1995 Research Publication

DRUG DEVELOPMENT RESEARCH 34:184-195 (1995)

Research Overview

Impact of Biophysical Parameters on the Biological Assessment of Peptide Nucleic Acids, Antisense

Inhibitors of Gene Expression Stewart A. Noble, Michele A, Bonham, john E. Bisi, David A. Bruckenstein,

Pamela H. Brown, Stephen C. Brown, Rodolfo Cadilla, Micheal D. Gaul, jeffery C. Hanvey, C. Fred Hassman, John A. Josey, Michael J. Luzzio, Philip M. Myers, Adrian J. Pipe,

Daniel j. Ricca, Charles W. Su, Cynthia 1. Stevenson, Stephen A. Thomson, Robert W. Wiethe, and lee E. Babiss

Departments of Medicinal Chemistry (S.A.N., R.C., M.D.C., C.f.H.,).A.J., M.I.L., D.I.R., S.A.T., R.W.W.), Bioanalytical and Structural Chemistry (S.C.B., P.M.M., C.W.S.), Cell Biology (M.A.B., I.E.B., D.A.B., P.H.B., I.C.H., L.E.B.), and Oligomer Development (C.L.S), Glaxo Research Institute, Research Triangle Park, North Carolina; Department of Medicinal Chemistry I (A.I.P.1, CldXO Group Research,

Crecnford, United Kingdom

ABSTRACT Peptide nucleic acids (PNA) are oligodeoxynucleotide (ODN) analogs in which the sugar phosphate backbone of the ODN has been replaced by one derived from units of N-eth- ylaminoglycine. PNAs recognize DNA and RNA in a sequence specific manner and form complexes that can be characterized by biophysical methods. The binding motif i s context dependent; homopyrimidine PNAs combine with complementary polypurine targets to form stoichiometric 2:l complexes, whereas PNAs containing both purine and pyrimidine bases afford a 1 :I heteroduplex with rnis-match sensitivity comparable to that found in dsDNA. These complexes mediate the antigene and antisense effects of PNAs via the steric blockade of enzyme complexes responsible for DNA transcription, cDNA synthesis, and RNA translation. PNAs, like ODNs, are taken up by cells via endocytosis leading to their entrapment within intracytoplasmic vesicles. Under circumstances where agent delivery i s solved by cell microinjection, PNAs can effect selective inhibition of endogenous and exogenous genes. The impact of biophysical parameters on the biological assessment of PNAs as antisense inhibitors of gene expression i s presented and discussed. Q 1995 Wiley-Liss, Inc.

Key Words: peptide nucleic acid, oligodeoxynucleotide, antisense, triplex, S W O T Ag

INTRODUCTION lignancy. This latter prospect is particularly appealing since it offers the opportunity for therapeutic inter- The inhibition of gene expression mediated by

dsDNA and mKNA is a powerful tool for the study of cell control mechanisms and developmental biology, and has potential applicatio11 as a novel therapeutic strategy for the treatment of human disease and ma-

oligonucleotide birldirlg to genetic ternplates sucll as vention a general strategy, in con-

Address reprint requests to Stewart A. Noble, Department of Medicinal chemistry, Claxo Research Institute [R&D 3.42451, Five Moore Drive, Research Triangle Park, NC 27709.

0 1995 Wiley-Liss, Inc.

INHIBITORS OF GENE EXPRESSION 185

trast to classical approaches to drug discovery which are too often reliant on serendipitous finding and in- volve the identification and design of compounds directed against a series of unrelated proteins, such as enzymes, receptors, or ion channels. In theory drugs directed against the genetic templates, targeting the initiating events in the amplifying cascade of gcne expression, ought to be more efficient than those targeting the inhibition of gene products.

Natural products and synthetic ligands which cx- ert their biological effects as a consequence of their ability to bind or modify DNA are known and some, such as doxorubicin, mitoxantrone, and cisplatin, have found application as anticancer chemotherapeu- tics. Unfortunately these compounds are unable to recognize specific gene sequences and, therefore, act as indiscriminate anti-proliferative agents. In contrast, a synthetic oligodeoxynucleotide (ODN) which recognizes its complementary secpience via Watson- Crick base pairing, can provide absolute specificity of action, since statistically the sequence defined by any linear combination of the four naturally occurring nu- clcotide bases adenine (A), guanine (G), cytosine (C), and thymine (T) to afford an oligonucleotide of‘ 17 residues in length, occurs just once at random in the entire human genome [Helene and Toulme, 19891. Assessment of the therapeutic potential of sequence specific strategies is a growing field of scientific en- deavor and is the subject of several recent reviews [Milligan et al., 1993; Matteucci and Rischofberger, 1991; Weintraub, 19901. In the context of cancer gene targets, the bcr-abl (chronic myelogenous leukemia) [Smetsers et a]., 1994; Lewalle and Martitat, 1993; Szczylik et al., 19911, Ha-ras (many human cancers) [Gray et a]., 1993; Helene, 1991; Saison-Rehmoaras et al., 19911, myb (T-cell and myeloid leukemia) [Citro et al., 1992; Ratajczak ct al., 1992; Calabretta et al., 19911, myc (human leukemias and solid tumors) [Wickstrorn, 1991; McManaway et al., 19901, and bcl-2 (B-cell lymphoma) [Kitada et al., 19931 onco- genes are important candidates. Further discussion of this work is beyond the scope of this article.

