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A new DNA quadruplex
JULI FEIGON
DNA STRUCTURES
A new DNA quadruplex Oligonucleotides containing runs of cytosines adopt a new kind of four-stranded structure which can be described as mutually intercalating duplexes. Thirty or so years ago, biophysical chemists found evidence that DNA chains are not conlined to forming antiparallel duplexes - depending on their sequence, they can also form triplexes, quadruplexes and parallel duplexes with non-canonical base pairs [l]. In the past few years, these novel structures have been rediscovered and redefined [24]. The new interest in nonstandard DNA structures stems from the possible biological roles suggested for them, but the impetus behind the recent work comes from technological developments - high resolution NMR spectroscopy and single crystal X-ray diffraction have made it possible for the first time to determine the structures in detail. Now, a new and unpredicted DNA structure has come along. This is a four-stranded structure formed by the mutual intercalation - hence its name ‘i-DNA’ - of two hemiprotonated dC.dC duplexes at acid pH. Gehring et al. [ 51 have recently reported the structure of the first example of i-DNA, formed by four strands of the oligonucleotide d(TQ. The structure is most clearly illustrated by the ‘pencil model’ shown in Figure 1. The four strands can be considered ;astwo pairs; in each pair, two d(TC5) strands base pair in a parallel duplex with five CC base pairs (Fig. 2). The two duplexes come together to form an antiparallel quadruplex, with the base pairs of the two duplexes interdigitated and the long axes through the base planes approximately at right angles to each other. The antiparallel arrangement of the two intercalated duplexes results in an alternating head-to-head and tail-to-tail stacking of neighboring base pairs. The model structur~ewas determined by analysis of twodimensional lH and. 31P NMR spectra and symmetry considerations. Peaks in the spectra were sequentially assigned to specific atoms in a model-independent way from a 1H3lP hetlero-TOCSY (total correlation spectr oscopy: see box) spectrum [6]. The unexpected NOE (nuclear Overhauser effect: see box) crosspeaks from Tl-C&2<5<3C4 are explained by the proposed model structure (Fig. 1). Independent evidence for a fourstranded structure ‘was obtained by the concentrationdependent migration of d(TC5) on polyactylamide gels. Determination of the i-DNA structure was both helped and hindered by its high degree of symmetry. As all four strands are equivalent, the NMR spectra are relatively sim ple, with only one resonance for each set of four equivalent, non-exchangeable resonances from the four strands and live exchangeable C + imino resonances. The symmetry allowed the authors to rule out all non-symmetrical structures. The &safdvantages of so much symmetry are that each crosspe.ak also defines four distances in the molecule, and tlie“distinction between intra- and interstrand NOES is modlel-dependent (for example, an NOE @
Current
Biology
5’
3 1993 Current Biology
Fig. 1. Proposed i-DNA model structure of the d(TCs) quadruplex. Arrows indicate the axes of symmetry. The terminal T bases may not be base paired. (Adapted from 151).
from ~3 to C4 can be to either the C4 base-paired to C3, the ~4 intercalated above or the C4 in the same strand). This ultimately limits the accuracy of the calculated structure of the d(TC5) tetramer. Nonetheless, the arguments presented by Gehring et al. for the i-DNA structure are convincing. I-DNA differs from Watson-Crick B-DNA in its base pairing, in the parallel strand orientation of the two duplexes, and of course in the interdigitation of the two duplexes to form a quadruplex. Thirty years ago it was suggested that, at acid pH, poly(C) would form a parallel-stranded duplex with C + .C base pairs [7]. Recent NMR studies 1993,
Vol
3 No
9
611
612
Current
Biology
1993, Vol 3 No 9
5’H--
C -H5” Schematic of a part of a DNA strand, showing short interproton distances leading to NOES in NOESY spectra of DNA. Only the shortest interproton distances are indicated by the dashed lines, but the Hl I, H2’ and H2” protons are all within NOE distance of the base H8 and H6 protons.
e
0
H2”
Schematic of a part of a DNA strand illustrating the scalar connectivities between the phosphates and the deoxyribose
have shown, unexpectedly, that oligonucleotides contain ing the sequence d(CGA) form parallel duplexes containing C+.C base pairs, at low pH [ES].What is even more surprising about the acid d(TC5) structure is the mutual intercalation of the duplexes. When planar aromatic drugs intercalate into DNA, the maximum occupancy is one drug per two base pairs [ 911;in i-DNA, there is necessarily intercalation between each base pair. The mechanism of drug intercalation is easy to understand; it is much more diffcult to imagine how four strands come together to form this interdigitated quadruplex. In the Nh4R spectra, only the quadruplex (tid possibly some single strand) is observed, but the polykylamide gels indicate that at lower concentrations a duplex is also formed.
