Anal. Chem. 1003, 65, 3545-3549
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AC RESEARCH
ACCELERATED ARTICLES
Characterization of Antisense Binding Properties of Peptide Nucleic Acids by Capillary Gel Electrophoresis Donald J. Rose Bioanalytical and Structural Chemistry Department, Glaxo Research Institute, 5 Moore Driue, Research Triangle Park, North Carolina 27709
The binding of peptide nucleic acids (PNAs),novel antisense agents, to their complementary oligonucleotide is characterized by capillary gel electrophoresis (CGE). The ability of CGE to resolve the free and bound species enables the measurement of relative binding kinetics and the stoichiometry of binding. The binding kinetics depend on the relative sequence orientation of the target oligonucleotides. The stoichiometry of binding is 1:l for the PNA-oligodeoxynucleotide heteroduplex whereas the stoichiometry for the PNAoligoribonucleotide is more complicated. INTRODUCTION Antisense therapy is based on the base-specificrecognition of a sequence of DNA or RNA by an antisense agent. Binding of an antisense agent to its target prevents the expression of proteins which can contribute to a disease state.13 These agents, usually short segments of a single-stranded nucleic acid oligomer (oligonucleotide), can be targeted against different sites within the cell. In the nucleus, the agent can bind to double-stranded DNA by triplex or D-loop formation and prevent transcription into messenger RNA (mRNA). Alternatively, in the cytoplasm, the agent can bind to mRNA and interfere with the translation process, which prevents specific protein expression. In either case, specific, highaffinity binding is important for the success of an antisense agent. Although holding much promise for treatment of disease, effective antisense therapy requires delivery of the antisense (1) Zamecnik, P. C.; Stephenson, M. L. R o c . Natl. Acad. Sci. U.S.A. 1978, 75, 280. (2) Stephenson, M. L.; Zamecnik, P. C. R o c . Natl. Acad. Sci. U.S.A. 1978, 75, 285. ( 3 ) Uhlmann, E.; Peyman, A. Chern. Rev. 1990,90, 544-584. 0003-2700/93/0365-3545$04.0010
agent to the appropriate cells, transport of the agent across the cellular (and nuclear) membrane(s), and an in vivo halflife sufficient to produce the desired result. One obvious antisense agent is a short strand of DNA, referred to as an oligodeoxynucleotide(ODN). However, because of the phosphodiester backbone, ODNs are susceptible to hydrolytic cleavage by nucleases in the cell. As a result, DNA analogs have been developed by replacing atoms in the phosphodiester backbone. For example, the nonbridging phosphate oxygen can be replaced with one or two sulfurs or a methyl group to produce a phosphorothioate,’phosphorodithioate,6 or methyl phosphonatee linkage, respectively, all of which are less susceptible to nuclease action. A radical departure from these types of modifications is replacement of the entire sugar-phosphate backbone with an polyamide backbone to produce a peptide nucleic acid (PNA), as shown in Figure 1.7 PNAs have been shown to be capable of binding to a complementary strand of DNA or RNA and represent a potential antisense agent.7~8However, because of their unique chemical structure, for example, the complete lack of charge in the oligomer backbone, the nature of the PNA binding is important in assessing the viability of this antisense agent. Characterization of antisense binding has traditionally been accomplished by measuring the melting temperature (T,)of the double-stranded duplex. In this method, a solution of the double-stranded duplex in a spectrophotometer cuvette (4) Eckstein, F. Annu. Reu. Biochern. 1985,54, 376. (5) Marshall, W. 5.; Caruthers, M. H. Science 1993,259, 1564-1669. (6)Ts’O, P. 0.P.; Miller, P. 5.; Aurelian, L.; Murakami, A.; Agris, C.; Blake, K. R.; Lin, S.-B.;Lee, B. L.; Smith, C. C. Ann. N.Y.Acad. Sci. 1988,507, 220. (7) Nielson,P. E.;Egholm, M.;Berg, R. H.;Buchardt, 0.Science 1991, 254,1497-1500. (8) Hanvey, J. C.; Peffer, N. J.; Bisi, J. E.; Thompson, S.A.; Cadilla, R.; Josey, J. A.; Ricca, D. J.; Hassman, C. F.; Bonham, M. A.; Au, K. G.; Carter, S.G.; Bruckenstein,D. A.; Boyd, A. L.: Noble, S. A.: Babiss, L. E. Science 1992,258, 1481-1485. 0 1993 American Chemical Soclety
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 24, DECEMBER 15, 1993
PNA
DNA
”,’
“L
H -
OH
NH,‘ Flgure 1. Structure of a deoxyribonucleic acid (DNA) and a peptide nucleic acid (PNA) with B = purine or pyrimidine base (A, C, T, or G).