Two mechanisms by which ODNs can effect the sequence specific inhibition of gene expression have been described. The first involves binding of the ODN to the major groove of a preformed &DNA duplex, resulting in the formation of a stable, collinear “triple helix” or “triplex” [Le Doan et al., 1987; Moser and Dervan, 19873. Recognition is mediated by Hoogsteen or reverse Hoogstecn base pairs. The presence of a triplex structure within a dsDKA has been shown to effect inhibition of transcription elongation in vitro and in vivo [Maher et al., 1992; Gri- goriev et al., 1992; Postel et al., 19911. hiechanisti-

cally, triple helix formation is restricted to target sequences containing a polypurine tract and a requirement for protonation of third strand cytosine residues. It is clearly desirable that a general solution be found whereby ODNs could be used to bind all four base pairs of an intact DNA duplex. Progress in pur- suit of this goal has been hard won. To date alternate straKd binding b y “switch back’ oligomers which form a triple helix in one orientation, then invert polarity arid switch over to the other strand, has allowed for modest extension of the code to mixed sequence of the type (purine),,,l(pyrimidine),, [ McCurdy et al., 1991; Horiie and Dervan, 19901. Work targeting pH independent recognition has been more successful and led to the introduction of carbocyclic 5-methyl- 2’-deoxycytidine [ Froehler and Ricca, 19921, MODA [Young et al., 19911, and P1 [Koh and Dervan, 19921 as C + surrogates. The seduction of the triple helix approach is that it has the potential to afford highly efficacious agents since the target gene is only present at one or two copies per cell.

The second approach, termed “antisense” (AS) involves the ODN agent participating in duplex formation, with target recognition mediated via Watson- Crick base pairing. In this case, targeting an ODN to RNA results in a localized heteroduplex and inhibition of gene expression, by either physical blockade of cnzyme complexes or by cleavage of the RNA target via the intervention of RNase H. The potential of AS ODNs to serve as code blocking agents was first rec- ognized by Zamecnik and Stephenson [ 19781 who demonstrated inhibition of Rous sarcoma virus replication in chick embryo fibroblasts o n addition of a tridecamer ODN with sequence complementarity to reiterative sequences in thc viral RNA genome. An- tisense agents can be targeted to a wide variety of sequences located on both pre-mRNA (hnRNA) in the nucleus and mRNA in both the nucleus and cyto- plasm. These sites include RNA splice donor and ac- ceptor sites, poly A cleavage and addition sites located on hnRNA, translation cap sites, and coding regions (exons) on mature cytoplasmic mRNA. Unlike triple helix formation, AS technology is conceptually appli- cable to any target sequence, but in practice requires that particularly sensitive functional sites be identi- fied by empirical mapping [Cowsert et al., 19931.

Whether the application is as a triplex or an- tisensc agent, it is vital that an ODN be able to reach its site ofactiori largely intact, accumulate to an effective concentration, selectively recognize the intended target, arid bind with sufficient affinity to elicit functional inhibition of the target gene. The problems associated with drug delivery arc formidable as ODNs are rapidly degraded in serum and the plasma mem-

186 NOBLE ET AL.

brane represents a natural barrier to the passage of polyanionic molecules of high molecular weight (4-6 kD). In this article we report characterization of the physicochemical and biological consequences of complexation hetween PNAs and genetic templates, and data which highlights drug delivery as a significant issue facing the research community.

PEPTIDE NUCLEIC ACIDS Recognition of dsDNA

A peptide riucleic acid (PNA) is an ODN analog in which the usual deoxyribose-phosphodiester back- hone has been replaced by one derived from N-eth- ylamiqoglycirie (Fig. 1). Initial reports pertaining to PNAs indicated that the PNA NII,T,,,L~s# could site- specifically recognize a single dA,,-dT,,j target contained within a 248 bp dsDNA restriction fragment via PNA-dA,, strand complex formation with concom- itant extrusion of a dT strand D-loop [Nielsen et al., 1991; Berg, 19911. Intrigued by the possibility that strand displacement by PNAs might form the basis of a general strategy for dsDNA recognition, we initi- ated work and showed that other polypyrimidine or pyrimidine-rich PNAs exhibit similar behavior. We also showed that binding of' either NH,T,,,L~s# or NH,CT,CAT,CT,CLys# to the transcribed strand of a G-free transcription cassette could effect 90-100% site specific termination of eukaryotic pol I1 transcription elongation in a dose dependent manner [ Hanvey et al., 19921. Interestingly, inhibition never exceeded 50% when NH~T,,L~s# was bound to the non-transcribed strand, suggesting that the KNA polymerase could bypass the D-loop in this instance.

