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IR SPSCTROSCOPVOF DNA
307
[ 16] I n f r a r e d S p e c t r o s c o p y o f D N A By E. TAILLANDIERand J.
LIQUIER
Introduction Compared to other techniques some of the advantages of infrared spectroscopy in the study of nucleic acids are (1) the possibility of obtaining data from samples under an extremely wide variety of physical states (solutions, gels, hydrated fibers or films, and crystals); (2) no limitation introduced by the size of the investigated molecule, that is, one can work as well with short oligonucleotides as with long polynucleotides, DNA fragments obtained by enzymatic or chemical cleavage, or high molecular weight native DNAs. (3) Infrared spectroscopy is a nondestructive technique, which requires only small amounts of samples [a good Fourier transform infrared (FT-IR) spectrum can be obtained with 2 0 D units of material]. (4) It gives vibrational information characteristic of the helical conformations of the nucleic acids. (5) The comparison of FT-IR spectra obtained in solution and in crystals permits correlations with precise structures known from X-ray crystal diffraction studies as well as NMR solution studies. Different types of information can be obtained by IR spectroscopy. For example, it is possible to characterize molecular geometry using what is usually called IR "marker bands" which are conformation sensitive (wavenumber and/or relative intensity and/or polarization of the absorption). These bands can show the existence of a conformational transition when different factors such as temperature, hydration, concentration, nature, and amount of counterions are varied (B --~ A, B --* C, B -* Z, helix --~ coil, etc.). In the case of mixtures of conformations, computer simulations based on "perfect" pure form spectra are used to evaluate quantitatively the amount of each geometric form in the sample, by reference with these marker bands. Another type of information which can be obtained from the FT-IR spectra concerns recognition of DNA sequences by a wide variety of molecules, such as oligonucleotides (triple-helical structures), drugs and proteins. Although less commonly employed in studying nucleic acids than its "sister" technique, Raman spectroscopy, infrared spectroscopy has been successfully used since the early work of Fraser and Fraser ~ by several i M. J. Fraser and D. B. Fraser, Nature (London) 167, 759 (1951). 2 H. Falk, K. A. Hartman, and R. C. Lord, J. Am. Chem. Soc. 85, 391 (1963). 3 H. T. Miles and J. Frasier, Biochem. Biophys. Res. Commun. 14, 21 0964).
METHODS IN ENZYMOLOGY, VOL. 211
Copyright© 1992by AcademicPress,Inc. All rightsof reproductionin any form r'-~erved.
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SPECTROSCOPIC METHODS FOR ANALYSIS OF D N A
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g r o u p s . 2-7 In this chapter we shall briefly present a general survey of the
FT-IR spectrum of a nucleic acid, describe the characterization of the different classic families of DNA conformations (A, B, and Z), and then consider how FT-IR spectroscopy can be used in the study of DNA recognition, particularly in the case of triple-stranded structures and D N A - d r u g interactions. General Features of F T - I R Spectra of Nucleic Acids Nucleic acid vibrations arise from different parts of the macromolecule, and the corresponding IR absorptions are detected in several regions of the spectrum. The main vibrations are observed in the spectral range between 1800 and 700 cm -~, and four domains can be distinguished: between 1800 and 1500 cm -~ absorption bands due to the stretching vibrations of double bonds in the base planes; between 1500 and 1250 cm -~ bands due to the base-sugar entities, strongly dependent particularly on the glycosidic torsion angle; between 1250 and 1000 cm -I strong absorptions of the phosphate groups and of the sugar; below 1000 cm -~ bands due to the vibrations of the phosphodiester chain coupled to vibrations of the sugar. Figure la presents a DNA FT-IR spectrum recorded in H20 solution. The enlarged area shows the spectral domain corresponding to the vibrations of the phosphate groups. On such a spectrum we can observe an important contribution of the solvent (H20), which makes it difficult to obtain data in the region around 1600 cm -~ and below 1000 cm-L To avoid this solvent problem, the spectrum of the same molecule is recorded in D20 solution (Fig. lb). Now we can easily study the FF-IR spectrum in the previously mentioned regions (see enlarged insets, Fig. 1). The combination of H20/D20 solutions covers the complete spectral range necessary for nucleic a d d studies. If a DNA film is exposed to an atmosphere with controlled hydration obtained by using saturated solutions of known salts, the H20 contribution is less important than in the case of an aqueous solution. Careful treatment of data allows one to obtain an infrared spectrum even in the 18001500 cm -~ region. When the film is exposed to D20 vapor, H20/D20 exchange occurs, and this can be followed on the IR spectrum. Important
4 M. Tsuboi, in "Applied Spectroscopy Reviews" (E. G. Brame, ed.), Vol. 3, p. 45. Dekker, New York, 1969. 5 j. Pilet and J. Brahms, Biopolymers 12, 387 (1973). H. Fdtzsche, H. Lang, and W. Pohle, Biochim. Biophys. Acta 432, 409 (1976). 7 E. Taillandier, J, Liquier, and J. A. Taboury, in "Advances in Infrared and Raman Spectroscopy" (R. J. H. Clark and R. E. Hester, ecls.), Vol. 12, p. 65. Heyden, London, 1985.
[ 16]
IR SPECTROSCOPY OF D N A
309
!
O
b
f
O
8
O
\
FIO. 1. FT-IR spectra of DNA in solution. (a) H20 solution; (b) D20 solution. Enlarged parts of the spectra present the absorptions involving mainly the vibrations of the phosphate groups (top), the double bonds of the bases in their plane (bottom left), and the sugarphosphate backbone (bottom right).
310
SPECTROSCOPIC METHODSFORANALYSISOF DNA
[ 16]
modifications of the DNA spectrum can be observed that reflect the exchange of labile hydrogens of the bases, in particular the NH groups of thymines and the NH2 groups of adenines, guanines, and cytosines. Exchange ofhydrogens attached to C-8 of the purine base moieties is slow and observable only at elevated temperatures. In the solid state, DNA can be studied by IR spectroscopy of microcrystals. Microscopes especially designed for IR spectroscopy that contain mirror all reflecting Cassegrain type optics are now commercially available and can be coupled to FT-IR spectrophotometers. Such systems need a more sensitive detector, usually a nitrogen-cooled MCT detector. An example is given in Fig. 2, which presents the micro-FT-IR spectrum of
0
OD W 0
(0 N N
O~ O3
t~ tq
Flo. 2. Micro-Fl'-IRspectrumof a d(CGCGAATTCGCG)crystal.
[ 16]
IR SPECTROSCOPYOF DNA
311
d(CGCGAATTCGCG). In this case the crystal area analyzed by Fr-IR microspectroscopy is about 50 × 50 am. Characterization of Nucleic Acid Secondary Structures The main IR marker bands of A-, B-, and Z-DNA discussed below have been summarized in Table I. Polynucleotide and oligonucleotide conformations determined by IR spectroscopy are presented in Table II.
Right-Hand A- and B-DNA Conformations In low ionic strength solutions, native DNAs and most synthetic double-strand poly- and oligonucleotides adopt a B-form conformation. Under the same conditions double-stranded RNAs adopt an A-type conformation. Figure 3 presents the FT-IR spectra obtained in H20 solutions of two synthetic nucleic acids with regularly alternating A-U sequences, the first TABLE I MAIN INFRARED MARKER BANDS OF A-, B-, AND Z-DNA CONFORMATIONS
Conformation A
B
Z
Assignment
1705
1715
1418 1401 1375
1425
1695 1433 1408
In-plane base double bond stretching A-T bases Deoxyribose
1375 1355
1335 1335
1344 1328
1275
1281
1240 1188
1225
1265 1215
899 877 864 806
894
1065 929
1320
830-840
dA.dG anti dA.dG syn dT dA dG T G Antisymmetric phosphate stretching Deoxyribose Deoxyribose
Exact positions of bands are within - 2 cm-~ of these numbers, depending, in particular, on base sequence of DNA
T A B L E II CONFORMATIONS DETERMINED BY INFRARED SPECTROSCOPY a
Polymer Polynucleotides Native D N A
Poly[d(A-T).poly[d(A-T)]
Poly(dA).poly(dT)
Counterion
Na + Li + Na + Cs +, Li + N i 2+ Na +
poly(dT)
Na +
Poly(dA).poly(rU)
Na +
Poly(rA).poly(rU) Poly[d(2NH2A-T)].poly[d(2NH2A-T)]
Na ÷ Na +
Poly[d(A-C)].poly[d(G-T)]
Na +
Poly[d(G-C)].poly[d(G-C)]
Li + Ni 2+ Na ÷
Poly[d(m5C-G)]"poly[d(m5C-G)] Poly(dG).poly(dC)
M g 2+, Co 2+, Ni 2+ Na + Na +
Poly[d(I-Br5C)-poly[d(I-Br5C)]
Na +
Poly(rA).
