin symmetrically, meaning they were capable of forming their own hairpin structures, as observed previously [41]. Thus, when targeting hairpin structures, ideal hybridization probes should be complementary in an unsymmetrical fashion, ideally pairing with nucleotides in the loop as well as along one side of the stem [21]. This

strategy was adopted recently in the targeting of an RNA hairpin element within a mRNA (vide infra).

Quadruplex DNA targets The quadruplex motif consists of four guanine bases simultaneously hydrogen-bonded to one another to form a quasi-square planar structure (Fig. 4). [42,43] Consecutive G-tetrads can stack on top of one another and metal cations can be bound between adjacent tetrads to balance the negative electrostatic potential of the carbonyl oxygens directed toward the interior of the tetrad. Aside from the novel structure of the G-quartet, there is growing interest in this non-duplex secondary structure because of its purported role in several biological contexts [44], including telomere stabilization [43] and protein-mRNA interactions [45]. In addition, numerous in vitro selection experiments performed on randomized DNA and RNA libraries have yielded high-affinity aptamers for both protein [45–47] and small-molecule targets [48] in which the aptamer contains a highly G-rich sequence which likely folds into a quadruplex structure. Recent results indicate that G-quadruplex structures are present in vivo [37,44] making them potential targets for therapeutic compounds [49].

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One of the simplest G-quadruplex structures is the thrombin-binding aptamer, a 15 nucleotide DNA sequence that folds into an intra-molecular G-quadruplex (Fig. 4) and exhibits nanomolar affinity for the protein thrombin [47,50]. A short PNA heptamer was synthesized and targeted to the central seven nucleotides of the quadruplex [51]. The PNA readily hybridized to the quadruplex, forming a PNA-DNA duplex of surprisingly high stability (∆G298 = −11.7 kcal mol−1) considering that there were only seven base pairs present. Further experiments revealed that the overhanging four nucleotides on either end of the duplex were contributing 2.3 kcal mol−1 to the stability. This effect was also seen with other duplex and overhang sequences, indicating that it is not specific to the PNA-DNA hybrid used in this study. These results could have important ramifications for PNA probes, because the high affinity allows the use of short oligomers to form stable hybrids, and virtually every biological target will be longer than the PNA, meaning there will almost always be overhanging nucleotides present to potentially stabilize the hybrid even further. One additional noteworthy result from this study involved the effect of ionic strength on hybridization of the PNA probe to the DNA quadruplex. Early work with PNA showed that its affinity for cDNA targets exhibited very modest dependence on the ionic strength of the buffer used for hybridization [52]. This was attributed to the uncharged PNA backbone and has been perceived as a major advantage of PNA relative to hybridization agents based on anionic, DNA-like backbones. However, whereas the PNA-DNA hybrid stability might be relatively insensitive to changes in ionic strength, the stability of all competing secondary structure in the DNA target will likely be acutely sensitive to the ionic strength. This effect was demonstrated clearly with the DNA quadruplex target, where a buffer containing 10 mM potassium phosphate and 100 mM KCl yielded a PNA-DNA hybrid with a melting temperature (Tm) of 34°C and a ∆G = −8.3 kcal mol−1. When the KCl concentration was reduced to 10 mM KCl, the Tm and ∆G increased to 55°C and –11.7 kcal mol−1, respectively. The less favorable ∆G for the higher ionic strength buffer is due almost entirely to stabilization of the quadruplex structure by the added potassium ions. In terms of the diagram shown in Fig. 1, this lowers the free energy of the target without significantly stabilizing the hybrid, therefore reducing the free energy of hybridization. Of broader significance is the fact that most applications will involve targeting of PNA to relatively long DNA or RNA sequences. These will almost certainly be capable of folding into secondary and tertiary structures whose stabilities will depend acutely on ionic strength. Therefore, one can expect a

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significant ionic strength dependence for PNA hybridization to long DNA or RNA targets.