We and others have further characterized the nature of the complex responsible for these effects. Flow linear dichroisrn (LD) arid circular dichroism (CD) spectroscopy reveal that NH,T~LYS# associated with poly(dA) to form a PNA:DNA complex with 2:l stoichiometry, reflecting a right-handed helical con- formation [Kim et al., 19931. The stoichiometry of binding to nucleic acid templates is best evaluated under conditions wlicre the total strand concentration is kept constant [Job, 19281. As shown in Figure 2A, the homopyrimidine PNA, N H ~ ( T C ) ~ T ~ # forms a 2:1 stoichiometric complex with complementary oligomers, in a manner which is indcpendcnt of strand orientation, a property which is attributable to the achiral nature of the PNA. A thermal melting study of the 2:l complex formed between the PNA, NH,(TC),T,# and d(A,(GA),) is shown in Figure 3. The data indicates that the complcx has an apparent Tm of 86°C. Interpretation of this data should be un- dertaken with some care, however, since siich mea-

A B

Figure 1. Comparison of the structure of a peptide nucleic acid (PNA) (A) with that of an oligodeoxynucleotide (ODN) (B). PNAs are derived from ODNs by replacement of the sugar-phosphate repeating unit by a backbone formed by linkage of N-ethylami- noglycine monomers. PNA and ODN repeating units are shown in brackets; B represents either adenine, thymine, guanine, or cytosine.

surements can only be related to thermodynamic properties under circumstances where the system is in equilih-ium. The fact that significant hysteresis is apparent when the solution is cooled at O.l"C/min, indicates that the high Tm does not reflect extraordi- nary binding affinity, but is simply an artifact induced by slow kinetics.

In order to provide data in support of slow kinetics we have performed experiments to assess the extent and rate of D-loop formation [Peffer et al., 19931. D-loop formation was monitored by band shift assay of a &DNA restriction fragment bearing the target sequence under conditions of varying ionic strength at 25°C. The results indicate that D-loop formation occurs rcadily in a low ionic strength solution (such as TE buffer), but that the extent of strand invasion decreases rapidly as the concentration of NaCl is raised, diminishing to only 10% formation in 1 h at 100 mM NaC1. The fact that strand invasion is inhib- ited by moderate salt concentration implies that DNA breathing is essential for D-loop formation. It is of note that the extent of D-loop formation at 100 mM NaCl could he enhanced to 50% strand invasion under conditions of prolonged incubation (24 11). Al- though slow to form, the resulting D-loop was found to he stable for up to 5 h and unaffected by raising ionic strength. Dissociation studies indicate that the PNA off-rate from DNA is slow, lending credence to our claim that the inhibition of trailscription we ob- serve is the consequence of termination, rather than pausing of the polymerase at the D-loop. In contrast, when a triplex-forming ODN was used in similar experiments, the inhibition of transcription seen was


: 0 W N

PNA-(TCTCTCTCTCTTTTT) PNA-(GTACGTCTTCAC) 1 . 0 , I ' I I . I , ,

0.7 1 0.6&

0.5 1

A

0.4 " " " " ' 1 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0

L 4

a 0.6 OD(266)p OD(280)p A

0 . 4 ' " " " ' I '

0.0 0.2 0 . 4 0 . 6 0 . 8 1 . 0

mole f rac t ion ODN mole f rac t ion ODN

Figure 2. Job plots of PNA complexed with complementary nucleic acids. A The polypyrirnidine PNA NHJTC)~T~# forms a 2 : l stoichiometric complex (0.33 mol fraction nucleic acid) with antiparallel complementary DNA and RNA oligomers and with DNA in the parallel orientation. B: The mixed sequence PNA NH~CTACC- TCTTCACoil forms a 1 :I stoichiometric complex (0.5 mol fraction

nucleic acid) with either d(CATGCACAAGTG) [PI or dCTCAA- CACGTAC) [apl. Complexes were annealed in 20 m M NaP04,140 m M NaCI, 0.25 mM EDTA, pH 7.2; absorbance was monitored as a function of oligonucleotide mole fraction under conditions where total strand concentration was maintained at 8.3 pM.

