NMR Spectroscopy - American Chemical Society

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P N M R Studies of D N A Conformation and

Downloaded by CALIFORNIA INST OF TECHNOLOGY on March 23, 2018 | https://pubs.acs.org Publication Date: July 9, 1982 | doi: 10.1021/bk-1982-0191.ch013

Dynamics JACK S. COHEN and CHI-WAN CHEN National Institutes of Health, National Institute of Child Health and Human Development, Developmental Pharmacology Branch, Bethesda, MD 20205 31

P NMR i s a valuable tool in elucidating the conformational and dynamic properties of DNA in solution. Recent work on natural DNA and synthetic polydeoxynucleotides of specific sequence are described* It i s advantageous to study medium length DNA derived either from nucleosomes (145 base p a i r s ) or from sonication of synthetic polymers, i n order to resolve sharper s i g n a l s . The P NMR c h a r a c t e r i s t i c s of a l t e r n a t i n g B-DNÂ and Z-DNA are delineated and compared. 31

Different conformations of DNA in fibers have been known for some time (1). However a l l of the models o r i g i n a l l y proposed had uniform backbone conformations with mononucleotide repeat units. Evidence for the existence of variant structures of DNA with non-uniform backbone conformations has become available onTy comparatively r e c e n t l y . This evidence has derived mainly from X-ray studies on oligonucleotide c r y s t a l s ( 2 , 3 ) , and from NMR studies of DN As i n solution ( 4 , 5 , 6 ) . These variant structures have altered our view of double stranded DNA as a r i g i d rod in which only the base-sequence v a r i e s . While Watson-Crick base p a i r i n g is considered to be common to a l l these structures, the new features are not only the handedness, but the concept that the base sequence determines the conformation of the phospho-diester backbone. The c r y s t a l structure of d(AT) showed an alternating sugar-phosphate backbone conformation, although the molecules were not h e l i c a l l y arranged (2). The c r y s t a l structures of d( CG ) (3) and d(CG) (7,8) exhibited an a l t e r n a t i n g backbone conformation with a left-handed sense. These observations were made in the s o l i d - s t a t e on small oligonucleotide crystals, and X-ray fiber d i f f r a c t i o n patterns also indicate the formation of a left-handed variant structure for some sequences in DNA fibers ( 9 ) · Observation of the t r a n s i t i o n of r i g h t - to left-handed °NA -ill s o l u t i o n . however, requires a spectroscopic technique. C i r c u l a r dichroism (CD) i s sensitive to the B- to Z-DNA t r a n s i t i o n , as o r i g i n a l l y observed by Pohl and Jovin i n 1972 for poly(dGdC )#poly(dGdC ) in 2

3

This chapter not subject to U.S. copyright. Published 1982 American Chemical Society. Levy; NMR Spectroscopy: New Methods and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

2

N M R SPECTROSCOPY

250 0 1

high salt c o n c e n t r a t i o n s (Η>>· P NMR s p e c t r o s c o p y is emerging as an important technique for the elucidation of the s t r u c t u r e s and dynamics o f these c o n f o r m a t i o n a l v a r i a n t s o f DNA i n s o l u t i o n . Z-DNA

to

The t r a n s i t i o n from presumed right-handed Β-DNA presumed l e f t - h a n d e d Z-DNA i n s o l u t i o n has been "3 1