Physical state
Form
S, F H FL FL S, F H
B A C B
FL
A
FL FH S, F H FL S, F H FL S FL S S low salt S high salt S, F H FL FH F FH, S low salt FL, S high salt FH F FH FL S low salt S high salt
C Z B H H A H A A B Z B A C Z B Z Z Z B+ A A B Z
Refs.
b b c
d,e d,e f d
e,g g h h h h h i i j j k j
l,m,n Lm, n o n p q q
Oligonucleotides d(CCGCGG)
Na + Na ÷ Na +
d(m5CGCGm5CG) d(m5CGGCm5CG) d(CBr8GCGCBr8G) d(CBr8~CBr8G) d(CCCGC~)
Na + Na + Na + Na + Na +
d(C2NI-12ACGTG)
Ni 2+ Na
d(m5CGCAm5CGTGCG)
Na
d(m5CGTAmSCG)
Na
d(CGGCCG) d(CGm5CGCG)
312
F F S low salt S high salt C F F F F FH FL F FH FL FH FL FH FL
Z Z B Z B-Z Z Z Z Z B A Z B Z B Z B Z
r s t t t r r u r v v v w w w w x x
TABLE II (continued) CONFORMATIONS DETERMINED BY INFRARED SPECTROSCOPYa
Polymer d(GGTATACC) d(CGCGAATTCGCG) d(ACATGT)
Counterion
d(AAAAATTTTT)
Mg 2+ Mg 2+ Na Ni 2+ Na +
r(A-U)6, r(A-U)s
Na +
Physical state C C F F FH FL S
Form B+ A B+ A B Z B H A
Refs. y p t,s t,s g g h
a S, Solution; FH, film high hydration; FL, film low hydration; F, film any hydration; C, crystal; and H, neteronomous. b j. filet and J. Brahms, Biopolymers 12, 387 (1973); H. Fritzsche, A. Rupprecht, and M. Rupprecht, Nucleic Acids Res. 12, 9165 (1984). ¢ J. Brahms, J. Pilet, P. L. Tran Thi, and R. L. Hill, Proc. Natl. Acad. Sci. U.S.A. 70, 3352 (1973). a S. Adam, J. Liquier, J. A. Taboury, and E. Taillandier, Biochemistry 25, 3220 (1986). e j. Pilet, J. Blicharski, and J. Brahms, Biochemistry 14, 1869 (1975). f S. Adam, P. Bourtayre, J. Liquier, J. A. Taboury, and E. Taillandier, Biopolymers 26, 251 (1987). g E. Taillandier, J. P. Ridoux, J. Liquier, W. Leupin, W. A. Denny, Y. Wang and W. L. Peticolas, Biochemistry 26, 3361 (1987). h j. Liquier, A. Akhebat, E. Taillandier, F. Ceolin, T. Huynh Dinh, and J. Igolen, Spectrochim. Acta 47A, No. 2, 177 (1991). F. B. Howard, C. W. Chen, J. S. Cohen, and H. T. Miles, Biochem. Biophys. Res. Commun. 3, 848 (1984). J E. Taillandier, J. A. Tahoury, S. Adam, and J. Liquier, Biochemistry 23, 5703 (1984). k D. H. Loprete and K. A. Hartman, J. Biomol. Struct. Dyn. 7, 347 (1989). E. Taillandier, J. A. Tahoury, J. Liquier, P. Sautiere, and M. Couppez, Biochimie 63, 895 (1981 ). " J. Pilet and M. Leng, Proc. Natl. Acad. Sci. U.S.A. 79, 26 (1982); P. B. Keller and K. A. Hartman, Nucleic Acids Res. 14, 8167 (1986). n j. A. Tahoury, J. Liquier, and E. TaiUandier, Can. J. Chem. 63, 1904 (1985). o j. A. Tahoury, P. Boutayre, J. Liquier, and E. Taillandier, Nucleic Acids Res. 12, 4247 (1984). P E. Taillandier, in "Structure and Methods" Vol. 3 "DNA and RNA" (R. H. Sarma and M. H. Sarma, eds.), p. 73. Adenine Press, 1990. q B. Hartman, J. Pilet, M. Ptak, J. Ramstein, B. Malfoy, and M. Leng, Nucleic Acids Res. 10, 3261 (1982). ' S. Adam, J. A. Taboury, E. Taillandier, A. Popinel, T. Huynh Dinh, and J. Igolen, J. Biomol. Struct. Dyn. 3, 873 (1986). s S. Pother, T. Huynh Dinh, J. M. Neumann, S. Tran Dinh, S. Adam, J. Tahoury, E. Taillandier, and J. Igolen, Nucleic Acids Res. 14, 1107 (1986). t L. Urpi, J. P. Ridoux, J. Liquier, N. Verdaguer, I. Fita, J. A. Subirana, F. Iglesias, T. Huynh Dinh, J. Igolen, and E. Taillandier, Nucleic Acids Res. 17, 6669 (1989). u j. A. Taboury, E. Taillandier, T. Huynh Dinh, andJ. Igolen, J. Biomol. Struct. Dyn. 2, 1185 (1985). E. Taillandier, W. L. Peticolas, S. Adam, T. Huynh-Dinh, and J. Igolen, Speetrochim. Acta 46, 107 (1990). w j. A. Tahoury, S. Adam, E. Taillandier, J. M. Neumann, S. Tran Dinh, T. Huynh Dinh, B. Langlois d'Estaintot, and J. Igolen, Nucleic Acids Res. 12, 6291 (1984). x E. Taillandier, S. Adam, J. P. Ridoux, and J. Liquier, Nucleic Acids Res. 16, 5621 (1988). r j. Liquier, E. Taillandier, W. L. Peticolas, and G. A. Thomas, J. Biomol. Struct. Dyn. 8, 295 (1990).
313
314
SPECTROSCOPIC METHODS FOR ANALYSIS OF D N A
C]
[ 16]
m ~.q
0
W i.q
-
N
t~
b
t#l tM
FIG. 3. FT-IR spectra of H20 solutions of d(A-U). (a) and r(A-U)8 (b). The enlarged area between 1350 and 1270 cm -I shows absorptions characteristic of A ( \ \ \ \ \ \ ) and B (/////) geometries.