Table 1. Test Sequences for the Binding Studies zHN-TTTTTT-Kft d(GCCAAAAAAGCC) 2”-GTACGTCTTCAC-OH d(GTGAAGACGTAC) d(CATGCAGAAGTG) r(GTGAAGACGTAC)
Ts-LyS PNA flanked dA, ODN mixed-sequence PNA antiparallel ODN parallel ODN antipadallel ORN (oligoribonucleotide)
is gradually heated until a change in t h e UV absorbance occurs due t o the hypochromicity difference between the duplex and the dissociated single strands. Previous work has characterized preformed DNA-DNA duplexes using capillary gel electrophoresis (CGE).9 This paper shows t h e application of CGE as a technique for measuring t h e binding of P N A t o oligonucleotides in terms of relative binding kinetics and binding stoichiometry.
porollel OON
I a’
A
,
__c
antiporollel ODN
b , b‘
. ontiporallel ORN
EXPERIMENTAL SECTION Capillary electrophoresis was carried out on an AB1 270A or 270A-HT instrument (Applied Biosystems, Foster City, CA) using gel-filled capillaries (Micro-Gel 100,ABI, 50-pm i.d., 50cm overall, 30 cm to detector) with buffer reservoir solutions provided by the manufacturer (75 mM Tris-phosphate pH 7.0, 10% methanol). All samples and sample components (in mixtures), unless otherwise stated, were 20 pM in 25 mM NaP04-50 mM NaCl pH 7.0 buffer. Sample concentrations were estimated by measuring UV absorbance a t 260 nm in a cell of known path length and using extinction coefficients derived from DNA and RNA. Sample introduction was by electromigration (10 s, 5 kV). Unless otherwise noted, the capillary was thermostated at 30 “C. The general experimental procedure involved mixing an aliquot of a PNA stock solution with an aliquot of an oligonucleotide stock solution, introducing a portion of the sample into the gel column, and letting the sample migrate (-15 kV) through the column. The area of the separated components was measured (260 nm) as a function of elapsed time after mixing or of oligonuc1eotide:PNA ratios. The PNAs and complementary nucleic acid oligomers (5’ to 3’) are shown in Table 1. The Ts-LyS PNA has an amide block on the C terminus and a free amine on the N terminus (see Figure l), resulting in an overall net charge of +2, whereas the mixedsequence PNA has no terminal lysine, resulting in a net charge (9) Chen, J. W.; Cohen, A. S.; Karger, B. L. J . Chromatogr. 1991,559,
295-305.
I
I
I 9
I
18
26
35
Migration Time (rnin)
Figure 2. Electropherogramsof the parallel ODN (a),antiparallel ODN (b), antiparallel ORN (c), and their respective oligonucleotide-PNA complex (a’, b’, c’). Concentrationof each component(oligonucleotide or PNA) is 20 wM. Peaks marked with asterisks represent minor bound
species.
of 0 a t neutral pH. The ODNs complementary to Ts-Lys were made with flanking groups to ensure no bridging between oligomers (e.g., one d& spanning two Ts-Lys molecules). The PNAs were synthesized in-house whereas the oligonucleotides were contract synthesized (Research Genetics, Huntsville, AL).
