Molecular Biology, Vol. 37, No. 2, 2003, pp. 223–231. Translated from Molekulyarnaya Biologiya, Vol. 37, No. 2, 2003, pp. 255–265. Original Russian Text Copyright © 2003 by Shchyolkina, Borisova, Livshits, Jovin.
UDC 577.113
Parallel-Stranded DNA with Natural Base Sequences A. K. Shchyolkina1, O. F. Borisova1, M. A. Livshits1, and T. M. Jovin2 1
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 11999 Russia; E-mail:
[email protected] 2 Max-Planck Institute for Biophysical Chemistry, Goettingen, D-37070, Germany Received June 21, 2002
Abstract—Noncanonical parallel-stranded DNA double helices (ps-DNA) of natural nucleotide sequences are usually less stable than the canonical antiparallel-stranded DNA structures, which ensures reliable cell functioning. However, recent data indicate a possible role of ps-DNA in DNA loops or in regions of trinucleotide repeats connected with neurodegenerative diseases. The review surveys recent studies on the effect of nucleotide sequence on preference of one or other type of DNA duplex. (1) Ps-DNA of mixed AT/GC composition was found to have conformational and thermodynamic properties drastically different from those of a Watson– Crick double helix. Its stability depends strongly on the specific sequence in a manner peculiar to the ps double helix, because of the energy disadvantage of the AT/GC contacts. The AT/GC boundary facilitated flipping of A and T out of the ps double helix. Proton acceptor groups of bases are exposed into both grooves of the psDNA and are accessible to solvent and ligands, including proteins. (2) DNA regions containing natural minor bases isoguanine and isomethylisocytosine were shown to form ps-DNA with transÄí-, trans isoGC, and trans iso5meCG pairs exceeding in stability a related canonical duplex. (3) Nucleotide sequence dG(GT)4G from yeast telomeres and microsatellites was demonstrated to form novel ps-DNA with GG and TT base pairing. Unlike d(GT)n- and d(GnTm) sequences able to form quadruplexes, the dG(GT)4G sequence formed no alternative double- or multistranded structures in a wide range of experimental conditions, thus suggesting that the nucleotide context governs the observed structural polymorphism of the d(GT)n sequence. The possible biological role of ps-DNA and the prospects of its study are discussed. Key words: parallel-stranded DNA, thermodynamic parameters of formation, structure, biological role
INTRODUCTION Conformational polymorphism of DNA is now extending further and further beyond the Watson–Crick double helix [1]. In addition to the families of the A, B, and Z forms—double helices with antiparallel orientation of strands—existence of DNA double helix with parallel strands (ps-DNA) was proven. Such mutual orientation of strands and non-Watson–Crick base pairing can arise at low pH, as a consequence of chemical modifications of nucleotide bases or sugar-phosphate backbone, or upon ligand binding [2–7]. Formation of ps-DNA by natural base sequences at neutral pH and physiological ion conditions reported in the pioneer studies [8–10] is of particular interest. This is connected with elucidation of a supposed biological role of ps-DNA, specifically in biosynthesis [11], regulation of cell processes via formation of ps-DNA in DNA loops, and in structural organization of single-stranded viral DNA [12, 13]. Recently an NMR study of alternative DNA structures associated with the genetic instability of trinucleotide repeats, which may be of etiological significance in the onset and progression of Friedreich’s ataxia, has revealed formation of a ps (GAA)((CTT) duplex with GC and AT base pairs [14]. Other trinucleotide repeats also connected with neurodegenerative diseases formed
self-complementary ps-DNA d(GGA)n with GG and AA pairs [15] and ps-DNA d(CGA)n with C+C, GG, and AA pairs [16, 17]. Stretches of ps-DNA can arise within repetitive Watson–Crick DNA in looped structures and may serve as a structural factor that enhances or determines the length variability of the trinucleotide repeats important for etiology of this type of diseases. This review considers recent data on the properties of ps-DNA with natural base sequences and on role of nucleotide sequence as a major factor determining the stability of ps-DNA. PS-DNA WITH AT, GC COMPLEMENTARITY Mirror repeats able to form ps-DNA with AT, GC pairs are abundant in genomes of various organisms [11]. Here we consider the structural features and thermodynamic stability of such a ps-DNA as compared with those of a Watson–Crick double helix. The base pair schemes have been established with Raman and FTIR spectroscopy [18, 19] and then verified by NMR [3, 14]. AT pairs with two H-bonds were found to be of reversed Watson–Crick type. Bases of a GC pair in psDNA are somewhat shifted relative to one another and also form two H-bonds (“sheared” reversed Watson–
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Cl
Cl transAT
Cl
Cl transGC Fig. 1. Schemes of AT and GC pairs in ps-DNA.