[16]
IRsPECTROSCOPYOF DNA
315
one (Fig. 3a) with deoxy sugars, the second one (Fig. 3b) with ribo sugars: d(A-U)n and r(A-U)8. Besides the important modifications induced on the IR spectrum by the 0-2' deletion, differences between the spectra are due to the conformations adopted in solution by these two nucleic acids, namely, B and A. Some of the characteristic absorptions are shown hatched on Fig. 3, and the wavenumbers are given in Table I. In particular the phosphate antisymmetric stretching vibration is found at 1226 cm -~ in the B form and at 1245 cm -~ in the A form, and a band reflecting the base pairing of the nucleic acid is shifted from 1716 to 1712 cm -~. The A-DNA conformation can also be observed in DNAs by varying the water content of the nucleic acid sample. An easy way to achieve this is to decrease the relative humidity to which a DNA film is exposed. The decrease of the relative humidity (R.H.) from 100 to 58% R.H. will induce for some DNA sequences a B ---, A conformational transition, followed by subsequent disorganization of the double-helical structure if the hydration is further decreased. Some DNA sequences will not undergo this transition; for example, the A- conformation is not observed by decreasing the water content of a poly(dA), poly(dT) sample. On the contrary, the alternating polymer poly[d(A-T)] readily adopts this conformation, as can be seen in Fig. 4. We present in Fig. 4 a series of F r - I R spectra o f a poly [d(A-T)] film (1 Na + per phosphate counterion) exposed to decreasing relative humidifies. The displayed spectral region (1000-800 cm -~) contains several IR marker bands of the A and B geometries corresponding to vibrations of the phosphodiester chain coupled to deoxyribose vibrations. Both sets of characteristic bands have been shown hatched on Fig. 4. The B ~ A transition is reflected by the disappearance of the band located at 841 cm -~ and the emergence of a doublet at 880-865 cm -~ and of a band at 808 cm -t. The two A- and B-DNA conformations can also be characterized by a pattern of bands reflecting the vibrations of the bases coupled to vibrations of the sugar and located between 1250 and 1400 cm-L Figure 5 presents the FT-IR signatures of four double-stranded polynucleotides in this spectral region: from top to bottom poly(dA).poly(dT), poly(rA).poly(dT), poly(dA) • poly(rU), and poly(rA) •poly(rU). All spectra have been recorded in H20 solutions, and the marker bands for the A-form strands (riboses) and B-form strands (deoxyribose) are shown hatched (Fig. 5). During the B ---, A transition we observe the shift of a band involving the N-3 H bending vibration ofthymines (or uracils)from 1281 to 1275 cm -~ and the opposite displacements of an adenine band found at 1344 cm -~ and a thymine band found at 1328 cm -1 in the B form to lower and higher wavenumbers, respectively, around 1335 cm -~ in the A form. Another possibility for characterizing DNA conformations is the use of
316
SPECTROSCOPIC METHODSFOR ANALYSISOF DNA
[ 16]
¢11
,7
FIO. 4. B ---,A conformational transition ofpoly[d(A-T)] followed by FT-IR spectroscopy. The DNA film is exposed to decreasing relative humidities. (Top) B form, R.H. 100% (///). (Bottom) A form, R.H. 58% ( \ \ \ \ ) . Bands characteristic of both geometries have been hatched.
polarized infrared radiation. If the sample presents a preferential orientation axis (e.g., in the case of stretched films or fibers) the infrared spectra obtained with an incident radiation having an electric field E polarized parallelly or perpendicularly to the orientation axis of the molecule will be different. These dichroic spectra can be used to compute orientations of transition dipole moments in the molecule and in simple cases give information concerning geometric parameters) ,9 Such computations can be 8 D. B. Fraser, J. Chem. Phys. 21, 1511 (1953). 9 R. L. Rill, Biopolymers 11, 1929 (1972).
[ 16]
IR SPECTROSCOPYOF DNA
317
in
°Z,) N
FIG. 5. FT-IR spectra between 1400 and 1250cm-m of H20 solutions. (a) Poly(dA).poly(dT); (b) poly(rA).poly(dT); (c) poly(dA).poly(rU); (d) poly(rA).poly(rU). (I//I)B form; (\\\\)A form.
318
SPECTROSCOPIC METHODS FOR ANALYSIS OF D N A
[ 16]
:i
I\ CI
B "B
b
C ,
I
i
FIG. 6. Infrared polarized spectra of oriented films of poly[d(A-T)]. (a) 1 Na + per nucleotide, R.H. 5896, A form; (b) 1 Na + per nucleotide, R.H. 9896, B form; (c) 1 Cs+ per nuclt~tide, R.H. 7696, C form. ( m ) Electric vector of incident fight perpendicular to orientation axis; ( - - - ) electric vector of incident light parallel to orientation axis. I, Antisymmetric ohosohate stretching vibration; II, symmetric phosphate stretching vibration.
[16]
IR SPECTROSCOPYOF DNA
319
TABLE III POSITION AND POLARIZATIONOF PHOSPHATE GROUP VIBRATION ABSORPTION BANDS IN DEOXYRIBOPOLYNUCLEOTIDES
Antisymmetric stretching
Symmetric stretching
Conformation
v
Polarization
v
Polarization
A B C Z
1241 1225 1231 1215
± 0 II 0
1089 1089 1089 1090
II ± ± ±
performed on absorption bands involving the antisymmetric and symmetric motions of the phosphate groups. 5 Figure 6 gives an example of such polarized infrared spectra in the case of a poly[d(A-T)] film. I° The spectral region presented displays the strong absorptions of the phosphate groups. In Fig. 6 spectra (solid lines) have been obtained with polarization of the incident light perpendicular to the orientation axis, and spectra (dashed lines) have also been obtained with a parallelly polarized incident radiation. We can see the complete inversion of the polarization of the symmetric stretching vibration of phosphates (located around 1090 cm -l) when the polymer undergoes the B ---, A transition (Figs. 6a and 6b). Simultaneously the antisymmetric stretching vibration located around 1230 cm -I from nondichroic becomes perpendicularly polarized. In the case of the C form obtained in the presence of Li + or Cs ÷ this band becomes parallelly polarized (Fig. 6c). The results concerning these bands are presented in Table III. As previously stated in the introduction, these conformation-sensitive IR bands can be used to evaluate the amount of the two A and B helical geometries when both are present in the sample. Figure 7 presents the micro-FT-IR spectrum in the region of the phosphate-sugar backbone vibrations of a d(GGTATACC) crystal (Fig. 7b), along with two reference spectra of native DNA in the A (Fig. 7a) and B conformations (Fig. 7c). The crystal spectrum presents absorptions characteristic of both conformations (882, 861, and 812 cm -1 for the A form, 834 cm -I for the B form). The amount of B conformation has been estimated to be 14% and inter10 S. Adam, P. Bourtayre, J. Liquier, J. A. Taboury, and E. Taillandier, Biopolymers 26, 251 (1987).
320
[16]
SPECTROSCOPIC METHODS FOR ANALYSIS OF DNA
t~ tO 00 C¢
C= va
O0
b O1 tD
N
FIG. 7. FT-IR spectra of(a) A-form DNA (film at 58% R.H.); (b) d(GGTATACC) crystal; (c) B-form DNA (film at 98% R.H.). ( \ \ \ \ ) A-form bands; (////) B-form bands.
preted in agreement with a model in which one B-form octamer is trapped in a tunnel formed by six A-form octamers. 1t,~2 lIj. Doucet, J. P. Benoit, W. B. T Cruse, T. Prange, andO. Kennard, Nature(London)337, 190 (1989). ~2J. Liquier, E. Taillandier, W. L. Peficolas, andG. A. Thomas, Z Biomol. Strua. Dyn. 8, 295 (1990).