0 .65

0.6

0 . 5 5

E 0

0 . 5 v n 0

0.45

0.4

I " I " '

p(GTACGTCTTCAC) and 1 d(GTGAAGACGTAC) [1:1]

t 0.35 I L a

L - - . A , , 1 1 1 1 > ~, 1 , , ~, , , 1 , , I , , , , , -1 . 1 0 2 0 3 0 4 0 5 0 6 0 7 0 80 9 0

TEMP ("C)

Figure 3. Thermal melting studies on PNA-ODN complexes. The 2:l stoichiometric complex formed by association of the polypyrimidine PNA N H ~ ( T C ) ~ T ~ # with d(A,(GA),) i s dissociated by heating [T, = 86"CI and anneals [T, = 5O"Cl on cooling. Marked hysteresis is apparent and is associated with slow kinetics. In con-

trast the 1 : I stoichiometric complex formed by association of the PNA NH~CTACGTCTTCACOH with d(CTGAAGACCTAC) i s highly reversible with T, = 48°C. In this case the curves are essentially superimposable, but have, for the sake of clarity, been slightly displaced in the figure.

transient; complete inhibition could not be achieved due to the dissociation of the O D N from the DNA [Young et al., 19911.

Recognition of ssDNA and R N A

We have previously reported that the PNA N I - I ~ T ~ ~ L Y S # can inhibit the progression of RNase H- MMLV reverse transcriptasc (RT) as a consequence of its site specific association with an rA,, target embcd-

ded in a 268-1)ase RNA transcript [Hanvey et a]., 19921. We have since demonstrated that the ability of PNAs to inhibit RT is not restricted to A,-T, se- qucnces and appears to be general phenomenon. In- terestinglj-, we have also observed that NH,T,,LYS# can combine with the sequence rGA,GA, to effect inhibition of RT, indicating that the binding of PNAs to KNA can occur at sites of imperfect sequence homology. The specificity of target recognition is gov-

188 NOBLE El AL.

erned by absolute binding affinity and relative binding affinity to other closely related sequences [Woolf et al., 1992; Roberts and Crothers, 19911. In principle shorter ODNs (11-15 nucleotides) are better able to discriminate between a complementary template and near sequence neighbors bearing a single mismatch since specificity decreases as mismatch effects are over-ridden by the net binding affinity associated with longer oligomers.

We synthesized the PNA NH,GTACGTCT,- CACOH [Thomson et al., submitted; Egholm et al., 1992, 1993a] in order that we might fiirther charac- terize the nature of complexation with single stranded templates and examine the influence of base pair mismatch on PIKA target recognition. Job plot analysis (Fig. 2B) indicates that the PNA NH~GTACGTC- T,CACOH forms a 1:1 stoichiometric complex with complementary oligodeoxynucleotides in either the parallel (PEA NH, terminus binds to ODN 5' end) or antiparallel (PNA NH, terminus binds to ODN 3' end) orientation. Thermal melting of the latter complex reveals a cooperative, reversible S shaped transition with Tm = 48°C (Fig. 3) compared to 49.5"C for the ODN:ODN duplex control (Table 1). The reversible nature of the thcrmal melting curves is in marked contrast to that of the homopyrimidine sequence and presumably reflects the faster kinetics of this system. To assess this we performed further op- tical experiments in which changes in ahsorbance were used to derive kinetic parameters. As shown in Figure 4, the PNA:ODN anti-parallel complex has association and dissociation lifetimes of 2.9 min and 12.1 min, respectively, under conditions where the 0DN:ODN complex achieves equilibrium within seconds. Interestingly, similar studies performed on the homopyrimidine sequences indicated association lifetimes measured in hours (data not shown). The diver- gence between ODN and PNA kinetic parameters is of note since a slower rate of sequence sampling might lead to biological effects arising from site occupancy at sequences of imperfect homology. Inspection of the data presented in Table 1 reveals that PNA binding in the antiparallel orientation is preferred, as noted by Egholm et al. L1993bI. This appears to be a general property for PNAs as is the enhanced binding affinity for RNA, relative to ODN controls. Although not generally seen, it is of note that heating the PNA NH,GTACGTCT,CACOH in isolation also resulted in an observable transition, indicating the presence of self associated, higher order structure.

Prompted by the inhibitory effects of ionic strength on strand invasion of dsDNA we have examined the dependence of PNA binding to ssDNA as a function of salt concentration. The data presented in

TABLE 1 . Tm Analysis of PNA and ODN Binding to Complementary ODNs and RNA'

Oligonucleotide PNNON ODN/ON (ON) (TrnVCI) (Tm [ T I )

d(CATGCAG AAGTG) 44.0 [PI 51.5 d(CTGAAG ACGTAC) 48.5 LAPI 49.5 r(G UCAACACC U AC) 60.0 50.0 - 33.0a -

*The PNA NH~GTACCTCTTCACWI/ON complex was annealed in 20 m M NaPO,, 140 mM NaCI, 0.25 mM EDTA, pH 7.2, at 8.3 pM total strands. Samples were ramped from 1 O-90°C, cooled from 90-1 O"C, and then heated from 10-90°C at O.S"C/min. Tms are reported ?1 .O"C. The ODN d(CACTTCTCCATC)/ON complex was annealed and Tm determined as previously described. [PI denotes that the PNA binds the ODN in the parallel orientation. [APl denotes the PNA bind5 the O D N in the antiparallel orientation. "Heating a solution of the PNA NH~CTACCTCTTCACOH 14.1 pM] in buffer indicated a transition at 33.0"C.