Crick GC) regardless of the base context (Fig. 1). Eval-
uation of the effect of the GC pair on the stability of psDNA with a mixed AT, GC composition had led to the following remarkable inconsistency. Incorporation of four separate GC pairs in a 25-bp AT sequence was found to destabilize ps-DNA dramatically [20]. Correspondingly, nucleotide sequences with isolated GC pairs were shown to preferentially form imperfect aps duplexes rather than perfect ps ones [21]. Nevertheless, unlike the above sequences, mixed sequences comprising blocks of two or three G and C bases did form psDNA [9, 22, 23]. Such an unusually strong sequence dependence of ps-DNA stability required explanation in terms of its structure and thermodynamics of formation. Experimental data evidenced the higher stability of at least the 3'-terminal GC pair as compared with AT [23]. A reasonable assumption was made that the stacking contact of AT and GC pairs, or AT/GC boundary, was particularly disadvantageous in energy. In that case, blocks of two or more consecutive GC pairs would decrease the number of such contacts and increase ps-DNA stability. For a detailed study, a set of linked oligonucleotides able to fold back into monomolecular ps double helices—hairpins—with the same AT/GC composition but different nucleotide sequences was designed and synthesized [24]:
ps-N1:
3'-d(CTATAGGGAT)-L-d(ATCCCTATAG)-3',
ps-N2:
3'-d(CTGAGTAGAT)-L-d(ATCTACTCAG)-3',
ps-N4:
3'-d(CTGATAGGAT)-L-d(ATCCTATCAG)-3' as well as oligonucleotides
ps-AT:
3'-d(AT)5-L1-(AT)5-3',
ps-N6
3'-d(TTATAGGGAT)-L-d(ATCCCTATAA)-3',
ps-N7
3'-d(TTGAGTAGAT)-L-d(ATCTACTCAA)-3',
aps-GA/TC:
5'-d(GA)5-L-(TC)5-3', where L: -(CH2CH2O)3-, L1: -(CH3)6-.
Predominance of intramolecular 10-bp-long hairpins over alternative intermolecular duplexes was achieved by the choice of experimental conditions. It is noteworthy that homogeneity of ps-DNA species necessary for a quantitative study was thoroughly verified with a set of fluorescence methods. Circular dichroism (CD) spectra of the ps-DNA with mixed AT/GC composition have no major distinctions from those of related aps-DNA. This allowed one to suppose that there are no drastic differences in nearest-neighbor base pair interactions between ps and aps double helices. Molecular modeling described below supported the assumption that the mutual orientation of base pairs in ps-DNA is similar to that in aps-DNA. Difference CD spectra of ps- and aps-DNA revealed one specific feature of CD spectra of ps-DNA: a positive low intensity band around 290 nm. This band was accounted for by a contribution of guanine in the transGC pair. The melting absorbance profiles at 260 nm indicated that the thermostability was sequence-depen-
dent, and were used to determine the thermodynamic parameters of ps-DNA formation (Fig. 2). We faced a choice of a theoretical model for adequate description of thermal denaturation of ps hairpins: either a two-state model (a native and a fully melted hairpin) or multistate melting via a series of intermediate states with variable numbers n of native base pairs. In order to discriminate between the two mechanisms, nondenaturing electrophoresis of ps-N1 and aps-GA/TC hairpins was run in a linear temperature gradient (TGGE). The TGGE data provided experimental evidence for a multistate mode of the helix–coil transition of the ps-N1 hairpin, whereas the aps hairpin melted in an “all-or-none” mode [24]. A special statistical–mechanical formalism of the multistate transition (“heterogeneous zipper”) was developed, in which the melting process is described as proceeding step by step along the hairpin molecule. It takes into account the energy difference between transÄí- and transGC base pairs formation and an unfavorable energy contribution of the AT/GC boundary. The best global fit of the MOLECULAR BIOLOGY
