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29 Kaneda, Y.. Iwal, K. and Uchlda, T. (1989) J Bioi Chw. 264, 37 Kreuter, J., Alyautdm, R. N., Kharkewh, 1). A. and Ivanov, A. A. 12126-12129 (1995) Braitr Rex. 674. 171-174

30 Mumper, R. J., Dugud. J. G Anwer, K., Barron, M. K., Nitta. H. and RoIland, A. P. (1996) Pl~amr. Rex. 13, 701-709

31 BertIing, W. M. et al. (1991) Biot~chnol. .4ppl. Bi~chwt. 13, 39(GOS 32 Tomlinson, E. and RoIIand, A. P. (1995)j. Control. R&w 39.357-372 33 Davis, S. S., Douglas, S. J., IIlum. L.. Jones, P. D. E., Mak, E. and

MiiIIer, R. H. (1986) m Targeting c!f Drugs wfh Sytrthetic Syrtrw (NATO ASI Smrs, Vol. 113) (Grrgoriadts, G., Semor, J. and Poste, G., eds). pp. 123-146. Plenum Press

34 Seth, P. (1994) Biochm. Biophys. Rex. Cwwwr. 21I5, 1318-1324 35 IIIum, L. and Daws, S. S. (1987) Life Str. 40, 1553-1560 36 Moghinu. S. M. (1995) Adv. Dn1.y D&v. Rev. 17, 61-73

38 Hohnberg, E. et a/. (199O)J. Liyusw~c Rex. 1, 39%406 39 Moghxni, S. M., Porter, C. J. H., Mur, I. S., Illurn, L. and

Davis, S. S. (1991) Bi&em Biophys. R~s. Cwunun. 177, 861-866 40 Moghimi. S. M. (1995) Adv. Drtrf D&. REV. 17, 103-115 41 Weissleder, R., Ehzondo, G., Wxtenberg, J., Lee, A. S.,

Josephson, L. and Brady, T. J. (1990) Radiob,gy 175, 494-498 42 Hawley, A. E., Davis, S. S. and Illurn, L. (1995) Adv. Drug De/iv. Rev.

17, 129-148 43 Yamaguchi, T. and Mizushmu, Y. (1994) Crit. Rev. T/rev. Drq

Cmier Sysf. 11, 2 15-229

Peptide nucleic acids: expanding the scope of nucleic acid

recognition David R. Corey

Peptide nucleic acids (PNAs) are DNA analogs containing neutral amide backbone

linkages. PNAs are stable to degradation by enzymes and hybridize to complementary

sequences with higher affinity than analogous DNA oligomers. PNA synthesis

employs protocols derived from solidphase peptide synthesis, making the methodology

straightforward and flexible. PNAs are being incorporated into an expanding set of

applications, including genome mapping, the identification of mutations and

measurement of telomere length. The growth in the popularity of PNAs as a tool for

nucleic acid recognition should accelerate as the properties of PNAs become more

familiar.

The recognition of complementary DNA or RNA sequences by oligonucleotides is a central feature of biotechnology. Such recognition underlies widely used experimental techniques and diagnostic protocols, and makes it possible to consider antisense- or antigene-based inhibition as an approach to therapeu- tics. Given the pervasive use of oligonucleotides, improvements in the rate, affinity or specificity of oligonucleotide recognition are fundamentally important. Many laboratories have appreciated this fact and developed novel oligonucleotide chemistries to enhance hybridization, increase resistance to nuclease digestion and improve membrane permeability’. Most of the novel motifs contain relatively modest changes to either the nucleotide bases or to the phosphate backbone. However, one motif, peptide nucleic acids

D. R. Corey (corcy~2hou~~ie.su~tncd.edrr) is af the Howard Hughes Medica/ Inrfitute and the Dprtment ~JPhartnacolog~~, Utrivcnity qf Texas Southwestern Medictil Center nt Dallas, 5323 Harry Hitrcs Boulevard, Dallas, TX 75235, USA.