w A

m o n i t o r e d by P NMR f o r d(GC ) i n oligonucleotides (6), in 145 bp m a t e r i a l d e r i v e d from h i stone complexation and nuclease cleavage ( XX ) and in sonicated polymeric m a t e r i a l ( 1 2 ) . T h i s i s shown i n F i g . 1 ( a l l P NMR s p e c t r a are p r o t o n d e c o u p l e d ) , in which the Z-form i s represented by the d o u b l e t o f s h a r p e r peaks o f a p p r o x i m a t e l y equal areas i n hi.^h salt solution. These peaks r e p r e s e n t the a l t e r n a t i n g n a t u r e o f the GpC and CpG backbone c o n f o r m a t i o n s ( F i g . 2). The DNAs were s o n i c a t e d to provide sharper signals than i n the polymeric synthetic starting materials (5). It was shown t h a t these s o n i c a t e d p r o d u c t s save the same s a l t - i n d u c e d t r a n s i t i o n in CD spec t ra ( 12 ). It i s noteworthy t h a t peaks broadened and signal intensity was lost i n these s p e c t r a as a r e s u l t o f extensive aggregation at high salt concentrations. Attempts to follow this transition i n high Mg * c o n c e n t r a t i o n s or i n e t h a n o l s o l u t i o n s resulted in precipitation of the p o l y n u c l e o t i d e s a t the high concentrations (ca 1 rag/ml ) r e q u i r e d f o r P NMR observation. By c o n t r a s t CD s p e c t r a can be o b t a i n e d on much lower c o n c e n t r a t i o n s of m a t e r i a l . T h i s d i f f i c u l t y may be surmountable i n view of the f i n d i n g of Behe and F e l s e n f e l d ( 1 3 ) t h a t 5-methyl c y t i d i n e s u b s t i t u t e d f o r C i n d( GC) causes the salt c o n c e n t r a t i o n o f the m i d p o i n t o f the Β t o Ζ t r a n s i t i o n to be g r e a t l y r e d u c e d . T h i s has e n a b l e d us t o follow t h i s t r a n s i t i o n a t low s a l t c o n c e n t r a t i o n s by P NMR, w i t h no a g g r e g a t i o n o r p r e c i p i t a t i o n (JL£)« The lack of aggregation i s shown by the absence of line broadening. Two s i g n a l s of approximate a r e a 1:2 were observed on a d d i t i o n of 0.8 M NaCl and three P NMR s i g n a l s o f a p p r o x i m a t e l y e q u a l a r e a were o b s e r v e d at 5raM Mg concentration. The c h e m i c a l s h i f t o f the c e n t r a l peak appears t o be e q u i v a l e n t to t h a t of the s t a r t i n g m a t e r i a l , and t h i s t h i r d component might have been p r e s e n t i n the non-methylated copolymer but obscured by line broadening ( 1 2 ) , a l t h o u g h i t was a p p a r e n t l y absent i n the o t h e r m a t e r i a l i n the Z-form 3 1

2

3 1

3 1

3 1

ρ •

(6,11). The complexation of Mg with the p h o s p h o d i e s t e r s i n the Z' form d e s c r i b e d by Wang et aJL ( 1 5 ) might be expected to g i v e r i s e t o t h i s s h i f t e d phosphodiester s i g n a l . We a r e c u r r e n t l y a t t e m p t i n g t o c l a r i f y t h i s phenomenon. Allernatlng_B-DNA from

The sharp t r a n s i t i o n of d(GC) and i t s d e r i v a t i v e s Β t o Z-DNA i n h i g h s a l t , and the l a r g e s e p a r a t i o n

Levy; NMR Spectroscopy: New Methods and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 1982.


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6

Figure 1. Salt dependence of P NMR spectra at 109.3 MHz of sonicated poly(dGdC) - poly(aGaC) at 22°C, pH 6.5. Salt concentration was increased by adding crystalline NaCl. The doublet in the high form arises from an alternating structure, presumed to be Z-DNA (12). The size distribution of sonicated samples was determined by gel electrophoresis. 31


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Figure 2.

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Stereo view of the structure of Z-DNA (3). This structure is not necessarily the same as that present in solution.