Triplet repeat DNA One other set of reports concerning targeting of triplet repeat DNA sequences by PNA is worth describing [53–55]. Expansion of CAG triplet repeats has been linked to several genetic diseases, including the well known Huntington’s disease. The number of triplet repeats correlates inversely with age of onset and directly with disease severity. Methods that can accurately count the number of repeats are therefore of great interest. PNAs complementary to either the (CAG)n or (CTG)n were found to effectively invade the DNA target and hybridize to their intended sequences. When the PNA was conjugated to biotin, the resulting PNA-DNA complex was captured using streptavidin-coated magnetic beads, allowing isolation of genes having CAG repeats [54]. When linked to a fluorescent reporter, the PNA could be used to visualize sites of CAG repeats in cells using fluorescence in situ hybridization [53]. In addition, an antigene triplet repeat PNA reportedly blocks transcription of genes containing CAG triplet repeats [55], although strand invasion by the PNA was not demonstrated directly. What is interesting about these reports, in terms of hybridization, is the structure of the DNA target. In each case, a complementary DNA strand was present, meaning that the target could be a duplex. However, other reports have noted the tendency of CAG repeats to fold into hairpin structures [56], meaning that the target sequence for the PNA could actually be extruded from the duplex to form either a hairpin or cruciform structure. Although no experiments were performed to assess the secondary structure of the DNA when targeted with PNA, the success of these hybridizations illustrates the ability of PNA to invade a variety of DNA structures and sequences, beyond the original homopurine-homopyrimidine motifs.

RNA targets Several reports have been published pertaining to the targeting of PNA probes to folded RNA sequences. These include the RNA component of telomerase [57,58], various mRNAs [11–13,59], ribosomal RNA [60] and the transactivation response region (TAR) RNA from HIV [61,62]. Although results have been mixed, very little in-depth analysis of the effect of RNA structure on PNA hybridization has been provided. One exception is a recent report pertaining to antisense PNAs targeted to the Ha-Ras mRNA [13]. Prior work had shown that PNAs complementary to several different regions of the mRNA could arrest polypeptide chain elongation, meaning that an actively translating

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ribosome could be sterically blocked by the PNA [11]. In the more recent report, a PNA probe was shown to invade a stable hairpin within the RNA and block translation. Melting-temperature analysis with a model hairpin showed that overhanging nucleotides significantly stabilized the PNA-RNA hybrid, as reported previously for PNA-DNA hybrids. In addition, two transitions were observed in the melting curve, indicative of a lower temperature triplex and a higher temperature duplex form. The presence of a PNA2-RNA triplex was verified by mass spectrometry. The triplex was able to form because the PNA sequence contained eight consecutive pyrimidines, out of 13 total bases. The lower Tm for the triplex reflects the increased length of the duplex. Of broader importance in this work was the conclusion that when PNAs with high G-C content are used, ribosomes can be arrested at the site of hybridization, leading to a truncated protein. Extension of this design concept to other mRNAs or sequence contexts that only permit duplex formation remains to be demonstrated.

Outlook The sequestration of nucleic acid primary structure within complex 3D folds imposes both thermodynamic penalties and kinetic barriers to hybridization. High affinity probes such as PNA can recover at least some of the free energy through enhanced enthalpy or diminished entropy changes for binding, whereas kinetics can be accelerated through the addition of cationic substituents. Further progress in this field will rely on increasingly complex model systems where factors such as probe length, ionic strength and target selection can be further examined. In addition, advances in predicting the secondary and tertiary structure of natural nucleic acids will be vital because with greater knowledge of the 3D structure of the target comes greater likelihood that successful hybridization probes can be rationally designed for biological systems. In fact, it is the ‘hybridization’ of organic chemistry for the design and synthesis of PNA, coupled with the physical chemistry required to analyze the thermodynamics and kinetics of hybridization, that illustrates one approach to dealing with sequence-specific recognition of DNA and RNA. The excellent biostability of PNA provides additional motivation for its continued development for in vivo applications [63–65]. Although this review has focused on the properties of PNA oligomers, many of the principles can be extended to other high affinity analogues, such as locked nucleic acid [66–69]. Comparisons among several different backbone chemistries should be particularly useful for isolating those structural components that contribute most to a favorable hybridization property.

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Acknowledgements I would like to thank my collaborators and colleagues for their hard work and stimulating discussions, as well as the National Institutes of Health for their generous support of our research in this area.

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