(PNAs)‘,~, represents a dramatic departure from standard oligonucleotide chemistry, and it is becoming clear that imagination will be the primary limitation to the development of new applications for PNAs.

PNAs have been the subject of recent reviews+s, and I will focus here on practical considerations for their synthesis, on their physical properties and on how these properties offer significant advantages for devel- oping new applications for nucleic acid recognition.

PNA synthesis PNAs are DNA analogs in which 2-aminoethyl-

glycine linkages replace the normal phosphodiester backbones-s. A methylene carbonyl linker connects standard nucleotide bases to this backbone at the amino nitrogens (Fig. 1). This chemistry has three important consequences: firstly, in contrast to DNA or phosphorothioate oligonucleotides, PNAs are neutral molecules (I will address the influence of the absence of charge in subsequent sections); secondly, PNAs are achiral, which avoids the need to develop a

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stereoselective synthesis; and thirdly, PNA synthesis uses standard Boc (Ref. 6) or Fmoc (Ref. 7) protocols for solid-phase peptide synthesis.

Laboratories can obtain PNA monomers or ready- made oligomers from PerSeptive Biosystems (Framingham, MA, USA) or from suppliers holding a license fi-om PerSeptive Biosystems. PNA syntheses by either Boc or Fmoc protocols are straightforward using manual or automated protocols. Our laboratory finds manual synthesis to be simple, inexpensive and efflcient8. It lends itself readily to the production of chemically modified PNAs or the simultaneous synthesis offamilies of closely related PNAs. Standard pre- cautions, such as the use of tiesh, dry reagents during the coupling steps, generally lead to successful syntheses. Manual synthesis will be especially advanta- geous for laboratories that have small budgets for PNA synthesis or that wish to synthesize a pilot series ofPNAs to test the feasibility of a project*. Laboratories with the experience or the resources to support automated synthesis may prefer that approach, especially if they foresee the need for synthesis oflarge numbers ofPNAs.

As with peptide synthesis, the success of a particu- lar PNA synthesis will depend on the properties of the chosen sequence. For example, while in theory PNAs can incorporate any combination of nucleotide bases, we find that the presence of adjacent purines can lead to deletions of one or more residues in the product. In anticipation of this difficulty, we usually repeat the coupling of residues likely to be added inefficiently. We purify PNAs by reverse-phase high-pressure liquid chromatography8 and achieve yields and purity of product similar to those observed during the synthesis of peptides. PNAs exhibit excellent stability to the deprotection conditions required for either Boc or Fmoc synthesis.

It may be desirable to modify PNAs for a given application (Table 1). Modifications can be accom- plished by coupling amino acids during solid-phase synthesis or by attaching compounds that contain a carboxylic acid group to the exposed N-terminal amine. Alternatively, PNAs can be modified after synthesis by coupling to an introduced lysine or cysteine. The ease with which PNAs can be modified facilitates optimization for better solubility or for specific func- tional requirements. Once synthesized, the identity of PNAs and their derivatives can be confirmed by mass spectrometry.

Regardless of how PNAs are obtained, investigators should anticipate that several will be needed to achieve experimental aims. PNA properties, such as solubility and target recognition, will not always be predictable, especially for laboratories lacking experience with PNAs. We have generally needed to redesign our initial PNAs at least once before obtaining oligomers with the desired properties. Laboratories unwilling or unable to make this commitment of resources should critically re-examine their plans for PNAs, balancing

*Det&d protocols for the manual cynthem ofPNAs are available from the author upon request.

0 Base

0

‘V

? o=p-o- O=P-o- I

A d Base

0

v OR’ OR’

NH2 NH2 \ \

N N Base Base

0 03\NH NH

\ Base Base N N

A

-s- -s- 0 0

0 ‘+OH OH

Figure 1 Chemical structure of DNA (phosphate backbone; left) and PNA (amide backbone; right) oligomers.

the potential complications with the potential for useful insights.