Why should i-DNA be favoured over a parallel duplex? The authors argue that i-DNA is energetically favoured because of tie increased number of stacking and van der Waals interactions and more favorable electrostatic interactions. In DNA triplexes, neighboring C + .G.C triplets are destabilizing relative to TAT neighbors, presumably due to electrostatic repulsion from the N3 protonated C. The orientation of base pairs in i-DNA allows maximal separation of the N3 nitrogens and reverses the orientation of the carbonyl and amino dipoles in successive base pairs. Although d(TC5) is the only DNA oligonucleotide for which a three-dimensional i-DNA structure has been proposed, in a related study on imino and amino proton
DISPATCH
unimolecuat [ 121 quadruplexes, respectively, containing four G-quartets and the thymines in loops. /
Fig. 2. Structure data El indicate is shared equally
of a hemiprotonated C.C based that the proton of the protonated between the two base pairs.
Gehring et aE. [ 51 speculate that four repeats of the C-rich complementary strand could fold to form an intramolecular i-DNA structure. Although formation, of the i-DNA structure for d(TC5) requires low pH in vitro, the authors argue that there may be other conditions in which i-DNA could form at physiological PH. In spite of the philosophical attractiveness of this proposal for the structure of telomeres, it seems unlikely to me that i-DNA is the relevant structure in vivo. Nevertheless, at the very least, the i-DNA structure reminds us that we still have a lot to learn about the alphabet of DNA. pair. The NMR imino group
References 1.
SAENGER W:
Springer-Verlag;
exchange rates, Gueron and co-workers IlO] present evidence that d(C,& d(TC& d(Q), d(TC*), d(TC,T), d(T2CsT2), d(C*TC$ and d(TC3) also form tetrameric structures at low pH and for the latter four they report NMR spectra with the characteristic NOES observed for d(TCs). Thus, they argue that the i-DNA structure may be a general feature of C-rich strands at acid pH. These structures also have in common a much slower exchange rate for the imino protons than is observed for the imino protons of B-DNA The data indicate base-pair lifetimes of minutes, as opposed to the few millisecond lifetimes of Watson-Crick base pairs in B-DNA. It is perhaps worth noting that two other unusual DNA structures, Z-DNA [ll] and G-DNA [12,13], also have slowly exchanging imino resonances. The most important question for those scientists with a biological orientation is whether the i-DNA structure has a functional role in viva. This question also applies, of course, to the various other non-standard DNA structures that have been deiined, such as triplexes, G-DNA quadruplexes, parallel DNA, bent DNA, Z-DNA and so on. The Iirst approach to answering this question is always to see if the sequences that can form a particular non-standard structure occur in any interesting situations in viva. As with sequences that can form the other non-standard structures mentioned above, the answer for i-DNA is yes - in this case the repeat sequences found in telomeres. Telomeres are the structures at the ends of linear chromosomes and are composed of DNA and associated proteins. The telomeric DNA generally has a repeating G-rich sequence on one strand, with the consensus sequence d(T/A)1&;1-8, two repeats of which form a 3’ overhang beyond the telomeric duplex [ 141. An example is the Oxytricba repeat d(T*Gb), for which the base-paired complement would be d(Cd&). Oligonucleotides containing the Oxp-ick telomere repeat sequence readily form quadruplexes composed of Gquartets (G-DNA) 1[15]. NMR spectroscopic and X-ray crystallographic studies have shown that DNA oligonucleotides with one-and-a-half and three-and-a-half of these repeats form symmetrical bimolecular [12,16] and
2.
3.
4. 5.
6.
7. 8.
9. 10.
11.
12. 13.
14. 15.
16.
WEUS
Princ@Ces 1984.
of Nucleic
Acid
Structure.