Table 2 indicates that the binding of the PNA NH,GTACGTCT,CACOH to its cognate ODN in the antiparallel motif' is relatively insensitive to ionic strength; the complex being only slightly destabilized as judged b y depression of Tm by 2. Sac in response to a two order magnitude increase in salt concentration. For reasons presently unknown, thc extent of' desta- blization is more pronounced when binding is in the parallel orientation (ATm = -S.5"C), but is insuffi- cient to abrogate template binding. This behavior is in contrast to that noted for PYA strand invasion and opposite to the known effects of ionic strength on ds- DNA duplex stability, where ion pairing of the backbone markedly reduces inter-phosphate repulsion and results in significant stabilization of the structure (Table 2, ATmAntiparallcl = + 2O.S0C, ATml,arallel - - + 23°C).

The extent of Tm depression when the PNA NH,GTACGTCT,CACOH is bound to any one of twelve different single base mismatched ODNs at four separate sites within the context of the complementary anti parallel ODN is shown in Table 3 and is depicted graphically in Figure 5 . The data indicates that single mismatch substitution significantly desta- bilizes the complex in all cases, with Tm depression in a 9-18°C range. Once again, this would appear to be a general phenomenon, since we and others [Egholm et al., 1993bl have obtained similar results in different systems. The binding preference arid extent of duplex destabilization is striking and highly indicative of Wat- son-Crick base pairing. The fact that PNAs bind to RNA under physiological conditions and to complementary sequences of any base composition suggests that PNAs have considerable potential as antisense agents.

I N H I B I T O R S OF GENE EXPRESSION 189

r

k o H = 5.72 x 10" sec"

I

i $ 1 N 008 -

0 t 006 -

t k Q F F 3 1.38 !i see-'

0 04

O O Z L - ' ' " 1 ' ' I , , , I , , , , i . /

0 10 20 30 40 50 60

Minutes Figure 4. Binding kinetics of the PNA NH,GTACCTCTKACOH/ d(GTGAAGACCTAC) complex. Association kinetics were measured via changes in UV absorbance following hand mixing of the components at 25°C and total strand concentration of 4 pM. The observed curve fits (R = 0.98) to a single exponential having a lifetime of 2.9 min. Control experiments, data not shown, indi-

cate that the analogous ODN duplex reaches equilibrium within seconds. Dissociation kinetics were measured by hand mixing the preformed PNA-ODN complex with SDS micelles. The observed curve fits (R = 0.95) to a single exponential with a lifetime of 12.1 rnin.

TABLE 2. Trn Analysis of the Dependence of PNA-DNA Binding on Ionic Strength'

Complex Tm ("C) [NaCIl [mM]

Strand 1 Strand 2 10 50 100 500 1,000

NH~GTACCTCTTCACOH dKATGCACAACTC) IP1 47.0 46.0 45.5 41 .O 38.5 NH~CTACGTCTTCACOH d(CTCAACACGTAC1 [AP] 49.5 49.5 49.5 48.0 47.0 d(CTACGTC1 TCAC) d(CTCAACACCTAC) 35.5 45 .o 48.0 54.5 56.0 d(CACTTCTGCATG) d(CATGCACAAGTG) 31.5 42.5 4h.0 55.5 54.5

*The Strand 1/Strand 2 complex was annealed in 20 mM NaPO,, 0.25 rnM EDTA, pH 7.2, at 8.3 pM total strands in the presence of NaCl [lo-1,000 rnM1 as shown. Trn was determined as described in Table 1 and i s reported il .O"C. [PI denotes that the PNA binds the ODN in the parallel orientation. [AP] denotes that the PNA binds thc ODN in the antiparallel orientation.

Biological Assessment As noted above we have demonstrated that the

binding of a PNA to a RNA template results in site selective inhibition of' cDNA synthesis by reverse transcriptase. While such an assay can be used to evaluate the binding properties of PNAs, it was clearly important that we examine the potential of' PNAs to effect inhibition of RNA translation. Our initial experiments were conducted in vitro and focused upon the mixed sequence r(A,TCA,GA,), which is derived from SV40 T-antigen mRNA. The target site was placed downstream from the start of the interleukin-2 receptor a subunit gene cDNA and RNA gen- erated from the resulting plasrnid pCEM-IL2R-SV40. A PNA complementary to the target in the parallel orientation (NH,T,ACT,CT,#) was synthesized and

hybridized to the RNA prior to initiation of translation using rabbit reticulocyte lysate. Analysis of the resulting peptide fragments by PAGE revealed inhibition oE translation at a site which mapped precisely to that of the target sequence. Experiments in which the PNA NH,T,AT,CT,CT# was used as a sequence scrambled control, or in which the target was deleted, showed no effect. Using model systems we have subsequently shown that the PNA:RNA duplex does not serve as a substrate for RNase H, thus the observed inhibition most likely results from ribosome stalling/displacement as a consequence of the steric blockade of translation elongation.