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I R SPBCTROSCOPY Or D N A
321
Left-Hand Z-Conformation The first IR studies concerning left-hand conformations were devoted to the B --* Z reorientation of poly[d(G-C)] in films) T M In the presence of sodium counterions and in films this polymer undergoes a complete B ---, Z transition via decreasing the relative humidity. The marker bands of guanosines in a syn geometry were obtained first. Since then many other sequences have been shown to be able to adopt a Z conformation: regularly alternating purine-pyrimidine sequences, poly[d(A-C).poly[d(G-T)] ~6 and poly[d(A-T)]17; short oligomers containing AT base pairs, d(C2aminoACGTG), d(mSCGCAmSCGTGCG)) s and d(mSCGTAmsCG)19; and sequences containing GC base pairs out of regular purine-pyrimidine alternation, d(msCGGCmsCG), d(CBr8GGCBr8G), and d(CGCGC~). 2° A whole set of IR marker bands for GC and AT base pairs as well as the deoxyribose and the phosphate backbone in Z geometry has been obtained. The main IR Z-form marker bands are also summarized in Table I. Figure 8 presents the FT-IR spectra of the Z-conformations of three regularly alternating purine-pyrimidine polynucleotides. Main Z-form characteristic bands are dotted in Fig. 8. The incorporation of AT base pairs in a regularly alternating GC sequence makes the Z form of the nucleic acid more difficult to observe. In fact, for sequences such as poly[d(A-C)], poly[d(G-T)] and poly[d(A-T)] a simple decrease of hydration in the case of a film does not induce, as in the case of regularly alternating GC sequences, a B --, Z transition, but rather a B --, A transition. The Z form of these polynucleotides has been obtained by interactions with divalent metal ions, Ni 2+ and to a lesser extent Co 2+. We have shown by infrared spectroscopy that the hydrated nickel ions interact in a specific way with the N-7 groups of purines (guanines and/or adenines), stabilizing the syn geometry of the purine nucleosides and therefore the Z geometry of the DNA. 2~ This can be done using polynucleotides selectively deuterated on the C-8 H of the purine. A vibrational ~3E. Taillandier, J. A. Taboury, J. Liquier, P. Sautiere, and M. Couppez, Biochimie 63, 895 (1981). 14j. Pilet and M. Leng, Proc. Natl. Acad. Sci. U.S.A. 79, 26 (1982). 15j. A. Taboury, J. Liquier, and E. Taillandier, Can. J. Chem. 63, 1904 (1985). 16E. Taillandier, J. A. Taboury, S. Adam, and J. Liquier, Biochemistry 23, 5703 (1984). 17S. Adam, J. Liquier, J. A. Taboury, and E. Taillandier, Biochemistry 25, 3220 (1986). is j. A. Taboury, S. Adam, E. Taillandier, J. M. Neumann, S. Tran Dinh, T. Huynh Dinh, B. Langlois d'Estaintot, and J. Igolen, Nucleic Acids Res. 12, 6291 (1984). ,9 E. Taillandier, S. Adam, J. P. Ridoux, and J. Liquier, Nucleic Acids Res. 16, 5621 (1988). 20 S. Adam, J. A. Taboury, E. Taillandier, A. Popinel, T. Huynh Dinh, and J. Igolen, J. Biomol. Struct. Dyn. 3, 873 (1986). 21 S. Adam, P. Bourtayre, J. Liquier, and E. Taillandier, Nucleic Acids Res. 14, 3501 (1986).
322
SPECTROSCOPIC METHODS FOR ANALYSISOF DNA
[ 16]
N D O
W
om
g om
O 0
O ¢q
L
g,-
~o
, - /
FIO. 8. IR spectra of the Z form of regularly alternating purine-pyrimidine polynucleoi tides. (a) poly[d(G-C)]; (b) poly[d(A-C)]-poly[d(G-T)]; (c) poly[d(A-T)].
[ 16]
IR SPECTROSCOPYOF DNA
323
bending mode of the N-7 C-8 D group is sensitive to interactions on the N-7 site. When nickel is added to the DNA sample, shifts in the corresponding I R band, located around 1465 cm -1, reflect an interaction of the metal with the N-7 site. The coexistence of right-hand and left-hand geometries in the same molecule, for instance, in the case of B - Z junctions, can be studied using the IR marker bands of both conformations. 22,23 With a whole set of A, B, and Z characteristic IR absorptions, it is possible to study the conformational flexibility of an oligonucleotide sequence. For example, the d(CCGCGG) 24 sequence has been studied by FT-IR in solution and in crystal. Figure 9 presents the spectrum of the crystal (Fig. 9b) and of the solution form at low ionic strength (Fig. 9a) and high ionic strength (Fig. 9c). We clearly detect in spectra (Fig. 9a, c) the marker bands of the B form (1420, 1372, phosphate bands at 1220 and 1090 cm -1) and of the Z form (1410, 1360, 1320, 1264, phosphate bands at 1213, 1061, and 924 cm-~). In solution an increase in the ionic strength induces a complete B ~ Z transition of the sequence. The spectrum of the crystal, however, is quite different. It reflects neither a B nor a Z conformation. The deoxyribosebase geometry seems to be of the B type (1378 cm-I), whereas the phosphate-sugar backbone (924 cm -~), sugar (1413 cm -~), and phosphate group (1065 cm -1) vibrations reflect a Z form. The oligonucleotide seems to be trapped in an intermediate conformation. D N A Recognition Recognition in Major Groove by Specific Nucleic Acid Sequences: Triple-Strand Structures
The specific recognition of a predetermined DNA sequence in a longstrand double-helical polymer can be achieved by an oligonucleotide which forms a triple-strand structure. This is of particular interest in the case of oligonucleotides equipped with efficient DNA cleaving moieties at the 5' end. For example, metal chelates such as EDTA-Fe 25 or phenanthroline22S. Pochet, T. Huynh Dinh, J. M. Neumann, S. Tran Dinh, S. Adam, J. A. Taboury, E. Taillandier, and J. Igolen,NucleicAcids Res. 14, 1107(1986). 23S. Adam, J. P. Ridoux, P. Bourtayre, E. Taillandier, S. Pochet, T. Huynh Dinh, and J. Igolen, J. Biornol. Struct. Dyn. 6, 1 (1988). 24L. Urpi, J. P. Ridoux, J. Liquier, N. Verdaguer, I. Fita, J. A. Subirana, F. lglesias,T. Huynh Dinh, J. Igolen, and E. Taillandier, NucleicAcids Res. 17, 6669 (1989). 25H. E. Moserand P. B. Dervan, Science238, 654 (1987).
324
[ 16]
SPECTROSCOPIC M E T H O D S FOR ANALYSIS OF D N A
1
Q N N
C1
I11
W
M
O O
Fio. 9. Fr-IR spectra of d(CCGCGG). (a) Solution, low ionic strength, B form; (b) crystal, intermediate form; (c) solution, high ionic strength, Z form. (///) B-form bands; (dotted) Z-form bands.
[ 16]
IR SPECTROSCOPYOF DNA
325
copper~6 or eUipticin derivatives27 tethered to oligonucleotides might be used for sequence-specific recognition and cleavage of duplex DNA for chromosome analysis, gene mapping, and isolation via triple-helix formation. Interest in the study of the formation of triple helices also comes from the assumed existence of in vivo intramolecular triple-helical structures which may be formed by homopurine-homopyrimidine sequences. Such sequences are overrepresented in the eukaryotic genome and are often found within putative regulatory regions of genes and hot spots of recombination. A possible role for such triple helices in gene expression regulation has been proposed. 2s Infrared spectroscopy can be successfully used to detect the formation of the triple-strand structures. As noted previously, the spectral region between 1800 and 1500 cm -~ mainly contains absorptions due to double bonds of the bases in their planes. The positions and relative intensities of these absorptions are extremely sensitive to base pairing. The formation of a triple helix deeply modifies the base-base interaction schemes and is reflected in the IR spectrum in this region. Several base triplets may theoretically be formed. Figure 10 presents the characterization by VF-IR of three triplets, T-A-T on the left, C+-G-C in the middle, and G-G-C on the right. All spectra have been obtained in D20 solutions. For the T-A-T triplet, Fig. 10a presents the spectrum of double-strand poly(dA).poly(dT), and Fig. 10d that of triple-strand poly(dT).poly(dA)-poly(dT). The two spectra shown in Fig. 10b and 10c correspond to mixtures of poly(dA).poly(dT) with increasing amounts of an oligonucleotide, dT~0. An absorption located in the poly(dA).poly(dT) spectrum at 1622 cm -~ is assigned to an ND2 bending vibration coupled to a pyrimidic ring vibration of adenine, whereas the band located at 1641 cm -t corresponds to ring double-bond vibrations of thymines. When the amount of the third strand increases, these two bands progressively disappear and are no longer detected for the stoichiometric complex one double-strand poly(dA), poly(dT)-one singlestrand poly(dT), reflecting the formation of the triple helix. In the case of G-C base pairs, a triple-helical structure can be obtained starting with double-helical poly(dG).poly(dC) and a single poly(dC) strand, but only if the latter has been protonated. In this case a hydrogen bond can be established between the N-7 of the guanine initially involved 26 J.-C. Francois, T. Saison-Behmoaras, C. Barbier, M. Chassignol, N. T. Thuong, and C. Hrlrne, Proc. Natl. Acad. Sci. U.S.A. 86, 9702 (1989). 27 L. Perrouault, U. Asselin¢, C. Rivalle, N. T. Thuong, E. Bisagni, C. Giovannangeli, T. Le Doan, and C. Hrlrne, Nature (London) 344, 358 (1990). 28 R. D. Wells, D. A. Collier, J. C. Hanvey, M. Shimizu, and F. Wohlrab, FASEB J. 2, 2939 (1988).