RESULTS AND DISCUSSION Static Binding Studies. T h e first requirement for studying binding characteristics of PNAs is t h e separation of t h e bound species from t h e free species. Because the oligonucleotide has a lower molecular radius and nearly t h e same
ANALYTICAL CHEMISTRY, VOL. 65, NO. 24, DECEMBER 15, 1993 0RN:PNA complex
I
I
I
I
I
I
2
20
38
56
74
Migration Time (min)
Flgure 3. Back and forth migration of ORN-mixed-sequence PNA complex. See text for details.
charge as compared to the oligonucleotide-PNA complex, capillary gel electrophoresis was chosen as the separation system. Furthermore, free-solution capillary electrophoresis (CZE)was not used because (1)some of the positively charged PNAs adsorbed to the capillary surface, (2) it has less dependenceon mass (h4Was comparedto the gel counterpart, and (3) at neutral pH some of the ODNs had very low net mobilities, making the analysis prohibitively long (i.e,, the ODN mobility was of the same magnitude and opposite the electroosmotic flow). Figure 2 shows the capillary gel electropherogramsof the free oligonucleotides (ODN or ORN, Figure 2a-c) and the oligonucleotide bound to the mixedsequence PNA (Figure 2a'-c'). It should be noted that the free PNA did not migrate because of a zero net charge. Confirmatuion of the bound species was made by running samples of free and bound oligonucleotide through a gel column at 60 "C. In all cases, a single peak resulted and matched the migration time of the free oligonucleotide, indicating the later eluting peaks were not stable at elevated temperatures, presumably due to dissociation of the heteroduplex. Further confirmation for the existence of a bound complex is shown in Figure 3. In this experiment, a sample containing the mixed-sequence PNA and the antiparallel ORN was introduced into the capillary at the end opposite the autosampler and migrated past the detector into the
a 40000
30000
section of capillary within the thermostated (30 "C) oven compartment. Forty minutes after sample introduction, the high-voltage polarity was reversed and the capillary oven heated to 60 "C (see arrow, Figure 3) causing the sample components to reverse direction and migrate past the detector a second time. The first time past the detector, one major peak appears at -18 min, similar to that shown in Figure 2c'. However, during the reverse migration, a peak appears much earlier than expected, 10 min as compared to 22 min after reversal (a slight increase was expected due to reduced viscosity of the gel buffer at 60 "C). The peak appearing after the reversal is presumably due to free ORN produced from the dissociated complex. Because the backbone of the PNA does not confer a particular handedness, the mixed-sequence PNA can bind either of two oligonucleotides: binding of the antiparallel oligonucleotide involves alignment of the 3' end of the oligonucleotidewith the amino terminus of the PNA, whereas the parallel oligomer involves alignment of the 5' end with the amino terminus. Since gel electrophoresis separates species on the basis of molecular radius, the electropherograms in Figure 2 show mobility differences which may be due to differences in secondary or tertiary structure. For example, the free parallel ODN (Figure 2a) has a slightly lower electrophoretic mobility, shown as a longer migration time, than the antiparallel ODN (Figure 2b), even though the base compositionand charge are identical. However,upon binding mixed-sequence PNA, this mobility difference reverses such that the parallel ODN-PNA complexhas a significantlyhigher mobility than the antiparallel ODN-PNA complex (comparing Figure 2a' and b'). In the case of the RNA oligomer (antiparallel ORN, Figure 2c and c'), a significant decrease in mobility is seen relative to the ODN complexes,partly due to the lower mobility of the free ORN. In addition, two smaller bound species (marked with asterisks) appear which are not related to impurities in either sample. Since the formation of the oligonucleotide-PNA complex can be monitored by measuring the peak area of the free oligonucleotideand the peak of the complex, one can measure the stoichiometry of binding by titrating a known amount of oligonucleotide with PNA. Figure 4a shows the titration of the antiparallel ODN with the mixed-sequence PNA, the titration being monitored by the peak areas of the free ODN
b
r
ODN
55000
I 13750
I/" 0
r
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1000
pmol PNA added
1 500
-
0
ORN
x I
27500
0
3547
0
P
500
1000
1500
pmol PNA added
Flgure 4. Complexationtitratlon of antiparallel (a) 800 pmol of ODN and (b) 800 pmol of ORN wkh mixed-sequence PNA by monitorlng peak areas After each addition of oligonucleotide, the sample was heated for the unbound oligonucleotide (0)and the oligonucleotide-PNA complex (0). to 70 "C for 10 mln and allowed to cool to room temperature for 20 min.
