In addition to fomenting exciting medical research, these amazing molecules have inspired speculations relating to the origin of life on earth. Some scientists have suggested that PNAs or a very similar molecule may have formed the basis of an early kind of life at a time before proteins, DNA and RNA had evolved. Perhaps rather than creating novel life, artificial-life researchers will be re-creating our earliest ancestors

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Into the Groove

The story of PNA’s discovery begins in the early 1990s. To generate drugs with broader capabilities than antisense RNA, my colleagues Michael Egholm, Rolf H. Berg, and Ole Buchardt and I wanted to develop small molecules able to recognize double-stranded, or duplex, DNA having specific sequences of bases—no easy task. The difficulty has to do with the structure of the familiar DNA double helix. It is the bases—thymine (T), adenine (A), cytosine (C) and guanine (G)—that store information in DNA. (In RNA, thymine is replaced by the very similar molecule uracil, or U.) Pairs of these bases joined by hydrogen bonds form the “rungs” of the familiar DNA “ladder.” C binds with G, and A binds with T, in what is called Watson-Crick base-pairing. A compound that binds with a stretch of double-helical DNA having a characteristic base sequence would therefore be one that acts on any gene containing that particular sequence of bases on one of its strands. The task of recognition is relatively easy if a compound has to find a particular base sequence on single-stranded DNA or RNA. If two nucleic acid strands have complementary sequences, standard base-pairing can zip the two strands together. Thus, if one knows the sequence of a gene—from Human Genome Project data, for instance—producing a molecule to latch onto a section of the gene in a single strand is as simple as synthesizing the complementary sequence. In duplex DNA, however, the task of recognizing a sequence is more challenging because the atoms responsible for Watson-Crick pairing are already involved in the hydrogen bonds linking the two strands together and thus are not available for linking with another molecule. Yet cells contain numerous so-called gene-egulatory proteins that recognize sequences in duplex DNA to carry out their function of controlling gene expression. So the feat can be accomplished. If my group could find molecules capable of the task, the molecules could potentially serve as gene-regulating drugs. Gene expression takes place in two stages. First, in transcription, an enzyme constructsmessenger RNA (mRNA), which is a strand of RNA with a copy of the base sequence of one strand in the DNA helix. A molecular machine known as a ribosome, itself made of RNA and protein, carries out the second stage, translation of the mRNA into the protein coded by the gene. Antisense agents interfere with translation by binding to the mRNA. These compounds are typically small, chemically modified RNA or DNA molecules, designed with the appropriate sequence to identify their mRNA target by Watson-Crick base-pairing. By binding to its mRNA, the agent may trigger enzymes to degrade the RNA or may simply interfere physically with the mRNA’s functioning. Cells make use of proteins called transcription factors that recognize specific sequences in double-stranded DNA to control gene expression at the transcription stage. These proteins can repress a gene by obstructing the RNA polymerase enzyme that would otherwise transcribe the DNA’s sequence into mRNA, or they can activate a gene by helping the RNA polymerase to attach to the DNA and start transcription. Although these proteins offer a model of molecules capable of “reading” the DNA sequence from the outside of the helix, in the 1990s it was not yet possible for biochemists to start with a sequence and design a new protein to recognize it. A gene-regulatory protein recognizes its DNA sequence by having the correct overall shape and chemical composition on its surface to bind with the sequence in the so-called major groove of the DNA, which provides access to the base pairs that run along the center of the double helix. But the structure of the protein’s active surface depends on how its chain of amino acids folds up, a process that researchers cannot model with any accuracy. Some progress has been made since then by taking the lead from gene-regulatory proteins that include zinc-finger domains, which are lengths of about 30 amino acids that fold around a zinc ion, forming a characteristic “finger” structure that can fit in the major groove with a few amino acids lined up with the DNA’s bases.Researchers have developed artificial proteins with zinc fingers, but in general it is still difficult to program a sequence of amino acids to match even a relatively short DNA sequence. A discovery dating back to 1957, only four years after the discovery of the DNA double helix, provides another approach. That year Gary Felsenfeld, Alexander Rich and David Davies, all then at the National Institute of Mental Health, created triple helix structures in which a nucleic acid strand attaches itself in the major groove of a duplex nucleic acid molecule. The extra strand exploits a different kind of bonding of the base pairs T-A and C-G called Hoogsteen pairing, after Karst Hoogsteen [see box on preceding page]. Each position along the triplex thus has a triplet of bases in which a T binds to a T-A pair (T-A=T, where the “=” indicates the Hoogsteen pairing) or a C binds to a C-G unit (C-G=C). This structure, however, can form only when the extra strand is a homopyrimidine— made entirely of C and T (or U, in RNA)— because each Hoogsteen pair requires a G or an A on the strand of the double helix. In 1987 the late Claude Hélène, then at the National Museum of Natural History in Paris, and Peter B. Dervan of the California Institute of Technology independently demonstrated that the triple helix structure could indeed be exploited to design oligonucleotides (DNA strands about 15 nucleotides long) that read the sequence in double-stranded DNA and bind their Hoogsteen complementary target.