326
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SPECTROSCOVIC METHODS FOR ANALYSISOF D N A
0
IR SPSCTROSCOPVOF DNA
307
[ 16] I n f r a r e d S p e c t r o s c o p y o f D N A By E. TAILLANDIERand J.
LIQUIER
Introduction Compared to other techniques some of the advantages of infrared spectroscopy in the study of nucleic acids are (1) the possibility of obtaining data from samples under an extremely wide variety of physical states (solutions, gels, hydrated fibers or films, and crystals); (2) no limitation introduced by the size of the investigated molecule, that is, one can work as well with short oligonucleotides as with long polynucleotides, DNA fragments obtained by enzymatic or chemical cleavage, or high molecular weight native DNAs. (3) Infrared spectroscopy is a nondestructive technique, which requires only small amounts of samples [a good Fourier transform infrared (FT-IR) spectrum can be obtained with 2 0 D units of material]. (4) It gives vibrational information characteristic of the helical conformations of the nucleic acids. (5) The comparison of FT-IR spectra obtained in solution and in crystals permits correlations with precise structures known from X-ray crystal diffraction studies as well as NMR solution studies. Different types of information can be obtained by IR spectroscopy. For example, it is possible to characterize molecular geometry using what is usually called IR "marker bands" which are conformation sensitive (wavenumber and/or relative intensity and/or polarization of the absorption). These bands can show the existence of a conformational transition when different factors such as temperature, hydration, concentration, nature, and amount of counterions are varied (B --~ A, B --* C, B -* Z, helix --~ coil, etc.). In the case of mixtures of conformations, computer simulations based on "perfect" pure form spectra are used to evaluate quantitatively the amount of each geometric form in the sample, by reference with these marker bands. Another type of information which can be obtained from the FT-IR spectra concerns recognition of DNA sequences by a wide variety of molecules, such as oligonucleotides (triple-helical structures), drugs and proteins. Although less commonly employed in studying nucleic acids than its "sister" technique, Raman spectroscopy, infrared spectroscopy has been successfully used since the early work of Fraser and Fraser ~ by several i M. J. Fraser and D. B. Fraser, Nature (London) 167, 759 (1951). 2 H. Falk, K. A. Hartman, and R. C. Lord, J. Am. Chem. Soc. 85, 391 (1963). 3 H. T. Miles and J. Frasier, Biochem. Biophys. Res. Commun. 14, 21 0964).
METHODS IN ENZYMOLOGY, VOL. 211
Copyright© 1992by AcademicPress,Inc. All rightsof reproductionin any form r'-~erved.
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SPECTROSCOPIC METHODS FOR ANALYSIS OF D N A
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g r o u p s . 2-7 In this chapter we shall briefly present a general survey of the
FT-IR spectrum of a nucleic acid, describe the characterization of the different classic families of DNA conformations (A, B, and Z), and then consider how FT-IR spectroscopy can be used in the study of DNA recognition, particularly in the case of triple-stranded structures and D N A - d r u g interactions. General Features of F T - I R Spectra of Nucleic Acids Nucleic acid vibrations arise from different parts of the macromolecule, and the corresponding IR absorptions are detected in several regions of the spectrum. The main vibrations are observed in the spectral range between 1800 and 700 cm -~, and four domains can be distinguished: between 1800 and 1500 cm -~ absorption bands due to the stretching vibrations of double bonds in the base planes; between 1500 and 1250 cm -~ bands due to the base-sugar entities, strongly dependent particularly on the glycosidic torsion angle; between 1250 and 1000 cm -I strong absorptions of the phosphate groups and of the sugar; below 1000 cm -~ bands due to the vibrations of the phosphodiester chain coupled to vibrations of the sugar. Figure la presents a DNA FT-IR spectrum recorded in H20 solution. The enlarged area shows the spectral domain corresponding to the vibrations of the phosphate groups. On such a spectrum we can observe an important contribution of the solvent (H20), which makes it difficult to obtain data in the region around 1600 cm -~ and below 1000 cm-L To avoid this solvent problem, the spectrum of the same molecule is recorded in D20 solution (Fig. lb). Now we can easily study the FF-IR spectrum in the previously mentioned regions (see enlarged insets, Fig. 1). The combination of H20/D20 solutions covers the complete spectral range necessary for nucleic a d d studies. If a DNA film is exposed to an atmosphere with controlled hydration obtained by using saturated solutions of known salts, the H20 contribution is less important than in the case of an aqueous solution. Careful treatment of data allows one to obtain an infrared spectrum even in the 18001500 cm -~ region. When the film is exposed to D20 vapor, H20/D20 exchange occurs, and this can be followed on the IR spectrum. Important
4 M. Tsuboi, in "Applied Spectroscopy Reviews" (E. G. Brame, ed.), Vol. 3, p. 45. Dekker, New York, 1969. 5 j. Pilet and J. Brahms, Biopolymers 12, 387 (1973). H. Fdtzsche, H. Lang, and W. Pohle, Biochim. Biophys. Acta 432, 409 (1976). 7 E. Taillandier, J, Liquier, and J. A. Taboury, in "Advances in Infrared and Raman Spectroscopy" (R. J. H. Clark and R. E. Hester, ecls.), Vol. 12, p. 65. Heyden, London, 1985.
[ 16]
IR SPECTROSCOPY OF D N A
309
!
O
b
f
O
8
O
\
FIO. 1. FT-IR spectra of DNA in solution. (a) H20 solution; (b) D20 solution. Enlarged parts of the spectra present the absorptions involving mainly the vibrations of the phosphate groups (top), the double bonds of the bases in their plane (bottom left), and the sugarphosphate backbone (bottom right).
310
SPECTROSCOPIC METHODSFORANALYSISOF DNA
[ 16]
modifications of the DNA spectrum can be observed that reflect the exchange of labile hydrogens of the bases, in particular the NH groups of thymines and the NH2 groups of adenines, guanines, and cytosines. Exchange ofhydrogens attached to C-8 of the purine base moieties is slow and observable only at elevated temperatures. In the solid state, DNA can be studied by IR spectroscopy of microcrystals. Microscopes especially designed for IR spectroscopy that contain mirror all reflecting Cassegrain type optics are now commercially available and can be coupled to FT-IR spectrophotometers. Such systems need a more sensitive detector, usually a nitrogen-cooled MCT detector. An example is given in Fig. 2, which presents the micro-FT-IR spectrum of
0
OD W 0
(0 N N
O~ O3
t~ tq
Flo. 2. Micro-Fl'-IRspectrumof a d(CGCGAATTCGCG)crystal.
[ 16]
IR SPECTROSCOPYOF DNA
311
d(CGCGAATTCGCG). In this case the crystal area analyzed by Fr-IR microspectroscopy is about 50 × 50 am. Characterization of Nucleic Acid Secondary Structures The main IR marker bands of A-, B-, and Z-DNA discussed below have been summarized in Table I. Polynucleotide and oligonucleotide conformations determined by IR spectroscopy are presented in Table II.
Right-Hand A- and B-DNA Conformations In low ionic strength solutions, native DNAs and most synthetic double-strand poly- and oligonucleotides adopt a B-form conformation. Under the same conditions double-stranded RNAs adopt an A-type conformation. Figure 3 presents the FT-IR spectra obtained in H20 solutions of two synthetic nucleic acids with regularly alternating A-U sequences, the first TABLE I MAIN INFRARED MARKER BANDS OF A-, B-, AND Z-DNA CONFORMATIONS
Conformation A
B
Z
Assignment
1705
1715
1418 1401 1375
1425
1695 1433 1408
In-plane base double bond stretching A-T bases Deoxyribose
1375 1355
1335 1335
1344 1328
1275
1281
1240 1188
1225
1265 1215
899 877 864 806
894
1065 929
1320
830-840
dA.dG anti dA.dG syn dT dA dG T G Antisymmetric phosphate stretching Deoxyribose Deoxyribose
Exact positions of bands are within - 2 cm-~ of these numbers, depending, in particular, on base sequence of DNA
T A B L E II CONFORMATIONS DETERMINED BY INFRARED SPECTROSCOPY a
Polymer Polynucleotides Native D N A
Poly[d(A-T).poly[d(A-T)]
Poly(dA).poly(dT)
Counterion
Na + Li + Na + Cs +, Li + N i 2+ Na +
poly(dT)
Na +
Poly(dA).poly(rU)
Na +
Poly(rA).poly(rU) Poly[d(2NH2A-T)].poly[d(2NH2A-T)]
Na ÷ Na +
Poly[d(A-C)].poly[d(G-T)]
Na +
Poly[d(G-C)].poly[d(G-C)]
Li + Ni 2+ Na ÷
Poly[d(m5C-G)]"poly[d(m5C-G)] Poly(dG).poly(dC)
M g 2+, Co 2+, Ni 2+ Na + Na +
Poly[d(I-Br5C)-poly[d(I-Br5C)]
Na +
Poly(rA).