Physical properties of PNAs In contrast to DNA and RNA, which contain nega-

tively charged linkages, the PNA backbone is neutral. In spite of this dramatic alteration, PNAs recognize complementary DNA and RNA by Watson-Crick pairing”, validating the initial modeling by Nielsen et al.2 PNAs lack 3’ to 5’ polarity and can bind in either parallel or antiparallel fashion, with the antiparallel mode being preferred”. Nuclear magnetic resonance studies demonstrate that PNAs form a complex with RNA that is similar to an A-type helixZ3, whereas a PNA-DNA d u pl ex has elements of both A- and B- type structureZ4. Crystallography has revealed that the 2: 1 PNA-DNA complex forms a unique multistranded triplex structure and confirms that both Hoogstein and Watson-Crick base-pairing are present25, and that a PNA-PNA complex possesses a unique structureZ6.

Table 1. Modifications to PNAs

Type of modification Aim/use Refs

Acridine Oligomer stabilization Protein Specific DNA cleavage i Modified backbone - 9

chemistries DNA 10-12 Peptide Protein-kinase-C substrate Peptide His&based purification ;i Peptide Metal-dependent DNA cleavage bis-PNA Enhanced hybridization E Biotin Affinity purification 17-19 Fluorescein Visualize hybridization 20,21

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Hybridization of DNA oligonucleotides to DNA and RNA is destabilized by electrostatic repulsion between the negatively charged phosphate backbones of the complementary strands. By contrast, the absence of charge repulsion in PNA-DNA or PNA-RNA duplexes increases the melting temperature (T,,,) and reduces the dependence of T,,, on the concentration of mono- or divalent cations2. The lack of repulsion enhances the rate of PNA hybridization to sequences containing inverted repeats within double-stranded supercoiled DNA, increasing the association constant 50 000 times (kaPNA = 5 x 10s M-l s-l)

relative to that for analogous DNA oligomers27. The enhanced rate and affinity of hybridization are significant because they are responsible for the surprising ability of PNAs to perform strand invasion of complementary sequences within relaxed double-stranded DNA, an intriguing phenomenon that is discussed below. In addition, the efficient hybridization at inverted repeats suggests that PNAs can recognize secondary structure effectively within double-stranded DNA. Hybridization of PNAs to single-stranded DNA is also rapid (k, = lo6 M-l s-l; Ref. 22), although in this case the rate of association of PNAs is equivalent to that observed for hybridization of DNA oligonucleotides to complementary sequences, due to the fact that the negative charge of only one target strand is present to hinder hybridization by DNA oligomers. Enhanced recognition also occurs with PNAs immobilized on surfaces, and Wang et al. have shown that support-bound PNAs can be used to detect hybridization eventsa*.

One might expect that tight binding of PNAs to complementary sequences would also increase binding to similar (but not identical) sequences, reducing the sequence specificity of PNA recognition. As with DNA hybridization, however, selective recognition can be achieved by balancing oligomer length and incubation temperature. Moreover, selective hybridization ofPNAs is encouraged by PNA-DNA hybridization being less tolerant of base mismatches than DNA-DNA hybridization. For example, a single mismatch within a 16 bp PNA-DNA duplex can reduce the T,,, by up to 15°C (Ref. 22). This high level of discrimination has allowed the development of several PNA-based strategies for the analysis of point mutation+32.

High-affinity binding provides clear advantages for molecular recognition and the development of new applications for PNAs. For example, l l-13-nucleotide PNAs inhibit the activity of telomerase, a ribonucleo- protein that extends telomere ends using an essential RNA template, while the analogous DNA oligomers do not33 (Fig. 2). Presumably, binding by the latter to the telomerase template is too weak to block polym- erization. Inhibition is highly dependent on the complementarity of the PNAs to the telomerase template, and PNA scanning, in which relatively short PNAs probe overlapping nucleotide sequences, has identified nucleotides within the telomerase template that are determinants for optimal inhibition (S. E. Hamilton and D. R. Corey, unpublished).