We now report the results of experiments de- signed to ~ s s e s s the antisense potential of PNAs to effect inhibition of gene expression in whole cells. In

NOBLE ET AL. 190

~

TABLE 3. Mismatch Sensitivity of PNA-DNA Recognition*

Oligonucleotide Tm ("C)" ATm ('TIb

d(GTGAAGACGTAC) 48.5 -

dlGTGAAGACGCAC) 37.5 -11.0 d(GTCAACACCCAC) 39.0 -9.5

d(GTG AAG ACTTAC) 31 .O - 17.5

d(GTCAAC ACCAAC) 36.0 -12.5

d(GTCAAG ACATAC) 31.5 -17.0 d(CTGAACACCTAC) 30.5 -18.0

d(GTGAAGAAGTAC) 30.5 - 18.0 d(GTCAACAGGTAC) 34.0 - 14.5

d(CTG ACGACGTAC) 29.5 - 19.0 d(GTGACG ACGTAC) 35.5 -13.0 d(GTGATGACGTAC) 39.5 -9.0

d(GTGAAG ATGTAC) 39.0 -9.5

*The site and identity of single base pair rnismdtches in ODNs which are otherwise complementary to the PNA NH~GTACCTCTTCACUH are denoted by bolded typeface. "The PNA NH~GTACGTCTTCACO~ON complex wd5 annealed in 20 rnM NaPO,, 140 mM NaCI, 0.25 rnM EDTA, pH 7.2, at 8.3 pM total strands. Samples were ramped from 10-90°C, cooled from 90-10°C, and then heated from 10-90°C at 0.5Wmin. Trns are reported 2 1 . O T . bThe extent of Tm depression is calculated with reference to the Trn of the complementary PNA-ODN complex (48.5"C) and is presented graphically in Figure 5.

mismatch c preparation we asscssed the stability of a test PNA, I~H,T,L~s#, in a variety of' biological matrices and established that the compound was not prone to rapid metabolism. Confident in the knowledge that agent degradation would riot limit our experimental approach we examined the ability of cloned rat embryo fibroblast (CREF) cells to internalize a fluorcscently tagged PNA. incubation of CREF cells with the PNA Fla-NH(TC),T,# (20 pM) resulted in a predominant cytoplasmidperinuclear punctate fluorescent pattern, as shown in Figure 6A. We have observed a similar pattern with PNAs of varying length and sequence composition in a panel of different cell types. This purictate staining is reminiscent of the behavior of S-ODNs (Fig. 6B). We have found that the uptake efficiency of a fluorescently labeled PNA is typically no better and often worse than that of its ODN coun- terpart, as reflected by thc four-fold higher PNA concentration employed in the experiment shown in Fig- ure 6A. Although uptake data is reported at the 4-h time point, no improvement in the extent, or redis- tribution of the fluorescence was apparent on prolonged incubation (24 h). In a parallel series of cxper- iments we have demonstrated that cytoplasmic microinjection of fluorescently tagged PNAs and ODNs into viable cells results in their rapid translo- cation into distinct intranuclear compartments (data not shown). Thus it would appear that incubation of cells in PNA or O D N containing solutions results in

Figure 5. Influence of base pair mismatches on PNA target recognition. The PNA NH~GTACGTC~TCACOH forms a 1:l stoichiometric complex w i th d(CTCAAGACGTAC) having Trn = 48.5"C. The figure shows the extent o f Tm depression when the PNA i s bound to any o f twelve single base pair mismatched ODNs at four separate sites within the complementary ODN sequence and is a graphical representation of the data presented in Table 3.

their sequestration in intracytoplasmic endosomes. The fact that we have never observed nuclear staining on cell incubation suggests that leakage of cithcr PNAs or O D N from the endosoma1 compartment is not a significant process. i t is reasonable to assume that endosomal entrapment might limit the ability of PNAs and O D N s to participate in sequence specific events. The fact that we have Failed to demonstrate reproducible inhibition of T-Ag or secreted alkaline phosphatase reporter gene expression in a variety of cell types and feeding paradigms (using PNAs at closes up to 10 pM), is supportive of this notion.

To circumvent problems associated with agent delivery we have employed cell microinjection of temperature sensitive tsa 8 cells LJat and Sharp, 19891 to assess the ability of PNAs to inhibit endogenous T-Ag expression. Tsa 8 cells were incubated at 39°C for 48 h prior to inicroinjection such that levels of


Figure 6. Fluorescent microscopy of cloned rat embryo fibroblasts (CREF) cells incubated with either (A) the fluorescein tagged PNA FIahH(TC),T;Lys# (20 FM) or (B) the fluorescein tagged S-ODN FI-S-d((TC),T,) (5 FM). The punctate staining indicates that the majority of the fluorescent material is sequestered within intracytoplasmic vesicles and i s thus unable to freely participate in sequence specific processes.