Physical state
Form
S, F H FL FL S, F H
B A C B
FL
A
FL FH S, F H FL S, F H FL S FL S S low salt S high salt S, F H FL FH F FH, S low salt FL, S high salt FH F FH FL S low salt S high salt
C Z B H H A H A A B Z B A C Z B Z Z Z B+ A A B Z
Refs.
b b c
d,e d,e f d
e,g g h h h h h i i j j k j
l,m,n Lm, n o n p q q
Oligonucleotides d(CCGCGG)
Na + Na ÷ Na +
d(m5CGCGm5CG) d(m5CGGCm5CG) d(CBr8GCGCBr8G) d(CBr8~CBr8G) d(CCCGC~)
Na + Na + Na + Na + Na +
d(C2NI-12ACGTG)
Ni 2+ Na
d(m5CGCAm5CGTGCG)
Na
d(m5CGTAmSCG)
Na
d(CGGCCG) d(CGm5CGCG)
312
F F S low salt S high salt C F F F F FH FL F FH FL FH FL FH FL
Z Z B Z B-Z Z Z Z Z B A Z B Z B Z B Z
r s t t t r r u r v v v w w w w x x
TABLE II (continued) CONFORMATIONS DETERMINED BY INFRARED SPECTROSCOPYa
Polymer d(GGTATACC) d(CGCGAATTCGCG) d(ACATGT)
Counterion
d(AAAAATTTTT)
Mg 2+ Mg 2+ Na Ni 2+ Na +
r(A-U)6, r(A-U)s
Na +
Physical state C C F F FH FL S
Form B+ A B+ A B Z B H A
Refs. y p t,s t,s g g h
a S, Solution; FH, film high hydration; FL, film low hydration; F, film any hydration; C, crystal; and H, neteronomous. b j. filet and J. Brahms, Biopolymers 12, 387 (1973); H. Fritzsche, A. Rupprecht, and M. Rupprecht, Nucleic Acids Res. 12, 9165 (1984). ¢ J. Brahms, J. Pilet, P. L. Tran Thi, and R. L. Hill, Proc. Natl. Acad. Sci. U.S.A. 70, 3352 (1973). a S. Adam, J. Liquier, J. A. Taboury, and E. Taillandier, Biochemistry 25, 3220 (1986). e j. Pilet, J. Blicharski, and J. Brahms, Biochemistry 14, 1869 (1975). f S. Adam, P. Bourtayre, J. Liquier, J. A. Taboury, and E. Taillandier, Biopolymers 26, 251 (1987). g E. Taillandier, J. P. Ridoux, J. Liquier, W. Leupin, W. A. Denny, Y. Wang and W. L. Peticolas, Biochemistry 26, 3361 (1987). h j. Liquier, A. Akhebat, E. Taillandier, F. Ceolin, T. Huynh Dinh, and J. Igolen, Spectrochim. Acta 47A, No. 2, 177 (1991). F. B. Howard, C. W. Chen, J. S. Cohen, and H. T. Miles, Biochem. Biophys. Res. Commun. 3, 848 (1984). J E. Taillandier, J. A. Tahoury, S. Adam, and J. Liquier, Biochemistry 23, 5703 (1984). k D. H. Loprete and K. A. Hartman, J. Biomol. Struct. Dyn. 7, 347 (1989). E. Taillandier, J. A. Tahoury, J. Liquier, P. Sautiere, and M. Couppez, Biochimie 63, 895 (1981 ). " J. Pilet and M. Leng, Proc. Natl. Acad. Sci. U.S.A. 79, 26 (1982); P. B. Keller and K. A. Hartman, Nucleic Acids Res. 14, 8167 (1986). n j. A. Tahoury, J. Liquier, and E. TaiUandier, Can. J. Chem. 63, 1904 (1985). o j. A. Tahoury, P. Boutayre, J. Liquier, and E. Taillandier, Nucleic Acids Res. 12, 4247 (1984). P E. Taillandier, in "Structure and Methods" Vol. 3 "DNA and RNA" (R. H. Sarma and M. H. Sarma, eds.), p. 73. Adenine Press, 1990. q B. Hartman, J. Pilet, M. Ptak, J. Ramstein, B. Malfoy, and M. Leng, Nucleic Acids Res. 10, 3261 (1982). ' S. Adam, J. A. Taboury, E. Taillandier, A. Popinel, T. Huynh Dinh, and J. Igolen, J. Biomol. Struct. Dyn. 3, 873 (1986). s S. Pother, T. Huynh Dinh, J. M. Neumann, S. Tran Dinh, S. Adam, J. Tahoury, E. Taillandier, and J. Igolen, Nucleic Acids Res. 14, 1107 (1986). t L. Urpi, J. P. Ridoux, J. Liquier, N. Verdaguer, I. Fita, J. A. Subirana, F. Iglesias, T. Huynh Dinh, J. Igolen, and E. Taillandier, Nucleic Acids Res. 17, 6669 (1989). u j. A. Taboury, E. Taillandier, T. Huynh Dinh, andJ. Igolen, J. Biomol. Struct. Dyn. 2, 1185 (1985). E. Taillandier, W. L. Peticolas, S. Adam, T. Huynh-Dinh, and J. Igolen, Speetrochim. Acta 46, 107 (1990). w j. A. Tahoury, S. Adam, E. Taillandier, J. M. Neumann, S. Tran Dinh, T. Huynh Dinh, B. Langlois d'Estaintot, and J. Igolen, Nucleic Acids Res. 12, 6291 (1984). x E. Taillandier, S. Adam, J. P. Ridoux, and J. Liquier, Nucleic Acids Res. 16, 5621 (1988). r j. Liquier, E. Taillandier, W. L. Peticolas, and G. A. Thomas, J. Biomol. Struct. Dyn. 8, 295 (1990).
313
314
SPECTROSCOPIC METHODS FOR ANALYSIS OF D N A
C]
[ 16]
m ~.q
0
W i.q
-
N
t~
b
t#l tM
FIG. 3. FT-IR spectra of H20 solutions of d(A-U). (a) and r(A-U)8 (b). The enlarged area between 1350 and 1270 cm -I shows absorptions characteristic of A ( \ \ \ \ \ \ ) and B (/////) geometries.