TIBTECH JUNE 1997 (VOL 15)

While the neutral backbone linkages strengthen the binding of PNAs to complementary nucleic acid sequences, the lack of a phosphate backbone removes the electrostatic interactions essential for protein binding. This fact has several implications for the use of PNAs. The most obvious is that PNAs are not sus- ceptible to digestion by proteases or nucleases because enzymes cannot recognize their hybrid chemistry, affording PNAs a long half-life in serum34. More sub- tly, PNAs appear to have little ability to interact either sequence-specifically or otherwise with proteins that possess an affinity for DNA or RNA. For example, while short DNA duplexes analogous to the promoters for SP6 or T7 RNA polymerase are able to bind polymerase and inhibit transcription with high selec- tivity, and phosphorothioate duplexes are potent but non-sequence-selective inhibitors, PNA duplexes nei- ther bind measurably to polymerase nor inhibit tran- scriptionss. Unwanted interactions between proteins and nucleic acids are a major complication to the use of phosphorothioate oligomers in antisense research, and the lack of such interactions may improve the ability of PNAs to locate target sequences selectively within cells. Neutral PNAs are more hydrophobic than analogous DNA oligomers, and this can lead to difficulty solubilizing them at neutral pH, especially if the PNAs have a high purine content or if they have the potential to form secondary structures. Their solubility can be enhanced by attaching one or more positive charges to the PNA terminia.

To date, there have been no reports of the successful crossing of cell membranes by PNAs. Studies on phospholipid vesicles and whole cells have shown that PNAs do not readily cross membranes, suggesting that passive diffusion is not a viable mechanism for the introduction of PNAs into the nuclei of living cells36J7. However, the adaptability of PNA synthesis to the design of modified PNAs will allow the pursuit of multiple strategies for the introduction of PNAs and their derivatives into cells. While the current absence of techniques for intracellular delivery does not detract from the potential of PNAs for in vitro applications, the development of methods for their efficient uptake will be an outstanding achievement for the field.

PNA hybridization by strand invasion One of the most interesting and instructive proper-

ties of PNAs is their ability to recognize sequences within duplex DNA by strand invasion2x3 (Fig. 3). Strand invasion is doubly remarkable because PNAs must initiate recognition at target sequences that are already base-paired and then continue binding, overcoming entropic considerations favoring reformation of the parent duplex. Early studies recognized strand invasion by PNAs through gel retardation of hybridized complexes and through nuclease or chemical nicking of the displaced strandz. Complex formation has since been confirmed by electron microscopy3*, affinity cleavage15 and altered circular dichroisms’, and appears to be under kinetic contro14”.

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Initial work targeted PNAs to polypurine- polypyrimidine tracts. Surprisingly, PNAs did not hybridize by standard triplex formation, but formed four- stranded complexes consisting of a target DNA strand, a Watson-Crick-paired PNA strand, a Hoogstein- paired PNA strand, and a displaced DNA strand”. Upon recognition of this, Griffith et al. demonstrated enhanced recognition by bis-PNAs that contained two strands connected by a lysine-containing linking sequenceie. Presumably the initiation of strand invasion by PNAs at polypurine-polypyrimidine sequences is facilitated by rapid hybridization of PNAs to transiently unpaired duplex targets. I f this hybridization persists long enough (a variable that is dependent on exact complementarity of the PNA to the DNAJo), a second PNA can bind by Hoogstein pairing and stabilize the four-stranded complex. PNAs bind by strand invasion to sequences containing inverted repeat+7 and triplet repeats”, suggesting that sequences pos- sessing a tendency to form non-B-type structures are particularly suitable targets for recognition, and that strand invasion is not restricted to homopurine- homopyrimidine sequences. Recent studies have also shown that PNAs ofmixed sequence can invade sequences lacking secondary structure, albeit at a slower rateq’.