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of t h e s i g n a l s i n t h e P NMR spectra e n a b l e the Z-form to be r e a d i l y characterized by P NMR. O b s e r v a t i o n o f a l t e r n a t i n g B-DNA i s l e s s easy. The f i r s t evidence f o r the e x i s t e n c e o f two p h o s p h o d i e s t e r c o n f o r m a t i o n s i n p o l y ( d A d T ) * p o l y ( dAdT) i n s o l u t i o n was o b t a i n e d w i t h c a 145 bp m a t e r i a l derived from histone complexation and n u c l e a s e d i g e s t i o n ( F i g . 3) ( 5 ) . However, t h e s e p a r a t i o n o f the two components was o n l y 22 Hz i n t h i s c a s e . This s e p a r a t i o n c o u l d not be due t o t h e s i m p l e e f f e c t o f the differences i n the adenine and thymine bases because o f t h e i r d i s t a n c e s from t h e phosphorus atoms (5). C o n s e q u e n t l y t h e two components were a t t r i b u t e d to ApT and TpA a l t e r n a t i n g conformations ( F i g . 4 ) . In longer chain polymeric m a t e r i a l s these two r e s o n a n c e s c o u l d n o t be r e s o l v e d due t o b r o a d e r NMR signals (5). As mentioned e a r l i e r , the c r y s t a l structure of d(AT) also exhibited alternating backbone c o n f o r m a t i o n s (jj>), which c o u l d be extended i n the form of a double h e l i x t o s o - c a l l e d a l t e r n a t i n g B-DNA ( 1 6 ) . F u r t h e r e v i d e n c e f o r the a l t e r n a t i n g structure i n fibers o f d(AT )»d(AT ) was o b t a i n e d from s o l i d s t a t e P ΝMR s p e c t r a (17), which also showed a distinct d o u b l e t i n the p a r a l l e l o r i e n t a t i o n ( F i g . 5 ) . Similarly polyidAdT)·poly( dAdT ) p r e p a r e d by sonication also exhibited the doublet spectrum i n solution, as did sonicated p o l y ( dAdbr U )*poly( d A d b r U ), although p o l y ( dGdC )#poly( dGdC ) and p o l y ( dAdu )*polydAdU ) d i d n o t do so ( F i g . 6> (12). The Ρ NMR d o u b l e t was observed i n unmeited s o n i c a t e d samples as w e l l as i n 145 bp m a t e r i a l . A f u r t h e r i n d i c a t i o n that t h e two peaks r e p r e s e n t d i s t i n c t c o n f o r m a t i o n s i s t h a t they give s l i g h t l y d i f f e r e n t Ύ values ( I S ) . J 1


3 1

2

3 1

5

5

3

1

χ

That the f o r m a t i o n o f the a l t e r n a t i n g structure is completely r e v e r s i b l e has been shown f o r d( AT ) < 12,19) and f o r d( A b r U ) ( F i g . 7) ( 1 2 ) . In the l a t t e r case the d o u b l e t a l s o r e v e r s l b l y c o l l a p s e d t o a singlet at temperatures below c a 25*C. T h i s may indicate the n o n - a l t e r n a t i n g B-form i s p r e f e r e n t i a l l y s t a b i l i z e d a t lower temperature. However, i n the p h y s i o l o g i c a l temperature and s a l t c o n c e n t r a t i o n range d(AT ) does e x h i b i t t h e a l t e r n a t i o n . The C NMR spectrum o f 145 bp d(AT ), which exhibits d o u b l e t s f o r the C ' and C ' carbons ( F i g . 8> (20). This clearly extends the backbone a l t e r n a t i o n t o the sugar components and i n d i c a t e s t h a t the sugar a l t e r n a t i o n ( C ' endo, C ' endo ) seen i n the crystal of d( AT ) ( 2 ) , as well as that of d i G C ) ^ ^ ( 3 » Z , £ ) . maybe p r e s e r v e d i n s o l u t i o n . While p o l y ( d A d T ) does n o t c o n v e r t i n t o the Z-form in high s a l t , a s y s t e m a t i c change d i d o c c u r on a d d i t i o n of NaCl ( F i g . 9) ( 1 2 ) . An almost identical spectrum i n 1 M tetramethyiammoniura c h l o r i d e t o t h a t i n 1M NaCl has a l s o been o b s e r v e d i n a n independent study (21). The r e a s o n that the d o u b l e t was n o t r e s o l v e d i n 1M o r l o w e r NaCl concentrations i n this study presumably results from the higher molecular w e i g h t m a t e r i a l used, which g i v e s b r o a d e r s i g n a l s * In 5

l 3

2

3

3

2

2


NMR

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SPECTROSCOPY

j


CSF (Ml

I—•— —»—I—•—'—•—I—•— -2 -4 - 6 PPM 1

Figure 3. Temperature dependence of the Ρ NMR spectrum at 109.3 MHz of 145 bp poly(aAdT) hairpins in 10 mM tris-Cl (pH 8.0, 0.1 M NaCl, and 1 mM EOT A). (Reproduced, with permission, from Ref. 5. Copyright 1979, American Society of Biological Chemists.) 31



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Figure 4. Stereo view of structures of B-DNA and alternating B-DNA (16), superimposed as far as possible. The alternating structure is not necessarily the same as that present in solution.