RD, COLLIER DA, HANVEY JC, SHIMZU
New
M, WOHLRAB
York F: The
chemistry and biology of unusual DNA structures adopted by ohgopurine and oligopyrimidine sequences. FRSEB J 1988, 2~2939-2949. GUSCHLBAUER W, CHANTOT J-F, THLXE D: Pour-stranded nucleic acid structures 25 years later: from guanosine gels to telomere DNA J Biomol Strut Dyn 1990, 8:491-511. &PE K, JO~LN TM: Paraflel-stranded duplex DNA Meth Enzymol 1992, 211:193-220. GEHRING K, LEROY J-L, G~~RON M: A tetrameric DNA structure with protonated cytosinecytosine base pairs. Nature 1993, 363~561-565. KELLOGG GW, SZEWCUK AA, MOORE PB: Two-dimensional hetero-TOCSY-NOESY. Correlation of 3tP resonances with anomeric and aromatic tH resonances in RNA. / Am Cbem Sot 1992, 114:2727-2728. LANGR[DGE R, RICH A: Molecular structure of helical polycytidylic acid. Nature 1963, 198:725-728. ROBINSON
H, VAN DER MAKEL G, VAN BOOM
JH,
WANG
A H-J:
Unusual DNA conformation at low pH revealed by NMR: parallel-stranded DNA duplex with homo base pairs. Bio chemivy 1992, 31:1051Ck10517. WARING MJ: Complex formation between ethidium bromide and nucleic acids. J MoZ Biol 1965, 13:263-282. LEROY & GEHRING K, KE~~ANI A, GUBRON M: Acid muhimers of ohgodeoxycytidine strands: stoichiometry, base-pair characterization and proton exchange properties. Biochemistry 1993, 32:6019-6031. MIRAU PA, KURNS DR The unusual proton exchange properties of Z-form poly[d(G-C)]. Proc Natl Acad Sci USA 1985, 82:1594-1598. SMITH PW, FEIG~N J: Quadruplex structure of telomeric DNA oligonucleotides. Nature 1992, 356:164-168. SMITH FW, FEIGON J: Strand orientation in the DNA quadrttpIex formed horn the 03cyhicha telomere repeat oligonucleotide d(GqTpGq) in solution. Biocbemisty, in press. ZAKJAN V: Structure and function of telomeres. Annu Rev Genet 1989, 23:57!+604. WILLIAMSON fl, RAGHURA~~AN MK, CECH TR: Monovalent cationinduced structure of telomeric DNA the G-quartet model. Cell 1989, 59:871-880. KANG C, ZHANG X, RATLIFF R, MOVZIS R, RICH A Crystal structure of four-stranded O@ti&a telomeric DNA Nature 1992, 356:126131.
Juli Feigon, Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, California 90024-1569, USA
613
DNA STRUCTURES
A new DNA quadruplex Oligonucleotides containing runs of cytosines adopt a new kind of four-stranded structure which can be described as mutually intercalating duplexes. Thirty or so years ago, biophysical chemists found evidence that DNA chains are not conlined to forming antiparallel duplexes - depending on their sequence, they can also form triplexes, quadruplexes and parallel duplexes with non-canonical base pairs [l]. In the past few years, these novel structures have been rediscovered and redefined [24]. The new interest in nonstandard DNA structures stems from the possible biological roles suggested for them, but the impetus behind the recent work comes from technological developments - high resolution NMR spectroscopy and single crystal X-ray diffraction have made it possible for the first time to determine the structures in detail. Now, a new and unpredicted DNA structure has come along. This is a four-stranded structure formed by the mutual intercalation - hence its name ‘i-DNA’ - of two hemiprotonated dC.dC duplexes at acid pH. Gehring et al. [ 51 have recently reported the structure of the first example of i-DNA, formed by four strands of the oligonucleotide d(TQ. The structure is most clearly illustrated by the ‘pencil model’ shown in Figure 1. The four strands can be considered ;astwo pairs; in each pair, two d(TC5) strands base pair in a parallel duplex with five CC base pairs (Fig. 2). The two duplexes come together to form an antiparallel quadruplex, with the base pairs of the two duplexes interdigitated and the long axes through the base planes approximately at right angles to each other. The antiparallel arrangement of the two intercalated duplexes results in an alternating head-to-head and tail-to-tail stacking of neighboring base pairs. The model structur~ewas determined by analysis of twodimensional lH and. 31P NMR spectra and symmetry considerations. Peaks in the spectra were sequentially assigned to specific atoms in a model-independent way from a 1H3lP hetlero-TOCSY (total correlation spectr oscopy: see box) spectrum [6]. The unexpected NOE (nuclear Overhauser effect: see box) crosspeaks from Tl-C&2<5<3C4 are explained by the proposed model structure (Fig. 1). Independent evidence for a fourstranded structure ‘was obtained by the concentrationdependent migration of d(TC5) on polyactylamide gels. Determination of the i-DNA structure was both helped and hindered by its high degree of symmetry. As all four strands are equivalent, the NMR spectra are relatively sim ple, with only one resonance for each set of four equivalent, non-exchangeable resonances from the four strands and live exchangeable C + imino resonances. The symmetry allowed the authors to rule out all non-symmetrical structures. The &safdvantages of so much symmetry are that each crosspe.ak also defines four distances in the molecule, and tlie“distinction between intra- and interstrand NOES is modlel-dependent (for example, an NOE @
Current
Biology
5’
3 1993 Current Biology
Fig. 1. Proposed i-DNA model structure of the d(TCs) quadruplex. Arrows indicate the axes of symmetry. The terminal T bases may not be base paired. (Adapted from 151).