The addition of m&molar concentrations of divalent cations prevents strand invasion by PNAs in vitro, presumably because increased ionic strength stabilizes the parent duplex, and this has been thought to be a limitation on PNA use. However, it is important to emphasize that, in vitro, the cation concentration can be controlIed during hybridization. In vivo, the situ- ation is much more complex, and supercoiling, transcription, DNA secondary structure and ionic strength wiIl combine to dictate whether or not PNAs can bind

I increasing OINW

Figure 2 Effect on strand elongation by human telomerase of the addition of analogous PNA (0.0003, 0.003, 0.03, 0.3, 3.0 and 30.0 FM) and DNA (0.003, 0.03, 0.3, 3.0 and 33 PM) oligonucleotides as measured by the PCR-based TRAP assayas. Strand elon- gahon is inhibited by PNA, but not by the analogous DNA oligonucleotides. PNA and DNA oligomers were complementary to the RNA template of telomerase. Image sup plied by Anne Pitts (University of Texas Southwestern Medical Center, Dallas, USA).

by strand invasion. This belief is supported by comprehensive in vitro studies that reveal that supercoiling and ionic strength are critical determinants of the efficiency of strand invasionQ. As will be discussed below, AlIfrey and colleagues have reported that PNAs can recognize sequences within permeabilized cells,

Conditions that favor or disfavor strand invasion

Favor Secondary structure at target Supercoiling AT-rich regions Low ionic strength Transcription Potential for triplex pairing

Disfavor Relaxed target DNA High salt Divalent cations

Figure 3 Possible intermediates during strand invasion of duplex DNA. (1) Duplex target transiently becomes single-stranded. (2) PNA takes advan- tage of its ability to hybridize rapidly and stably to initiate binding. (3) PNA oligomer completes strand invasion; high-affinity PNA hybridization discourages reformation of the parent duplex. (4) Pairing at homopurine-homopyrrmidine sequences can be further stabilized by triplet formation to form a 2:l PNA-DNA complex. Condrtions that favor or disfavor strand invasion are shown.

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Table 2. Development of applications for PNAs

Application Year Refs.

DNA strand invasion 1991 2

Antisense inhibition 1992 3

Sl-Targeted DNA cleavage 1993 Inhibition of restriction-enzyme cleavage 1993 2

Mutation analysis 1994 Promoters of transcription 1994 t;

Delivery through blood-brain barrier 1995 Nucleic acid purification 1995 ;: DNA cleavage by attached nuclease 1995 8 Isolation of transcriptionally active genes 1995 19 Enhanced PCR amplification 1995 Blocking of transcription factor binding 1995 t;

Monitoring telomere length 1996 Inhibition of human telomerase 1996 :i Complementary assisted hybridization 1996 Detection of mutations 1996 Z,30 Genome cleavage 1996 Biosensors 1996 2”s” Arrest of transcription at CAG repeats 1996 43 Metal-dependent cleavage of DNA 1996 Transcription-mediated binding 1996 E Translation inhibition by PNA clamps 1996 In situ hybridization 1996 z’3 High-throughput screening of RNA 1996 Alternative to Southern blotting 1996 z:

Figure 4 Telomere recognition by PNAs - in situ hybridization of fluorescein-labeled PNA to telomeres. This image was produced by Steven Sherwood (Geron Corporation) using protocols adapted from Lansdorp et al.20 Low photobleaching and an excellent signal-to-noise ratio make it possible to quantitate telomeric repeats on individual chromosomes.

suggesting that strand invasion by PNAs can occur under conditions that are near-physiological43.