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theoretical curves to experimental melting profiles simultaneously for six ps-DNAs is shown in Fig. 2 with solid curves, and the resulting thermodynamic parameters of transÄí- and transGC formation as well as of the AT/GC boundary are given in Table 1. So, transGC is more stable than transÄí, while the energy loss (∆hj) is due to the AT/GC contact. The net effect of replacing k successive AT pairs in an AT tract with GC pairs is given by ∆∆G = k(∆gGG – ∆gAT) – 2∆hj ≈ (3k – 4) kJ mol–1 in 0.1 M LiCl at 3°ë. It follows that a single (isolated, k = 1) base pair replacement (AT/GC) destabilizes the ps double helix but is thermodynamically favorable for k ≥ 2. Figure 3 shows the theoretical melting curves of the six sequences simulated with the "heterogeneous zipper” model. Since hairpins ps-N1 to ps-N7 melt in a multistate rather than in a two-state mode, the melting curves plotted as a fraction of unpaired bases and as a fraction of fully melted hairpins are not coincident (compare Figs. 3a and 3b). Strikingly, the temperatures corresponding to 50% melted ps-N1 and ps-N2 hairpins differ by more than 13°ë. Such a strong dependence of the stability of double helices with the same AT, GC content on the nucleotide sequence is a peculiar feature of psDNA, whereby it differs much from the Watson– Crick double helix. Another important consequence of the disadvantage of the AT/GC contact in ps-DNA is facilitation of flipping out the isolated Aand T-bases at the boundary, which creates a novel motive for ligand binding to ps-DNA. What is the structural basis of the energy losses at the AT/GC contact in ps-DNA? The most probable cause is distortion of the sugar-phosphate backbone at this stacking contact, as the “sheared” transGC pair is not isomorphous to the transAT pair [19, 20]. Energy minimization of the ps-N1, ps-N2, and ps-N4 (Fig. 4) gave for the total internal energies the same order, psN1 > ps-N4 > ps-N2 as that inferred from the melting experiments. The derived internal energies of aps-N1 and aps-N2 structures appeared to be very similar and some ~100 kJ mol–1 more favorable than for ps-DNA. This difference is consistent with the thermodynamic data of spectroscopic experiments. The average helical twist and rise of the three ps-DNAs are close to those for B-DNA. The major significant differences
225
Absorbance at 260 nm 0.24
0.20
0.16
0.12
0.08
0
10
20
30
40
50 60 70 80 Temperature, °C
Fig. 2. Thermal denaturation curves of ps hairpins. Experimental curves (every fifth experimental point is indicated) in 0.1 M LiCl: ps-AT (䉭); ps-N1 (䊉); ps-N2 (䊊), ps-N4 (䊐); ps-N6 (䉬); ps-N7 (×). The solid curves are the fits to the data (see text).
between the two types of helix are mainly in the groove geometry: the two grooves of ps-DNA have practically the same width and depth. The proton acceptor groups of the bases N3(G), N7(G), O6(G), O2(C), N3(A), and O4(T) are easily accessible for solvent and ligands from both grooves of ps-DNA. The block of three successive guanines in ps-N1 forms a groove “pocket” exposing solvent-accessible acceptor groups N3(G), N7(G), and O6(G). Moreover, the O6(G) is presumably capable of specifically binding alkaline ions Li+ and Na+ [24], the property previously reported for crystals of nucleosides and nucleotides [25]. The TATA region in ps-N1 also presents
Table 1. Thermodynamic helix–coil transition parameters of trans AT and trans GC pairs and of the AT/GC boundary Oligonucleotides
∆hAT, kJ/mol
∆hGC, kJ/mol
∆hj, kJ/mol
∆sAT, kJ/(mol deg)
∆sGC, (∆gGC – ∆gAT)|13°C, kJ/(mol deg) kJ/mol
Six hairpins; 0.1 M LiCl ps-N1, ps-N2, ps-AT; 0.1 M NaCl
19.5 ± 0.1
22.7 ± 0.2
2.2 ± 0.1
58 ± 0.4
59 ± 0.4
3.1 ± 0.2
14.6
16.6
1.4
45
45
2.0
Note: Samples contained 10 mM Na phosphate buffer pH 7.0 or 8.0, or 10 mM Tris-HCl buffer pH 8. Parameters were determined with global fitting of the theoretical heterogeneous “zipper” curve simultaneously to melting curves of six oligonucletodes [24]. Enthalpy ∆hj of the AT/GC boundary is a part of the energy balance with a negative sign. MOLECULAR BIOLOGY
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PS-DNA WITH ë isoGC- OR iso5meCG PAIRS 1.0
The high stability of antiparallel DNA is provided by the cisGC base pair with three H-bonds isomorphic to the cisAT pair. By analogy, possible existence of a stable ps-DNA with transAT and trans isoGC pairs had been predicted, as the two pairs were isomorphic and the trans isoGC could have three H-bonds [12] (Fig. 5). Similarly, 5-methyl isocytosine can form trans Gm5isoC pair with three H-bonds isomorphic to transAT and trans isoGC. Ps-DNA comprising trans isoGC pairs [26], as well as ps-DNA with mixed transAT, trans isoGC, and trans m5isoCG pairs had been obtained in experiments [27], the structure of the latter had been studied in detail with NMR [28].