T-Ag protein might decay to undetectable levels. Cells were then microinjected with PNAs targeted to a site in the T-Ag coding sequence which Wagner et al. [I9931 had previously shown was sensitive to antisense intervention, returned to a permissive tern- perature (33”C), and scored for the reemergence of nascent T-Ag protein synthesis by indirect immuno- cytochemistry. Microinjection of the PNAs N H ~ A T ~ - CT,CAT,CT,CLys# (20-mer) and NH,CT,CAT,- CT,CLys# (15-mer) showed significant inhibition of T-Ag expression at estimated intracellular concentrations of 1 arid 5 p M, respectively, whereas the PNA NH,CT,CT,L~S# (10-mer) was without effect up to 10 pM. It is of note that an unmodified phosphodiester ODN (19-mer) targeted to the same site was unable to inhibit T-Ag expression to any extent. The specificity of the inhibition was confirmed by demonstrating strong cellul;ir staining for the control protein, 6-ga- lactosidase, which was derived from an expression plasmid coinjected with the PNA [Hanvey et al., 19921.

We have used the experimental design of Wagner et al. [1993] to assess the ability of‘ a variety of PNAs to inhibit thc exogenous expression of T-Ag in African green monkey kidney (CV-1) cells as a consequence of binding to sequences contained within the RNA 5’ untrarislated region. The T-Ag plasmid, pSV5080AAv1-11, was derived by restriction of pSV5080 with AvrII and subsequent ligation of a synthetic 51 bp DNA fragment (Fig. 7A). Microinjec- tion of pSVS080AAvII into CV-1 cells gave a level of T-Ag expression comparable to that obtained with

pSV5080. PNAs complementary to thc 51 base RNA insert were synthesized and assessed for dose dependent inhibition of T-Ag as previously described. A maximum intracellular dose of 20 p M was chosen i n light of our experience, requiring a needle concentration of 400 pM, assuming a 20-fold dilution on cell microinjection. We have found that our ability to conduct these studies has in large part been determined by PNA solubility in 50 inM HEPES, 80 ink1 KCI, pH 7.3; conditions which we routinely ernploy for cell microinjection. Inspection of the data presented in Figure 7B indicates that pyrimidine rich PNAs meet this solubility requirement whereas those with increasing piirine content fall progressively short. The effect is pronounced in thc ease of G-rich oligomers, e.g., NH,G,CT,GA,CL~S# (complementary to the p210 b3a2 bcr-abl splice site) and on occasion can preclude the evaluation of a compound, e. g., NH,ACA,GTG,ATCT,CL~S# (complementary to T-Ag intron). We have found that €”As are generally slow to hydrate, that solubility is aided by charged groups and diminished by the covalent attachment of hydrophobic moieties, such as fluorescein and by a propensity to self aggregate. This latter property is revealed by Raleigh scattering in the UV absorbance spectra of PNAs and is conceritratiori dependent (Fig. 8A,B). The nature of these complexes can be probed by ultracentrifugation which allows for the determination of molecular weight. The time course of a sedimentation velocity experiment for the PNA NH~T,LYS# at 5 p M is shown in Figure 8C. The data indicates that the I”A exists as

192 NOBLE ET AL.

5

PNA Solubilityb YO Inhibition of T-Agc PNA Oligomera [UMl [@ 2uM PNA]

391 448 151

404

300 395 342 360 269

96 Insoluble

98 99

86 95 100 100 100 95 59

51 49 42 17

Figure 7. A: The sequence of the 51 base pair synthetic insert that was used in the construction of pSV50804AvrII. Underlining in the antisense strand identifies sequences that as PNA oligomers were utilized to effect inhibition of exogenous T-Ag expression in cell microinjection assay. B: The inhibition of exogenous T-Ag expression in CV-1 cells via targeting PNAs to sites contained within the T-Ag 5'UTR. 'PNAs were prepared by the method of Thomson et al. (submitted), purified by reverse phase chroma- tography and characterised by electrospray mass spectrometry. Abbreviations: Fla, 5-(and 6-)carboxyfluorescein; IU, 5-iOdO- uracil; MeC, 5-methylcytosine; Lys, lysine; Clu, glutamic acid; #, C-terminal amide. bThe solubility of test PNAs under assay conditions was assessed prior to cell microinjection. Briefly, approx-

a high rnoleciilar aggregate which quickly sediments to the bottom of the cell and a lower molecular weight species which sinks more slowly, as evidenced by the rightward moving shoulder. Most PNA solutions we have examined by this technique indicate that large aggregates are present at PNA concentrations routinely used for biophysical, enzymatic, and biological evaluation of these agents. The true monomeric concentration of PNA solutions can, therefore, be far lower than those expected and disaggregation be- comes the rate limiting step for subsequent events.