from ~3 to C4 can be to either the C4 base-paired to C3, the ~4 intercalated above or the C4 in the same strand). This ultimately limits the accuracy of the calculated structure of the d(TC5) tetramer. Nonetheless, the arguments presented by Gehring et al. for the i-DNA structure are convincing. I-DNA differs from Watson-Crick B-DNA in its base pairing, in the parallel strand orientation of the two duplexes, and of course in the interdigitation of the two duplexes to form a quadruplex. Thirty years ago it was suggested that, at acid pH, poly(C) would form a parallel-stranded duplex with C + .C base pairs [7]. Recent NMR studies 1993,
Vol
3 No
9
611
612
Current
Biology
1993, Vol 3 No 9
5’H--
C -H5” Schematic of a part of a DNA strand, showing short interproton distances leading to NOES in NOESY spectra of DNA. Only the shortest interproton distances are indicated by the dashed lines, but the Hl I, H2’ and H2” protons are all within NOE distance of the base H8 and H6 protons.
e
0
H2”
Schematic of a part of a DNA strand illustrating the scalar connectivities between the phosphates and the deoxyribose
have shown, unexpectedly, that oligonucleotides contain ing the sequence d(CGA) form parallel duplexes containing C+.C base pairs, at low pH [ES].What is even more surprising about the acid d(TC5) structure is the mutual intercalation of the duplexes. When planar aromatic drugs intercalate into DNA, the maximum occupancy is one drug per two base pairs [ 911;in i-DNA, there is necessarily intercalation between each base pair. The mechanism of drug intercalation is easy to understand; it is much more diffcult to imagine how four strands come together to form this interdigitated quadruplex. In the Nh4R spectra, only the quadruplex (tid possibly some single strand) is observed, but the polykylamide gels indicate that at lower concentrations a duplex is also formed.
Why should i-DNA be favoured over a parallel duplex? The authors argue that i-DNA is energetically favoured because of tie increased number of stacking and van der Waals interactions and more favorable electrostatic interactions. In DNA triplexes, neighboring C + .G.C triplets are destabilizing relative to TAT neighbors, presumably due to electrostatic repulsion from the N3 protonated C. The orientation of base pairs in i-DNA allows maximal separation of the N3 nitrogens and reverses the orientation of the carbonyl and amino dipoles in successive base pairs. Although d(TC5) is the only DNA oligonucleotide for which a three-dimensional i-DNA structure has been proposed, in a related study on imino and amino proton
DISPATCH
unimolecuat [ 121 quadruplexes, respectively, containing four G-quartets and the thymines in loops. /
Fig. 2. Structure data El indicate is shared equally
of a hemiprotonated C.C based that the proton of the protonated between the two base pairs.
Gehring et aE. [ 51 speculate that four repeats of the C-rich complementary strand could fold to form an intramolecular i-DNA structure. Although formation, of the i-DNA structure for d(TC5) requires low pH in vitro, the authors argue that there may be other conditions in which i-DNA could form at physiological PH. In spite of the philosophical attractiveness of this proposal for the structure of telomeres, it seems unlikely to me that i-DNA is the relevant structure in vivo. Nevertheless, at the very least, the i-DNA structure reminds us that we still have a lot to learn about the alphabet of DNA. pair. The NMR imino group
References 1.