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 24,DECEMBER 15, 1993
I
L
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0
2
4
6
a
10
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Time (min)
Rgurr 5. Blnding kinetics of T M y s PNA-flanked dA6 ODN (2:l at 25 OC). Electropherogramshows unbound ODN peaks resutting from 10 sample lntroductbns (108, -5 kV each),with each introduction migrating for 1 min at -15 kV before the next Introduction (The run shown here was started after the tenth sample introduction). Inset shows peak areas (Including area of O W before PNA addition) as a functlon of tlme from mixing of ODN and PNA. The arrow indicates an unknown specks which dovebps as the free ODN decreases, possibly related to a bound species.
and the ODN-PNA complex. The equivalence point of the complexation titration, the point at which all ODN is bound by PNA, is obtained by extrapolating the straight segment of the free ODN curve to zero peak area. Extrapolation of the curve in Figure 4a shows 800 pmol of PNA was added to 800pmol of ODN, indicating a 1:1 binding stoichiometry and confirmation of a heteroduplex. (The titration of the parallel ODN proved difficult because of the extremely slow binding kinetics, as shown below). In contrast, titration of the RNA oliomer (antiparallel ORN) with PNA extrapolated to an equivalence point of 400 (Figure 4b) and 600 pmol during a duplicate titration (not shown). In addition, the ORN-PNA complex peak area increases even though the free ORN is not detectable. In other words, complex forms upon addition of PNA even though no free ORN is detectable. It appears that the RNA system is not well-behaved and that a complex equilibrium exists between the bound species, free ORN, and
a
25000
P
SO00
,
some unknown species, perhaps an aggregate with limited solubility and a stoichiometry greater than 1:l. Kinetic Binding Studies. In addition to static measurements of binding, as shown above, CGE was used to measure relative kinetics of interaction. Initial studies of binding kinetics by CGE involved mixing the PNA and ODN and analyzing the sample every 15 min, the time for the free, unbound ODN peak to appear at the detector. However, the binding kinetics were more rapid than the time scale of the experiment (i.e., no free ODN was detectable after the first sample introduction). Therefore, using the fact that sample zones introduced into gel capillaries remain well-defined because of low diffusion in the gel matrix, binding kinetics were measured by doing a series of samplings: a sample introduction (10 s,-5 kV) followed by a 1-minmigration (-15 kV). This produced a series of discrete zones within the gel, each representing a “snapshot” of the solution concentration of the sample components. The number of samplings was limited by the migration time of the free ODN from the first sampling. In other words, if a sampling was done while the zone from previous sampling was in the detection window, the peak area would be inaccurate because of the nonconstant velocity of the zone through the window. After the last sampling,the voltage was left on to migrate the sampled zones past the detector for integration. The binding kinetics of T6-Lys-flanked d& measured by this method is shown in Figure 5. It should be noted that no distinct peaks appeared in this electropherogram (or an analysis with only one sampling)which would represent the bound species. However, a broad feature does grown in over time next to the flanked d& peak (see arrow, Figure 5). Binding of the ODN to the PNA in 1:l stoichiometry should have produced a heteroduplex with a net charge of -9 (ODN = -11, PNA = +2) and a mass less than double that of the ODN. Such a complex should migrate within the normal time course of the experiment. The binding kinetics of the mixed sequence PNA proved to be a more well-behaved system since ODN-PNA heteroduplex migrated as a distinct zone, and therefore, ita increase could be monitored as well as the decrease in the free ODN. As noted above, the mixed-sequence PNA can bind either of two oligonucleotides, antiparallel (3’ to amine) and parallel (5’ to amine). The relative binding kinetics of these two ODNs is shown in Figure 6. In the case of the antiparallel ODN (Figure 6a), the kinetics of binding, the ”on-rate”, are faster than the time scale of the experiment (i.e., only the bound complex was seen in the time it took to mix the ODN and
f
I
4000 O 0
50
100
1 50
200
250 m 0
ElapsedTime (min)
50
100
150
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Elapsed Time(min)
Flguro 6. Relative binding kinetlcs of the mlxed-sequence PNA-ODN (1:l at 25 “C) by monitoring peak areas over tlme of both the free ODN (0)and the ODN-PNA complex (0)for (a) the armperalkl ODN and (b) theperallel ODN. The method for measuring the kinetics was as described In Flgum 5, except only slx sample lntroductlons WBTO done to measure short-term kinetics after which a serles of normel rune were done evety 30 min to monitor long-term klnetics.