[16]
IRsPECTROSCOPYOF DNA
315
one (Fig. 3a) with deoxy sugars, the second one (Fig. 3b) with ribo sugars: d(A-U)n and r(A-U)8. Besides the important modifications induced on the IR spectrum by the 0-2' deletion, differences between the spectra are due to the conformations adopted in solution by these two nucleic acids, namely, B and A. Some of the characteristic absorptions are shown hatched on Fig. 3, and the wavenumbers are given in Table I. In particular the phosphate antisymmetric stretching vibration is found at 1226 cm -~ in the B form and at 1245 cm -~ in the A form, and a band reflecting the base pairing of the nucleic acid is shifted from 1716 to 1712 cm -~. The A-DNA conformation can also be observed in DNAs by varying the water content of the nucleic acid sample. An easy way to achieve this is to decrease the relative humidity to which a DNA film is exposed. The decrease of the relative humidity (R.H.) from 100 to 58% R.H. will induce for some DNA sequences a B ---, A conformational transition, followed by subsequent disorganization of the double-helical structure if the hydration is further decreased. Some DNA sequences will not undergo this transition; for example, the A- conformation is not observed by decreasing the water content of a poly(dA), poly(dT) sample. On the contrary, the alternating polymer poly[d(A-T)] readily adopts this conformation, as can be seen in Fig. 4. We present in Fig. 4 a series of F r - I R spectra o f a poly [d(A-T)] film (1 Na + per phosphate counterion) exposed to decreasing relative humidifies. The displayed spectral region (1000-800 cm -~) contains several IR marker bands of the A and B geometries corresponding to vibrations of the phosphodiester chain coupled to deoxyribose vibrations. Both sets of characteristic bands have been shown hatched on Fig. 4. The B ~ A transition is reflected by the disappearance of the band located at 841 cm -~ and the emergence of a doublet at 880-865 cm -~ and of a band at 808 cm -t. The two A- and B-DNA conformations can also be characterized by a pattern of bands reflecting the vibrations of the bases coupled to vibrations of the sugar and located between 1250 and 1400 cm-L Figure 5 presents the FT-IR signatures of four double-stranded polynucleotides in this spectral region: from top to bottom poly(dA).poly(dT), poly(rA).poly(dT), poly(dA) • poly(rU), and poly(rA) •poly(rU). All spectra have been recorded in H20 solutions, and the marker bands for the A-form strands (riboses) and B-form strands (deoxyribose) are shown hatched (Fig. 5). During the B ---, A transition we observe the shift of a band involving the N-3 H bending vibration ofthymines (or uracils)from 1281 to 1275 cm -~ and the opposite displacements of an adenine band found at 1344 cm -~ and a thymine band found at 1328 cm -1 in the B form to lower and higher wavenumbers, respectively, around 1335 cm -~ in the A form. Another possibility for characterizing DNA conformations is the use of
316
SPECTROSCOPIC METHODSFOR ANALYSISOF DNA
[ 16]
¢11
,7
FIO. 4. B ---,A conformational transition ofpoly[d(A-T)] followed by FT-IR spectroscopy. The DNA film is exposed to decreasing relative humidities. (Top) B form, R.H. 100% (///). (Bottom) A form, R.H. 58% ( \ \ \ \ ) . Bands characteristic of both geometries have been hatched.
polarized infrared radiation. If the sample presents a preferential orientation axis (e.g., in the case of stretched films or fibers) the infrared spectra obtained with an incident radiation having an electric field E polarized parallelly or perpendicularly to the orientation axis of the molecule will be different. These dichroic spectra can be used to compute orientations of transition dipole moments in the molecule and in simple cases give information concerning geometric parameters) ,9 Such computations can be 8 D. B. Fraser, J. Chem. Phys. 21, 1511 (1953). 9 R. L. Rill, Biopolymers 11, 1929 (1972).
[ 16]
IR SPECTROSCOPYOF DNA
317
in
°Z,) N
FIG. 5. FT-IR spectra between 1400 and 1250cm-m of H20 solutions. (a) Poly(dA).poly(dT); (b) poly(rA).poly(dT); (c) poly(dA).poly(rU); (d) poly(rA).poly(rU). (I//I)B form; (\\\\)A form.
318
SPECTROSCOPIC METHODS FOR ANALYSIS OF D N A
[ 16]
:i
I\ CI
B "B
b
C ,
I
i
FIG. 6. Infrared polarized spectra of oriented films of poly[d(A-T)]. (a) 1 Na + per nucleotide, R.H. 5896, A form; (b) 1 Na + per nucleotide, R.H. 9896, B form; (c) 1 Cs+ per nuclt~tide, R.H. 7696, C form. ( m ) Electric vector of incident fight perpendicular to orientation axis; ( - - - ) electric vector of incident light parallel to orientation axis. I, Antisymmetric ohosohate stretching vibration; II, symmetric phosphate stretching vibration.
[16]
IR SPECTROSCOPYOF DNA
319
TABLE III POSITION AND POLARIZATIONOF PHOSPHATE GROUP VIBRATION ABSORPTION BANDS IN DEOXYRIBOPOLYNUCLEOTIDES
Antisymmetric stretching
Symmetric stretching
Conformation
v
Polarization
v
Polarization
A B C Z
1241 1225 1231 1215
± 0 II 0
1089 1089 1089 1090
II ± ± ±
performed on absorption bands involving the antisymmetric and symmetric motions of the phosphate groups. 5 Figure 6 gives an example of such polarized infrared spectra in the case of a poly[d(A-T)] film. I° The spectral region presented displays the strong absorptions of the phosphate groups. In Fig. 6 spectra (solid lines) have been obtained with polarization of the incident light perpendicular to the orientation axis, and spectra (dashed lines) have also been obtained with a parallelly polarized incident radiation. We can see the complete inversion of the polarization of the symmetric stretching vibration of phosphates (located around 1090 cm -l) when the polymer undergoes the B ---, A transition (Figs. 6a and 6b). Simultaneously the antisymmetric stretching vibration located around 1230 cm -I from nondichroic becomes perpendicularly polarized. In the case of the C form obtained in the presence of Li + or Cs ÷ this band becomes parallelly polarized (Fig. 6c). The results concerning these bands are presented in Table III. As previously stated in the introduction, these conformation-sensitive IR bands can be used to evaluate the amount of the two A and B helical geometries when both are present in the sample. Figure 7 presents the micro-FT-IR spectrum in the region of the phosphate-sugar backbone vibrations of a d(GGTATACC) crystal (Fig. 7b), along with two reference spectra of native DNA in the A (Fig. 7a) and B conformations (Fig. 7c). The crystal spectrum presents absorptions characteristic of both conformations (882, 861, and 812 cm -1 for the A form, 834 cm -I for the B form). The amount of B conformation has been estimated to be 14% and inter10 S. Adam, P. Bourtayre, J. Liquier, J. A. Taboury, and E. Taillandier, Biopolymers 26, 251 (1987).
320
[16]
SPECTROSCOPIC METHODS FOR ANALYSIS OF DNA
t~ tO 00 C¢
C= va
O0
b O1 tD
N
FIG. 7. FT-IR spectra of(a) A-form DNA (film at 58% R.H.); (b) d(GGTATACC) crystal; (c) B-form DNA (film at 98% R.H.). ( \ \ \ \ ) A-form bands; (////) B-form bands.
preted in agreement with a model in which one B-form octamer is trapped in a tunnel formed by six A-form octamers. 1t,~2 lIj. Doucet, J. P. Benoit, W. B. T Cruse, T. Prange, andO. Kennard, Nature(London)337, 190 (1989). ~2J. Liquier, E. Taillandier, W. L. Peficolas, andG. A. Thomas, Z Biomol. Strua. Dyn. 8, 295 (1990).