Applications for PNAs Standard DNA oligonucleotides already function

effectively in many methods, and so the question arises of whether the advantages of PNAs just@ the development of new protocols. To be more than just a laboratory curiosity, PNAs must either offer the potential for significant improvement of existing protocols or permit the development of new procedures that would not otherwise have been possible. It is reason- able to expect that PNAs might meet these requirements. The greater association constants for PNA hybridization will increase the rate of recognition and decrease the concentration of oligomer necessary, thus improving throughput and reproducibility while reducing cost. The enhanced affinity of PNA binding will allow recognition of binding sites at lower ionic strengths and higher temperatures than before. An increasing number of applications have been described (Table 2), confirming the potential for PNAs to facili- tate the development of novel methodologies. Two specific applications are offered below as case studies for the use of PNAs.

Determination of telomere size Standard protocols for telomere length determi-

nation employ Southern blot analysis of genomic DNA and provide a range for the telomere length of all chromosomes present. Methods have been developed to monitor the in situ hybridization of fluorescein-labeled DNA oligonucleotides to telomeric repeats, but these procedures provide only qualitative information. To monitor telomere length quantitatively, Lansdorp et al.

adopted the use of fluorescein-labeled PNAs20. This methodology yields clear results (Fig. 4), and the data obtained allow accurate estimates of telomere length to be made2”. It is likely that variations ofthis approach can be applied to other repetitive sequences to achieve similar results. In addition, PNA recognition has been exploited to iden+ efficient alternatives to Southern blot analysis for discriminating between hybridization to mutant and nonmutant target sequences2”s32. A similar report has described the use of PNAs for in situ hybridization to mRNA, and has shown that, relative to hybridization of DNA oligonucleotides, PNA hybridization is faster and requires a lower concentration of probes3.

Strand invasion of transcriptionally active genes Lansdorp et al. solved the problem of accessing

sequences within chromosomal DNA by denaturing the DNA. Findings by Allfrey and colleagues, however, suggest that strand invasion will occur spontan- eously at sequences within chromosomal DNA1”,“3. These studies targeted PNAs to triplet repeats of the nucleotides CAG and used this recognition to puri@ transcriptionally active DNA’” and to inhibit tran- scription43. These results indicate that efficient strand invasion is not restricted to polypurine-polypyrimidine

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sequences or inverted repeats but can occur at other sequences as well. CAG repeats have some tendency towards partially single-stranded structures, and secondary structure or active transcription may have facilitated the initiation of base-pairing at the target sequence. Interestingly, inhibition of transcription is also observed within permeabilized cells43, indicating that strand invasion can occur under physiological conditions of ionic strength. This result suggests that if PNAs can be delivered within cells then they will have the potential to be general sequence-specific regulators of gene expression.

Concluding remarks It is tempting to conclude that ‘anything DNA can

do, PNAs can do better’. This is a simplification, of course, since PNAs cannot act as primers nor as tem- plates for polymerases and they do not appear to have much potential to interact with proteins that recognize nucleic acids. However, PNAs do possess undeniable advantages for the recognition of complementary DNA and RNA sequences, and are a major advance in molecular recognition rather than being a mere curiosity. Exploiting these advantages will not always be simple. PNAs are a novel chemical motif, and investigators wishing to initiate PNA-dependent projects should consider their resources, the properties of PNAs and the applicability of conventional oligonucleotides. If the use of PNAs is not considered care- &lly, it is likely that the investigator will achieve little beyond complaints regarding poor solubility and high cost. However, for those willing to invest a sustained effort, PNAs provide the basis for significant techno- logical improvements and novel scientific insights.

Acknowledgements I thank Mieczyslaw Piatyszek (Geron Corporation,

Menlo Park, CA, USA), Donald Doyle and Susan Hamilton for thoughtful comments on this manuscript and Steven Sherwood and Geron Corporation for sup- plying Fig. 4. This work was supported by a grant from the Robert A. Welch Foundation (I-1244) and an award f?om the CaP CURE association. The author is an Assistant Investigator with the Howard Hughes Medical Institute.

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peptide nucleic acids: expanding the scope of nucleic acid recognition

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