SAENGER W:
Springer-Verlag;
exchange rates, Gueron and co-workers IlO] present evidence that d(C,& d(TC& d(Q), d(TC*), d(TC,T), d(T2CsT2), d(C*TC$ and d(TC3) also form tetrameric structures at low pH and for the latter four they report NMR spectra with the characteristic NOES observed for d(TCs). Thus, they argue that the i-DNA structure may be a general feature of C-rich strands at acid pH. These structures also have in common a much slower exchange rate for the imino protons than is observed for the imino protons of B-DNA The data indicate base-pair lifetimes of minutes, as opposed to the few millisecond lifetimes of Watson-Crick base pairs in B-DNA. It is perhaps worth noting that two other unusual DNA structures, Z-DNA [ll] and G-DNA [12,13], also have slowly exchanging imino resonances. The most important question for those scientists with a biological orientation is whether the i-DNA structure has a functional role in viva. This question also applies, of course, to the various other non-standard DNA structures that have been deiined, such as triplexes, G-DNA quadruplexes, parallel DNA, bent DNA, Z-DNA and so on. The Iirst approach to answering this question is always to see if the sequences that can form a particular non-standard structure occur in any interesting situations in viva. As with sequences that can form the other non-standard structures mentioned above, the answer for i-DNA is yes - in this case the repeat sequences found in telomeres. Telomeres are the structures at the ends of linear chromosomes and are composed of DNA and associated proteins. The telomeric DNA generally has a repeating G-rich sequence on one strand, with the consensus sequence d(T/A)1&;1-8, two repeats of which form a 3’ overhang beyond the telomeric duplex [ 141. An example is the Oxytricba repeat d(T*Gb), for which the base-paired complement would be d(Cd&). Oligonucleotides containing the Oxp-ick telomere repeat sequence readily form quadruplexes composed of Gquartets (G-DNA) 1[15]. NMR spectroscopic and X-ray crystallographic studies have shown that DNA oligonucleotides with one-and-a-half and three-and-a-half of these repeats form symmetrical bimolecular [12,16] and
2.
3.
4. 5.
6.
7. 8.
9. 10.
11.
12. 13.
14. 15.
16.
WEUS
Princ@Ces 1984.
of Nucleic
Acid
Structure.
RD, COLLIER DA, HANVEY JC, SHIMZU
New
M, WOHLRAB
York F: The
chemistry and biology of unusual DNA structures adopted by ohgopurine and oligopyrimidine sequences. FRSEB J 1988, 2~2939-2949. GUSCHLBAUER W, CHANTOT J-F, THLXE D: Pour-stranded nucleic acid structures 25 years later: from guanosine gels to telomere DNA J Biomol Strut Dyn 1990, 8:491-511. &PE K, JO~LN TM: Paraflel-stranded duplex DNA Meth Enzymol 1992, 211:193-220. GEHRING K, LEROY J-L, G~~RON M: A tetrameric DNA structure with protonated cytosinecytosine base pairs. Nature 1993, 363~561-565. KELLOGG GW, SZEWCUK AA, MOORE PB: Two-dimensional hetero-TOCSY-NOESY. Correlation of 3tP resonances with anomeric and aromatic tH resonances in RNA. / Am Cbem Sot 1992, 114:2727-2728. LANGR[DGE R, RICH A: Molecular structure of helical polycytidylic acid. Nature 1963, 198:725-728. ROBINSON
H, VAN DER MAKEL G, VAN BOOM
JH,
WANG
A H-J:
Unusual DNA conformation at low pH revealed by NMR: parallel-stranded DNA duplex with homo base pairs. Bio chemivy 1992, 31:1051Ck10517. WARING MJ: Complex formation between ethidium bromide and nucleic acids. J MoZ Biol 1965, 13:263-282. LEROY & GEHRING K, KE~~ANI A, GUBRON M: Acid muhimers of ohgodeoxycytidine strands: stoichiometry, base-pair characterization and proton exchange properties. Biochemistry 1993, 32:6019-6031. MIRAU PA, KURNS DR The unusual proton exchange properties of Z-form poly[d(G-C)]. Proc Natl Acad Sci USA 1985, 82:1594-1598. SMITH PW, FEIG~N J: Quadruplex structure of telomeric DNA oligonucleotides. Nature 1992, 356:164-168. SMITH FW, FEIGON J: Strand orientation in the DNA quadrttpIex formed horn the 03cyhicha telomere repeat oligonucleotide d(GqTpGq) in solution. Biocbemisty, in press. ZAKJAN V: Structure and function of telomeres. Annu Rev Genet 1989, 23:57!+604. WILLIAMSON fl, RAGHURA~~AN MK, CECH TR: Monovalent cationinduced structure of telomeric DNA the G-quartet model. Cell 1989, 59:871-880. KANG C, ZHANG X, RATLIFF R, MOVZIS R, RICH A Crystal structure of four-stranded O@ti&a telomeric DNA Nature 1992, 356:126131.
Juli Feigon, Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, California 90024-1569, USA
613