A Surprise Invasion

Inspired by this digital readout of the DNA double helix by groove-binding, triple helix–forming oligonucleotides, my group set out to synthesize a molecule that could do the same trick with fewer limitations. In particular, we hoped to find molecules that would not be limited to recognizing sequences made entirely of G and A. We also wanted our molecule to be neutral. The backbone of nucleic acids contains phosphate groups that carry a negative charge in solution. The repulsion caused by these negative charges on all three backbones weakens the binding of the third strand to the triplex. We therefore decided to base the design on amide chemistry, involving the same kind of bond as links amino acids in proteins. Well-established techniques using amide, or peptide, bonds allow convenient synthesis of highly stable, neutral molecules. The peptide nucleic acid molecule that we came up with has a peptidelike backbone made of a much simpler repeating unit than the sugar and phosphate of DNA and RNA. Each unit may have a standard nucleic acid base (T, A, C or G) linked to it or bases that have been modified for special purposes. The spacing between bases along a PNA is very close to that of DNA and RNA, enabling short PNA strands, or PNA oligomers, to form very stable duplex structures with DNA and RNA strands as well as with another PNA strand. The bases zip together with standard Watson-Crick bonding.When we tried targeting duplex DNA with homopyrimidine PNA, to our surprise the PNA did not bind in the DNA’s major groove as planned. Instead one PNA strand invaded the helix, displacing one of the DNA strands to form Watson-Crick bonds with its complement, and a second PNA strand formed Hoogsteen bonds to make a PNA-DNA=PNA triplex. The displaced length of DNA formed a single-stranded structure called a P-loop, alongside the triplex. This triplex-invasion binding mode has several very interesting biological consequences, because the triplex has great stability and the P-loop affects central biological processes such as transcription, DNA replication and gene repair. For instance, the P-loop structure can initiate RNA transcription of the DNA. Furthermore, the single-stranded loop can be exploited in applications such as protocols to diagnose genetic disorders: the DNA in a sample must first be amplified (copied a large number of times), and the loop can serve as a specific attachment point for the copying process. Other binding modes also occur, depending on the target DNA sequence and on how we modify the PNA’s bases. Of these, double duplex invasion is particularly interesting. In this mode, we prepare two pseudo complementary PNA oligomers—that is, their bases are modified  enough to prevent formation of a PNA-PNA duplex but not enough to disrupt their individual binding to an ordinary complementary DNA strand. The PNAs thus invade  ouble-stranded DNA and form two PNA-DNA duplexes. In contrast to triplex formation, which requires a long stretch of purines (A and G) in the target DNA, the double-duplex-invasion binding mode has less restrictive sequence requirements: with the present technology, the target sequence must contain at least 50 percent A-T base pairs. Even that constraint would be relaxed with discovery of suitable modified forms of the G and C bases. PNA binds in these ways to complementary RNA or DNA molecules with even greater specificity and affinity than that exhibited by natural DNA. PNA oligomers with fluorescent groups attached are thus attractive as probes to detect specific genes in diagnostic tests. For instance, so-called fluorescence in situ hybridization analyses highlight the positions on chromosomes where specific sequences are present.

 Prospects for Drugs

Many studies, in cell cultures as well as solutions in vitro, have demonstrated proof of concept for using PNA oligomers to suppress or activate the transcription, replication or repair of specific genes by binding to DNA in various ways. Researchers have also reported numerous experiments showing that PNA oligomers can function somewhat in the manner of antisense RNA interference, inhibiting gene expression at the translation stage, both in cell cultures and in a few studies with mice. PNA achieves these effects by physically blocking key processes involving RNA. In contrast, DNA or RNA oligomers used for RNA interference are assisted by enzymes in cells that break down the RNADNA or RNA-RNA duplexes that are formed. The RNA-PNA structure is unlikely to receive this kind of assistance because the enzymes cannot recognize such a foreign structure, although so far researchers have studied the question only for one of the relevant enzymes. Yet the alien nature of PNA oligomers also makes them exquisitely stable in biological environs— enzymes that break down other peptides do not recognize them, so PNAs have more time to encounter matching RNA and disable it. In some cases, blocking an RNA process can restore a healthy protein. Matthew Wood of the University of Oxford and his co-workers demonstrated in 2007 that PNA can exploit this effect. When they injected PNA into mice with muscular dystrophy, the injected muscles showed increased levels of the protein dystrophin, whose absence causes muscular dystrophy. The PNA prevented a bad segment of the dystrophin gene from being translated from RNA to protein, thus eliminating a debilitating mutation present in that segment while leaving intact enough of the dystrophin to function. PNA oligomers and conventional nucleic acids share a common problem of poor bioavailability because they are large and predominantly hydrophilic (water-loving) molecules, making it difficult for them to enter cells, whose walls are made of hydrophobic lipid membrane. Despite the great stability of PNAs, they do not remain in an animal for long, being quickly excreted in urine thanks to their hydrophilicity. For instance, half of the PNA in a mouse is gone in less than half an hour. Thus, the advent of PNAbased drugs awaits the development of suitable chemical modifications or pharmaceutical formulations (that is, mixtures with other substances) to improve PNA bioavailability. Indeed, the mainfocus of research into genetic medicines in general is work on overcoming the problem of delivery to cells in the body. Researchers believe that hurdle is the last obstacle holding back medical  breakthroughs in this field

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