(a) 0.8 0.6 0.4 0.2
Our study was aimed to compare the stability of psDNA with transAT- and trans isoGC or trans m5isoCG pairs with that of homologous antiparallel DNA. To evaluate the contributions of trans isoGC and trans m5isoCG pairs to the ps-DNA formation energy was also of interest. The self-folding oligonucleotides were studied:
0 1.0 (b) 0.8 0.6
ps-t1: 3'-d(TC)6-L-d(isoGA)6-3'; ps-t2: 3'-d(Tm5isoC)6-L-d(GA)6-3'; aps GA/TC: 5'-d(GA)5-L-d(TC)5-3',
0.4
where L: -(CH2CH2O)3-.
0.2
0
10
20
30
40
50
60 70 80 Temperature, °ë
Fig. 3. Theoretical curves simulated with the heterogeneous zipper multistate model, marked as the experimental curves in Fig. 2. (a) Fraction of unpaired bases. (b) Fraction of completely unpaired hairpins.
an original motif for binding of peptide antibiotics into both grooves of ps-DNA, unlike binding into one (glycosidic) groove of B-DNA [4]. Summarizing, the ps-DNA with AT, GC base pairing have the structural and dynamic characteristics which strikingly distinguish it from the antiparallel double helix and present novel structural motifs for recognition by proteins and other ligands. However, since these parallel helices are inferior to canonical ones in stability, one should expect formation of ps-DNAs preferably in the regions of mispaired aps duplexes. An intriguing question arises: are there natural nucleotide sequences which could form stable ps-DNA successfully competing with perfect antiparallel duplexes? We consider such a case in the next section.
The predominant formation of intramolecular hairpin structure rather than any intermolecular alternative structure was determined by a method based on the measurement of fluorescence polarization of intercalated ethidium bromide (EtBr) [29]. We also used EtBr as a structural probe for the properties of secondary structures. The ps-t1 and ps-t2 were found to support intercalation of 5–6 EtBr molecules per 12 bp readily, with an association constant similar to that for aps-DNA under the prevailing experimental conditions. We concluded that these ps double helices have pronounced flexibility. The thermodynamic parameters of the helix–coil transition of ps-t1 and ps-t2 were derived using a twostate model by fitting theoretical curves to experimental denaturation profiles (Table 2). The formation enthalpy and entropy of ps-t2 were markedly smaller than those of ps-t1. The calculated free energy of formation indicates a significantly greater thermodynamic stability of ps-t1 than of ps-t2. For comparison, the transition enthalpies and free energies of formation calculated for a single base stacking in ps-t1, pst2, and aps-GA/TC are presented in Table 2. Surprisingly, despite the lower stability of the transAT pair in ps-DNA than of the cisAT pair in aps-GA/TC, the average free energy of formation of ps-t1 per bp appeared to be much higher than that of the homologous aps-GA/TC. The values for ps-t2 and apsGA/TC were close. In conclusion, in the alternating GA/TC context the trans isoGC pair is more energyMOLECULAR BIOLOGY
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5'
ps-N1
5'
5'
5'
227
5'
ps-N4
5 ps-N2
Fig. 4. Ball-and-stick model of the ps-N1, ps-N4 and ps-N2 hairpins. The three GC pairs in ps-N1 are regularly stacked, showing some propeller twist. The groove in ps-N1 is wider than in the other two structures, opening most of the position of the three GC pairs.