[16]
I R SPBCTROSCOPY Or D N A
321
Left-Hand Z-Conformation The first IR studies concerning left-hand conformations were devoted to the B --* Z reorientation of poly[d(G-C)] in films) T M In the presence of sodium counterions and in films this polymer undergoes a complete B ---, Z transition via decreasing the relative humidity. The marker bands of guanosines in a syn geometry were obtained first. Since then many other sequences have been shown to be able to adopt a Z conformation: regularly alternating purine-pyrimidine sequences, poly[d(A-C).poly[d(G-T)] ~6 and poly[d(A-T)]17; short oligomers containing AT base pairs, d(C2aminoACGTG), d(mSCGCAmSCGTGCG)) s and d(mSCGTAmsCG)19; and sequences containing GC base pairs out of regular purine-pyrimidine alternation, d(msCGGCmsCG), d(CBr8GGCBr8G), and d(CGCGC~). 2° A whole set of IR marker bands for GC and AT base pairs as well as the deoxyribose and the phosphate backbone in Z geometry has been obtained. The main IR Z-form marker bands are also summarized in Table I. Figure 8 presents the FT-IR spectra of the Z-conformations of three regularly alternating purine-pyrimidine polynucleotides. Main Z-form characteristic bands are dotted in Fig. 8. The incorporation of AT base pairs in a regularly alternating GC sequence makes the Z form of the nucleic acid more difficult to observe. In fact, for sequences such as poly[d(A-C)], poly[d(G-T)] and poly[d(A-T)] a simple decrease of hydration in the case of a film does not induce, as in the case of regularly alternating GC sequences, a B --, Z transition, but rather a B --, A transition. The Z form of these polynucleotides has been obtained by interactions with divalent metal ions, Ni 2+ and to a lesser extent Co 2+. We have shown by infrared spectroscopy that the hydrated nickel ions interact in a specific way with the N-7 groups of purines (guanines and/or adenines), stabilizing the syn geometry of the purine nucleosides and therefore the Z geometry of the DNA. 2~ This can be done using polynucleotides selectively deuterated on the C-8 H of the purine. A vibrational ~3E. Taillandier, J. A. Taboury, J. Liquier, P. Sautiere, and M. Couppez, Biochimie 63, 895 (1981). 14j. Pilet and M. Leng, Proc. Natl. Acad. Sci. U.S.A. 79, 26 (1982). 15j. A. Taboury, J. Liquier, and E. Taillandier, Can. J. Chem. 63, 1904 (1985). 16E. Taillandier, J. A. Taboury, S. Adam, and J. Liquier, Biochemistry 23, 5703 (1984). 17S. Adam, J. Liquier, J. A. Taboury, and E. Taillandier, Biochemistry 25, 3220 (1986). is j. A. Taboury, S. Adam, E. Taillandier, J. M. Neumann, S. Tran Dinh, T. Huynh Dinh, B. Langlois d'Estaintot, and J. Igolen, Nucleic Acids Res. 12, 6291 (1984). ,9 E. Taillandier, S. Adam, J. P. Ridoux, and J. Liquier, Nucleic Acids Res. 16, 5621 (1988). 20 S. Adam, J. A. Taboury, E. Taillandier, A. Popinel, T. Huynh Dinh, and J. Igolen, J. Biomol. Struct. Dyn. 3, 873 (1986). 21 S. Adam, P. Bourtayre, J. Liquier, and E. Taillandier, Nucleic Acids Res. 14, 3501 (1986).
322
SPECTROSCOPIC METHODS FOR ANALYSISOF DNA
[ 16]
N D O
W
om
g om
O 0
O ¢q
L
g,-
~o
, - /
FIO. 8. IR spectra of the Z form of regularly alternating purine-pyrimidine polynucleoi tides. (a) poly[d(G-C)]; (b) poly[d(A-C)]-poly[d(G-T)]; (c) poly[d(A-T)].
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bending mode of the N-7 C-8 D group is sensitive to interactions on the N-7 site. When nickel is added to the DNA sample, shifts in the corresponding I R band, located around 1465 cm -1, reflect an interaction of the metal with the N-7 site. The coexistence of right-hand and left-hand geometries in the same molecule, for instance, in the case of B - Z junctions, can be studied using the IR marker bands of both conformations. 22,23 With a whole set of A, B, and Z characteristic IR absorptions, it is possible to study the conformational flexibility of an oligonucleotide sequence. For example, the d(CCGCGG) 24 sequence has been studied by FT-IR in solution and in crystal. Figure 9 presents the spectrum of the crystal (Fig. 9b) and of the solution form at low ionic strength (Fig. 9a) and high ionic strength (Fig. 9c). We clearly detect in spectra (Fig. 9a, c) the marker bands of the B form (1420, 1372, phosphate bands at 1220 and 1090 cm -1) and of the Z form (1410, 1360, 1320, 1264, phosphate bands at 1213, 1061, and 924 cm-~). In solution an increase in the ionic strength induces a complete B ~ Z transition of the sequence. The spectrum of the crystal, however, is quite different. It reflects neither a B nor a Z conformation. The deoxyribosebase geometry seems to be of the B type (1378 cm-I), whereas the phosphate-sugar backbone (924 cm -~), sugar (1413 cm -~), and phosphate group (1065 cm -1) vibrations reflect a Z form. The oligonucleotide seems to be trapped in an intermediate conformation. D N A Recognition Recognition in Major Groove by Specific Nucleic Acid Sequences: Triple-Strand Structures
The specific recognition of a predetermined DNA sequence in a longstrand double-helical polymer can be achieved by an oligonucleotide which forms a triple-strand structure. This is of particular interest in the case of oligonucleotides equipped with efficient DNA cleaving moieties at the 5' end. For example, metal chelates such as EDTA-Fe 25 or phenanthroline22S. Pochet, T. Huynh Dinh, J. M. Neumann, S. Tran Dinh, S. Adam, J. A. Taboury, E. Taillandier, and J. Igolen,NucleicAcids Res. 14, 1107(1986). 23S. Adam, J. P. Ridoux, P. Bourtayre, E. Taillandier, S. Pochet, T. Huynh Dinh, and J. Igolen, J. Biornol. Struct. Dyn. 6, 1 (1988). 24L. Urpi, J. P. Ridoux, J. Liquier, N. Verdaguer, I. Fita, J. A. Subirana, F. lglesias,T. Huynh Dinh, J. Igolen, and E. Taillandier, NucleicAcids Res. 17, 6669 (1989). 25H. E. Moserand P. B. Dervan, Science238, 654 (1987).
324
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Q N N
C1
I11
W
M
O O
Fio. 9. Fr-IR spectra of d(CCGCGG). (a) Solution, low ionic strength, B form; (b) crystal, intermediate form; (c) solution, high ionic strength, Z form. (///) B-form bands; (dotted) Z-form bands.
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copper~6 or eUipticin derivatives27 tethered to oligonucleotides might be used for sequence-specific recognition and cleavage of duplex DNA for chromosome analysis, gene mapping, and isolation via triple-helix formation. Interest in the study of the formation of triple helices also comes from the assumed existence of in vivo intramolecular triple-helical structures which may be formed by homopurine-homopyrimidine sequences. Such sequences are overrepresented in the eukaryotic genome and are often found within putative regulatory regions of genes and hot spots of recombination. A possible role for such triple helices in gene expression regulation has been proposed. 2s Infrared spectroscopy can be successfully used to detect the formation of the triple-strand structures. As noted previously, the spectral region between 1800 and 1500 cm -~ mainly contains absorptions due to double bonds of the bases in their planes. The positions and relative intensities of these absorptions are extremely sensitive to base pairing. The formation of a triple helix deeply modifies the base-base interaction schemes and is reflected in the IR spectrum in this region. Several base triplets may theoretically be formed. Figure 10 presents the characterization by VF-IR of three triplets, T-A-T on the left, C+-G-C in the middle, and G-G-C on the right. All spectra have been obtained in D20 solutions. For the T-A-T triplet, Fig. 10a presents the spectrum of double-strand poly(dA).poly(dT), and Fig. 10d that of triple-strand poly(dT).poly(dA)-poly(dT). The two spectra shown in Fig. 10b and 10c correspond to mixtures of poly(dA).poly(dT) with increasing amounts of an oligonucleotide, dT~0. An absorption located in the poly(dA).poly(dT) spectrum at 1622 cm -~ is assigned to an ND2 bending vibration coupled to a pyrimidic ring vibration of adenine, whereas the band located at 1641 cm -t corresponds to ring double-bond vibrations of thymines. When the amount of the third strand increases, these two bands progressively disappear and are no longer detected for the stoichiometric complex one double-strand poly(dA), poly(dT)-one singlestrand poly(dT), reflecting the formation of the triple helix. In the case of G-C base pairs, a triple-helical structure can be obtained starting with double-helical poly(dG).poly(dC) and a single poly(dC) strand, but only if the latter has been protonated. In this case a hydrogen bond can be established between the N-7 of the guanine initially involved 26 J.-C. Francois, T. Saison-Behmoaras, C. Barbier, M. Chassignol, N. T. Thuong, and C. Hrlrne, Proc. Natl. Acad. Sci. U.S.A. 86, 9702 (1989). 27 L. Perrouault, U. Asselin¢, C. Rivalle, N. T. Thuong, E. Bisagni, C. Giovannangeli, T. Le Doan, and C. Hrlrne, Nature (London) 344, 358 (1990). 28 R. D. Wells, D. A. Collier, J. C. Hanvey, M. Shimizu, and F. Wohlrab, FASEB J. 2, 2939 (1988).
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