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SPECTROSCOPY

Figure 5. Proton dipolar decoupled and cross polarized Ρ NMR spectra (24.3 MHz) at 25°C of poly(dAdT) · poly(dAdT) fibers (right) and salmon sperm DNA fibers (left). The top spectra are from the fibers oriented perpendicular to the mag netic field in a teflon holder, and the bottom spectra are from the parallel orienta tion. Humidity was controlled. (Reproduced, with permission, from Ref. 17. Copyright 1980, Macmillan.) 31



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Figure 6. P NMR spectra at 109.3 MHz of sonicated alternating purine-pyrimidine hairpin duplexes. Key: a, poly(dAdT), 30°C; b, polyfdAdbr^U), 31°C; c, poly(dAdU), 31°C; d, poly(dldC), 39°C; and e, poly(dGdC), 23°C; all in 0.1 M NaCl, 0.05 mM EDTA, pH 6-7 except b in 5 mM tris-Cl, 0.1 mM EDTA (12). 31

2.5

Figure 7.

3.5

4.5 ppm

5.5

6.5

2.5

3.5

4.5 ppm

5.5

6.5

Temperature dependence of the Ρ NMR spectra of sonicated (3 h) polyfdAdb^U) in 5 mM tris-Cl, 0.1 mM EDTA, (pH 6.8) (12). 31


Levy; NMR Spectroscopy: New Methods and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 1982. 13

Figure 8. Proton decoupled C NMR spectra at 67.9 MHz of 145 bp hairpinned poly(dAdT) (12 mg in 1 mL) at 32 °C. Peaks marked with a and b are from residual EDTA and ethanol. Total acquisition time, 62 h. Note the doubling of the C2' and C3' resonances (2§).


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[NaCl]

1

I

2

4

I 6

ppm Figure 9. Salt dependence of P NMR spectra at 109.3 MHz of sonicated (3 h) poly(dAdT), 22°C, pH 6.8. Crystalline NaCl was added to the desired concentration^). 31


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our work on s o n i c a t e d m a t e r i a l i t Is c l e a r t h a t the smaller size and narrower d i s t r i b u t i o n ( 2 2 ) g i v e superior resolution ( F i g . 10). A new conformational variant of poly(dAdT )·ροΙν( dAdT) has a l s o been c l a i m e d i n CsF s o l u t i o n from NMR ( 2 3 ) and CD r e s u l t s (24). Also, Patel e t a.1 ( 2 5 ) have i n t e r p r e t e d t h e e f f e c t s o f CsF to i n d i c a t e a f a s t n o n - c o o o e r a t i v e t r a n s i t i o n between a low s a l t form h a v i n g a s i n g l e t * P resonance and a h i g h s a l t form w i t h a d o u b l e t . While we agree that there i s such a t r a n s i t i o n , i t i s c l e a r from o u r r e s u l t s that CsF i n c r e a s e s the s e p a r a t i o n o f the doublet ( F i g . 11) which i s r e s o l v e d i n low s a l t f o r s m a l l e r s i z e d m a t e r i a l ( 5 , 1 2 , 1 8 ) . We a l s o attribute this phenomenon t o a g r a d u a l change i n the degree o f w i n d i n g o f the DNA double h e l i x ( 2 6 ) , and i n the case of p o l y ( d A d T ) . p o l y ( d A d T ) a l l forms a r e a l t e r n a t i n g o r zig-zag s t r u c t u r e s (18). From the s e p a r a t i o n o f the two components i t i s clear that interconversion between the two conformers r e p r e s e n t e d by the d o u b l e t would be v e r y slow ( r a t e

NMR Spectroscopy - American Chemical Society

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