Cl
hp-GT:
3'-GTGTGTGTGG 3'-GTGTGTGTGG
GT-quadruplex:
Cl
transC · isoG
Cl
3'-GTGTGTGTGT 3'-TGTGTGTGTG 3'-GTGTGTGTGT 3'-TGTGTGTGTG
Fig. 6. Schemes of the structures involving GT repeats.
stable DNA–RNA hybrids [27]. This, as well as the resistance against endonucleases [30], makes ps-DNA an attractive potential instrument in antisense and antigene strategies.
Cl
transG ·
m5isoC
Fig. 5. Schemes of trans isoGC and transG m5isoC pairs.
favorable than the trans m5isoCG pair and, strikingly, than the cisGC pair. Hence, isoguanine may promote ps-DNA formation owing to the high stability of the trans isoGC pair. Isoguanine and 5-methylcytosine are natural minor bases present in plant genomes. IsoG is also a product of oxidative damage of DNA leading to mutations. Formation of ps-DNA may temporarily protect DNA regions from either degradation or expression and/or may facilitate binding of certain ps-DNA-specific repair proteins. Apart from this, oligonucleotide sequences containing isoG were shown to form very MOLECULAR BIOLOGY
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PS-DNA FORMED BY TELOMERIC (GT)4G SEQUENCE Above we have demonstrated that certain oligonucleotide sequences can form ps-DNA that may exceed in stability even the aps counterpart, not to mention any alternative structures. Let us now address an issue whether a nucleotide sequence can form a ps-DNA that would have no competitive alternatives. We did observe such situation with a repetitive GT sequence flanked by guanines [32]. GT repeats are abundant in genomes of various organisms. Simple direct repeats may give rise to replication slippage or single-stranded loops. Being among the most common microsatellites, the GT
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Table 2. Thermodynamic parameters for the formation of parallel-stranded hairpins ps-t1 and ps-t2 Oligonucleotide ps-t1 ps-t2 aps GA/TC
∆H, kJ/mol
∆S, kJ/(mol deg)
Tm, °C
∆h, kJ/mol bp
∆g37°C, kJ/mol bp
–310 ± 10 –172 ± 8 –236 ± 8
–0.87 ± 0.03 –0.48 ± 0.03 –0.69 ± 0.02
83.5 ± 0.4 87.8 ± 0.2 67 ± 0.2
–28.2 ± 0.9 –15.6 ± 0.7 –26.1 ± 0.1
–3.4 ± 0.1 –2.0 ± 0.1 –2.1 ± 0.1
Note: Samples contained 0.1 M NaCl. Parameters were obtained from the best fits of theoretical curves in a two-state model to experimental melting curves.
repeats are very genetically unstable, most probably because of the DNA polymerase slippage. The dG(GT)nG repeats are present also in the yeast Saccharomyces cerevisiae telomeres. The protein regulating the telomere length, Tel2p, binds specifically to the single-stranded G2–3(TG)1–6T overhang and promotes the formation of some secondary structures sta-
bilized by GG pairs [33]. These data draw attention to a possible role of structures formed by GT repeats in regulation of cell processes. With the aim to study the conformational possibilities of GT repeats, oligonucleotides able to fold back into ps hairpins were synthesized [32]:
hp-GT: 3'-d(GTGTGTGTGG)-L-d(GGTGTGTGTG)-3', hp-SGT: 3'- d(G4STG4STG4STG4STGG)-L-d(GGTGTGTGTG)-3', where L: -(CH2CH2O)3-, 4ST : thiothymidine. Previously, oligonucleotides 3'-d(GT)5-(CH2)6d(GT)5-3' (parGT) and 5'-d(GT)5-(CH2)6-d(GT)5-3' (antiGT) have been shown to form quadruplex structures with two (parGT) or four (antiGT) molecules [33]. The structure designated as GT-quadruplex comprised all four strands in parallel orientation (Fig. 6). Guanines of the GT-quadruplex formed layers of Gquartets, while the terminal and probably inner thymines might bulge out of the quadruple helix. Unexpectedly, flanking of the GT repeat with guanines in hpGT (and, probably, the presence of a less hydrophobic linker resulted in equilibrium formation of a ps hairpin (Fig. 6). The following experimental evidence for a ps hairpin formation was obtained [32]. First, thermal denaturation experiments showed that the parameters of the helix–coil Table 3. Average enthalpy of the helix–coil transition of ps-DNAs of various base composition Base composition GG, TT Hetero AT Homo AT GG, AT GC, AT
Average ∆H per bp, kJ/mol bp
–13.8 –13.4…–14.6 –18.8 –13.8…–14.2 –14.2…–17.6, depending on nucleotide sequence AT, isoGC –28.2 5 AT, m isoCG –15.6
Reference [31] [35] [34] [36] [24] [29] [29]
Note: All structures were studied at pH > 7.3 and physiological NaCl concentrations.