ANAL.YTICAL CHEMISTRY, VOL. 65, NO. 24, DECEMBER 15, 1993 3549 ODNODN
duplex
I Elapsed
OONPNA
free
1
~
8
19
30
Migration Time (rnin)
Flgure 7. Rate of strand dlsplacement for the mlxed-sequence PNA displacing the complementary strand of the antiparallel ODN. All oligomers In equal concentration.
PNA and do the first injection, -30 s). Lowering the temperature of the sample to 5 "C slowed the rate of binding to the point where the first sample introduction showed free ODN in solution but the next sample introduction, 1.5min later, showed no free ODN detectable in solution (data not shown). The relative kinetics of the ODN-ODN duplex were even faster than the mixed-sequence PNA-ODN, whereas the RNA oligomer (antiparallel ORN) gave results similar to the mixed-sequence PNA-ODN case (data not shown). In contrast to the antiparallel ODN, the parallel ODN showed much slower kinetics (Figure 6b), with approximately half of the ODN bound by PNA after 2 h. This result points to very different binding kinetics which need to be considered when these compounds are examined in in vitro assays. Assays should be conducted in such as way as to allow for complete binding of a compound; otherwise a kinetically slow but high-
-
affinity compound may be deemed ineffective. This is born out in the melting temperature (T,) measurements. The affinity of these two ODNs is not dramatically different with a T,'s of 46.0 "C for the parallel and 49.5 "Cfor the antiparallel ODNs. In addition to the on-rate kinetics of a singlestrand binding another single strand, it is possible to measure the rate of strand displacement. Strand displacement may occur in antisense therapy where, instead of forming a triplex with the double-stranded DNA, the oligonucleotide displaces the complementarystrand and forms a D-loop. The rate of strand displacement, shown in Figure 7, is measured by preforming the ODN-ODN homoduplex (antiparallel ODN bound to its complementary ODN strand), adding PNA to the solution, and doing an analysis every 30 min. For each analysis, the ODN-ODN homoduplex, the displaced ODN, and the ODNPNA heteroduplex are monitored. As one would expect, this rate is much slower than the on-rate described earlier, since the PNA must overcome the energy of 12 base pairings between the ODNs to successfully bind to the antiparallel ODN. Also, this experiment shows the equilibrium is strongly in favor of the ODN-ODN homoduplex. The application of capillary gel electrophoresis to the interaction chemistry of antisense oligonucleotides has been demonstrated. The separation mechanism of capillary gel electrophoresis, mobility through a gel medium, enables the separation of the oligonucleotide from the bound complex. Because of the small dimensions of the separation system, little sample (fmol) is required for the analysis. In addition, the low diffusion of sample zones in the capillary permits multiple sample introductions for measuring binding kinetics. This rapid sampling scheme could also be applied to other systems where one is monitoring the change over time for a mixture of species. In terms of limitations, veryrapid binding kinetics cannot be measured due to the sampling rate. Furthermore, weak interactions between oligomers cannot be seen since the temperature within the capillary may cause melting (dissociation) of the bound oligomer strands. RECEIVEDfor review July 16, 1993. Accepted September 24, 1993." *Abstract published in Aduance ACS Abstracts, November 1,1993.