The data presented in Figure 7B indicates that the PNA NH,(TC),T~L~S# combines with its comple-

imately 2 mg of PNA was re-constituted to afford a solution of 400 pM PNA in 50 m M HEPES, 80 m M KCI, pH 7.3. The sample was incubated at 3PC for 30 min, centrifuged at 14 x l o 3 min-' for 10 rnin, and saturation solubility determined by UV absorbance. In general homopyrimidine PNA achieved the solubility criterion of 400 pM; mixed sequences fell progressively short with increasing purine content. 'Cell microinjection experiments were performed according to Wagner et al. t19931. Hornopyrirnidine PNAs could effect complete inhibition of T-Ag expression at 2 pM, whereas PNA containing all four bases are less effective. To what extent this differential activity reflects the stoichiometry of target bindinghecognition by PNAs i s currently unknown.

mentary target to effect complete and selective inhibition of' T-Ag protein synthesis at 2 p M . PNAs derived by replacement of cytosine by 5-methylcytosine and/or thymine by 5-iodouracil, removal or addition of lysine, or by length titration give similar results and in general appear to be more effective than PNAs derived from non-homopyrimidine sequences (Fig. 7B). It is interesting to note that whereas the extent of inhibition diminishes incrementally with length for mixed sequences, this effect is not apparent until reaching the eight residue stage in the context of the homopyrimidine PNAs. To what extent this reflects the ability of the latter to participate in higher order


A

2 0 0 250 3 0 0

Figure 8. Aggregation of PNA solutions. A: Raleigh scattering (absorbance above 300 nm) is observed in the U V absorbance spectra of the PNA NH,AC,CT~CTCCCTCTL~S# at 5 pM. B: Evidence for Raleigh scattering is not apparent on dilution of the sample to 700 nM PNA, indicating the dynamic equilibration between aggregated and monomeric states. C: Time course ultracentrifugation of the PNA NH~T~LYS# at a nominal concentration of 5 p,M in

structures is presently unclear. The results reported in Figure 7B are consistent with PNA inhibition via steric blockade, since agents which are known to act via steric blockade, such as the C-S-propynyl-2’-0- allyl-ODN d(paUpC),5(paU),) gave 50% inhibition of T-Ag expression at 5 p,M. In contrast the C-5-propynyl-Ei-ODN, 5-d((pUpC),pU,) whose effects are cata- lytically mediated via RNase H had an IC,, = 0.25 pM and was consistently more potent than any of the PNL4s tested. Whilst the data unequivocally demonstrate that PNAs can elicit antisense effects by targeting the 5’ UTR of RNA, it is also apparent that these results belie the physicochemical complexity of the events responsible for T-Ag inhibition.

CONCLUSIONS

PNAs are ODN analogs which recognize RNA and DNA in a sequence specific manner and form stable complexes that can be characterized by biophysical methods. The binding motif is context dependent; homopyrimidine PNAs combine with complementary polypurine targets to form stoichiornetric 2: 1 complexes; mixed sequence PNAs combine with complementary targets to afford a stoichiometric 1:l heteroduplex which is reversible and has mismatch

20 mM NaP04, 140 m M NaCI, 0.25 m M EDTA, pH 7.2, at 50,OOOg over 8 h leads to rapid sedimentation of an aggregated species, as judged by AZbo and a second species, which sediments more slowly as evidenced by the rightward moving shoulder, corre- sponding to a monomeric species. The arrow indicates the cen- trifugal vector. The base of the curvette forms the righthand ver- tical axis; the UV spike the meniscus of the solution.

sensitivity comparable to that found in dsDNA. These complexes mediate the in vitro antigene and antisense effects of PNAs via the steric blockade of enzyme complexes responsible for DNA transcription, cDNA synthesis, and RNA translation. PNAs, like ODNs, are taken up by cells via endocytosis, leading to their sequestration in cytoplasmic vesicles and are thus unable to freely participate in sequence specific processes. Under circumstances where the problems associated with agent delivery are solved by cell microinjection, PNAs can ef€ect the selective inhibition of both endogenous and exogenous genes in a manner consistent with biological intervention via a mecha- nism based on steric blockade. PNAs cxhibit a propensity to aggregate in dilute solution, which, com- bined with moderate intrinsic solubility in an aqueous environment, limit experimental approaches to assess the potential of PNL4s as antisense inhibitors of gene expression.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the gift of the ODN reagents d(paUpC),(paU),) and 5-d((pUpC),pU,5) which were received from Gilead Sciences, Foster City, CA, and thank M.D. Mat-

194 NOBLE ET AL.

teucci, K . W . Wagner and B.C. Froehler in this re- gard. Thanks are also due to R.D. Stonehouse and A. A. Birkbeck of Glaxo Group Research, Greenford, U. K. for technical assistance in the preparation of the PNAs NH,G,CT,GA,CL~S# and NH,ACA,GTG,- TCT,CLys# .

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