transition of hp-GT and hp-SGT were independent of their concentration. This testified to self-folding of the oligonucleotides into monomolecular hairpins. Secondly, probing of the hp-GT and hp-SGT with EtBr showed that the structure readily permits intercalation of 4–5 EtBr molecules in an anticooperative mode. This type of intercalation is characteristic of double rather than quadruple helices, which bind intercalators in a cooperative mode [33]. Binding of the first EtBr molecule is impeded because creation of an intercalation site by drawing G4 quartets apart requires appreciable energy, whereas the subsequent EtBr molecules readily intercalate into the already available sites. The next important question is: what is the state of thymines in the hp-GT hairpin, are they bulged out or involved in base pairing? Molecular modeling showed a possibility of formation of isomorphic GG and TT pairs; substitution of S4(T) for O4(T) did not prevent H-bonding in the TT pair (Fig. 7). Further insight into the structure of the hairpin is provided by a study of the specially designed hp-SGT. The melting of the thiothymidinecontaining pairs was separately monitored at its absorption maximum at 335 nm, while the melting of GG pairs was followed by absorbance at 260 nm (Fig. 8). The experiment indicated the involvement of thymines in base pairing and simultaneous participation of bases of both types in formation/denaturation of the hairpin structure. The values of the hyperchromic effect and of the formation enthalpy for this ps-DNA as well as energy minimization in the course of molecular modeling allowed a suggestion that the structure of the ps dG(GT)4G hairpin involves all the bases in 10 bp of two types, GG and TT, rather than 8 GT pairs with unpaired end guanines. MOLECULAR BIOLOGY
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Cl
0.0004
0.195
0.0003 0.190 0.0002 0.185
0.0001 0
0.180 0.066
0.0005
(b)
Fig. 7. Schemes of GG and TT pairs in ps-DNA.
Remarkably, the novel self-complementary psDNA dG(GT)4G has some peculiar structural features (Fig. 9). The average overlapping of base pairs is greater at the 5'-GT-3' step than at the 5'-TG-3' step, thereby defining a dinucleotide motif for the helix. The average value of formation enthalpy of dG(GT)4G is given in Table 3. This parameter is close to those of ps-DNAs with other complementarity, with the exception of some especially stable helices [34–36]. The dG(GT)4G sequence has another important property. Notwithstanding its medium stability, the ps dG(GT)4G has no alternative competing structures in a wide range of experimental conditions studied. Increasing the oligonucleotide or counterion concentration, changing the type of counterions did not lead to formation of any multistranded structures, similar to the GT-quadruplex, or to any other structure different from the monomolecular 10-bp ps hairpin. The dG(GT)4G sequence is unique in this respect. CONCLUDING REMARKS Ps-DNA is a polymorphic double helix with an outstandingly extensive repertoire of base complementarity. Ps-DNA containing natural base sequences at neutral pH usually yields in stability to canonical DNA. Nevertheless, there exist nucleotide sequences that form the ps-DNA exceeding in stability any alternative structures. There even exists a psDNA-forming sequence that shapes no alternative structures in a wide range of experimental conditions. Ps-DNA displays the structural and dynamical features that strikingly distinguish it from canonical double-stranded helices and presents unique strucMOLECULAR BIOLOGY
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Absorbance at 355 nm
0.064
Cl T:T-pair, H-bonds N3–O2
Derivative
Cl G:G-pair, H-bonds N2H2 –N3
0.0005
(a)
0.0004
0.062 0.0003
0.060 0.058
0.0002
Derivative
Absorbance at 260 nm
Cl
229
0.056 0.0001 0.054 0.052
0
10
20
30 40 50 Temperature, °ë
60
0 70
Fig. 8. Thermal denaturation curves of the hp-SGT hairpin at 260 nm (a) and 335 nm (b). Concentrations: 1.1 µM oligonucleotide, 0.1 M NaCl, 10 mM Na phosphate buffer, pH 7.4. Every third or fourth experimental point is indicated; solid curves are the theoretical fits. Left axis, absorbance (䊉); right axis, derivative of absorbance (䊊).
tural motifs for binding of proteins and other specific ligands. Though the possible biological role of ps-DNA is widely discussed in the literature, no convincing direct evidence for the ps-DNA functioning in vitro or in vivo has been found so far. The very stable ps-DNA structure comprising isoGC pairs may give new impetus to the search for ps-DNA-specific proteins. It seems essential that the studies on nucleoproteins and complexes of ps-DNA with other ligands be conducted with control experiments directly determining the mutual orientation of DNA strands. Indeed, some ligands may induce reorientation of the ps-DNA strands upon binding, resulting in a ligand complex with an imperfect antiparallel duplex [37]. Direct determination of the mutual orientation of DNA strands has so far been carried out for relatively short
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Fig. 9. Side and top views of the minimized ps-5'-d(GGTGTGTGTG) helix. Both grooves are of equal size. The ribbon following the sugar backbone reflects the higher twist at the GT step (~51.) compared with the TG step (~42.) [32].
duplexes either by covalent labeling of oligonucleotide ends with fluorophores, or ensured by forced orientation in ps hairpins [37, 38]. As for longer DNA sequences, the prospects of the ps-DNA study in complexes and investigation of its biological role might lie also in development of methods detecting the ps-DNA with fluorescing structural probes such as base analogs. The latter should (i) form pairs with complementary bases, structurally similar to natural ones without distorting or destabilizing the ps double helix, and (ii) display intense fluorescence sensitive to the base pair structure. First data on detection of noncanonical DNA structures were obtained using this approach [39], which may be promising for monitoring ps-DNA formation in real time in vitro and in vivo. ACKNOWLEDGMENTS Studies [24, 30, 32] were supported by the Russian Foundation for Basic Research (project 99-0449179), program for support of Russian leading scientific schools (00-15-97834), NATO Linkage grant HTECH LG 971252, and the Max Planck Society.
REFERENCES 1. Watson D., Crick F.H. 1953. A structure for deoxyribose nucleic acid. Nature. 171, 737–738. 2. Rippe K., Jovin T.M. 1992. Parallel-stranded duplex DNA. Methods Enzymol. 211 (Part A), 199–220. 3. Germann M.W., Zhou N., van de Sande J.H., Vogel H.J. 1995. Parallel-stranded duplex DNA: an NMR perspective. Methods Enzymol. 261, 207–226. 4. Shchyolkina A.K., Minchenkova L.E., Minyat E.E., Khomyakova Y.B., Ivanov V.I., Klement R., Jovin T.M. 1998. Distamycin-stabilized antiparallel-parallel combination (APC) DNA. J. Biomol. Struct. Dynam. 15, 823–839. 5. Cubero E., Avino A., de la Torre B.G., Frieden M., Eritja R., Luque F.J., Gonzalez C., Orosco M. 2002. Hoogsteen-based parallel-stranded duplexes of DNA. Effect of 8-amino-purine derivatives. J. Am. Chem. Soc. 124, 3133–3142. 6. Geinguenaud F., Liquier J., Brevnov M.G., Petrauskene O.V., Alexeev Y.I., Gromova E.S., Taillandier E. 2000. Parallel self-associated structures formed by T, C-rich sequences at acidic pH. Biochemistry. 39, 12650–12658. 7. Escude C., Mohammadi S., Sun J.-S., Nguen C.-H., Bisagni E., Liquier J., Taillandier E., Garestier T., Helene C. 1996. Ligand-induced formation of Hoogsteen-paired parallel DNA. Chemistry and Biology. 3, 57–65. MOLECULAR BIOLOGY
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