gkf601 4740..4750 - BHAnar02.pdf

H NMR determination of base-pair lifetimes in

oligonucleotides containing single base mismatches

Pratip K. Bhattacharya, Julie Cha and Jacqueline K. Barton*

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA

Received June 5, 2002; Revised and Accepted September 10, 2002

ABSTRACT

Proton nuclear magnetic resonance (NMR) spectro-

scopy is employed to characterize the kinetics of

base-pair opening in a series of 9mer duplexes con-

taining different single base mismatches. The imino

protons from the different mismatched, as well

as fully matched, duplexes are assigned from the

imino-imino region in the WATERGATE NOESY

spectra. The exchange kinetics of the imino protons

are measured from selective longitudinal relaxation

times. In the limit of inÆnite exchange catalyst con-

centration, the exchange times of the mismatch

imino protons extrapolate to much shorter lifetimes

than are commonly observed for an isolated GC

base pair. Different mismatches exhibit different

orders of base-pair lifetimes, e.g. a TT mismatch has

a shorter base-pair lifetime than a GG mismatch.

The effect of the mismatch was observed up to a

distance of two neighboring base pairs. This indi-

cates that disruption in the duplex caused by the

mismatch is quite localized. The overall order of

base-pair lifetimes in the selected sequence context

of the base pair is GC > GG > AA > CC > AT > TT.

Interestingly, the fully matched AT base pair has a

shorter base-pair lifetime relative to many of the

mismatches. Thus, in any given base pair, the

exchange lifetime can exhibit a strong dependence

on sequence context. These Ændings may be

relevant to the way mismatch recognition is

accomplished by proteins and small molecules.

INTRODUCTION

DNA mismatches, or non-complementary base pairs, arise

in vivo

as a result of the misincorporation of bases during

replication (1), heteroduplex formation during homologous

recombination (2), mutagenic chemicals (3,4), ionizing radi-

ation (5) and spontaneous deamination (6). These errors are

usually detected and eliminated by DNA polymerase and

postreplicative mismatch repair system (7±9). How these

DNA mismatches are detected by the repair machinery of the

cell requires an understanding at the molecular level.

Therefore, it is useful to characterize the structure, dynamics

and biochemistry of various mismatched base pairs in DNA

and to determine how they affect the structure of the double

helix in terms of both global and local perspectives.

The structures of several DNA duplexes containing mis-

matched base pairs have been characterized by X-ray

crystallography (10±12) and NMR methods (13±16). In all

of these structural studies, the mismatches were shown to have

minimal effect on the global conformation of the DNA; the

distortions produced are limited to the mismatched site and

neighboring base pairs.

H NMR studies show that the

mismatches GG (17±19), AA (20±22), TT (20±22), CC

(23,24), GA (25±28) and GT (29) are well stacked in the

helix and the bases remain in an intrahelix orientation. In fully

base-paired right-handed B-form DNA duplexes, there are

NOEs evident between base protons (H8 or H6) and the 5

Øanking sugar H1

and H2

¢¢

protons, allowing an NOE walk

from the 5

to the 3

end of the oligonucleotide. In the case of

these mismatched duplexes, the NOE walk is conserved, again

supporting the notion that the mismatches are inserted and

stacked well between the Øanking base pairs, and the

oligonucleotides adopt the classical B form duplex with

minimal local disruption. Additionally,

P NMR studies

support a B

conformation (17,19,20,23).

Much less is known about the dynamic properties of the

mismatched bases in the DNA duplex and about any possible

role of dynamics in mismatch recognition. The bases in DNA

move rapidly within the double helix, undergoing thermally

driven structural Øuctuations in solution. Since base motions

occur within a multidimensional potential well, determined by

a combination of base-stacking and base-pairing forces, it is

reasonable to expect that the motions of a mismatched base

pair in DNA should be different from that of the fully matched

pair; this dynamic difference may inØuence the interactions of

mismatched base pairs with repair enzymes. Furthermore, the

dynamics of mismatches, as well as fully matched base pairs,

may play a pivotal role in modulating charge transport through

DNA, which is a topic of considerable current interest

(30±38).

The dynamics of mismatched duplexes have been the focus

of spectroscopic studies by Millar and co-workers (39). Time-

resolved Øuorescence anisotropic decay measurements were

obtained for a series of oligonucleotides containing interven-

ing AP¥X base pairs, where AP is the Øuorescent adenine

analog 2-aminopurine, and X = A, T, G or C. This technique

allowed the detection of base motions in DNA on the

picosecond timescale. Motions such as helical twisting,

propeller twisting, base tilting and base rolling could poten-

tially alter the emission dipole of AP, thereby contributing to

*To whom correspondence should be addressed. Tel: +1 626 395 6075; Fax: +1 626 577 4976; Email: jkbarton@caltech.edu

4740±4750

Nucleic Acids Research, 2002, Vol. 30 No. 21

„

2002 Oxford University Press

changes in the decay of the Øuorescence anisotropy. AP pairs

differently with each of the different bases and these

differences in its relative pairing ability were reØected in the

internal dynamics.

A complementary method of probing base-pair dynamics is

through

H NMR studies of imino proton exchange rates.

Clearly, such studies probe base-pair motions on a much

slower timescale. The imino protons of the aromatic

heterocyclic base exchange with the solvent protons when

the hydrogen bond in the base pair is disrupted (40,41). The

chemical proton transfer step from the open state is usually

rate limiting, and a proton acceptor, the base catalyst, must be

added to the solution in order to accelerate the exchange close

to opening-limited conditions. Base-pair lifetimes are then

obtained by extrapolation of the exchange times to inÆnite

catalyst concentration, where the dissociation constant of a

base pair is estimated by comparing the exchange rates of the

imino proton in the base pair and in the mononucleoside. The

unknown factor in this comparison is the accessibility of the

imino proton in the open base pair, which is related to

properties of the open state. Measurements of the NMR

relaxation rates of the imino protons as a function of solvent

exchange have yielded lifetimes in the range of 1±40 ms for

fully paired bases in B-DNA (42). In general, AT base-pair

lifetimes have been found to be in the range of 1±5 ms at 15

∞

except for AT tracts where lifetimes >100 ms have been

observed (43). For GC base pairs, lifetimes ~10

longer than

for AT base pairs have been observed (42), as one might

expect, given the presence of an additional hydrogen bond in

the GC base pair. However, it is important to note that the

sequence composition (43,44), the charged state of the double

helix (45) and drug interactions (46) all serve to modulate

base-pair dynamics sensitively.

Here, we report a systematic study of the dynamics of single

base mismatches within DNA duplexes, through the measure-

ments of imino proton exchange. Using

H NMR, we have

determined the base-pair lifetimes of different mismatches in a

given sequence context and have compared the exchange

times with those of fully matched base pairs. We observe

strong sequence dependence of the base-pair opening times

and that rates are increased at the mismatched site and in the

directly neighboring site. These results underscore the

importance of DNA sequence and of sequence context in

governing base dynamics.

MATERIALS AND METHODS

Oligonucleotide preparation

Oligonucleotides were synthesized using standard phosphor-

amidite chemistry on an Applied Biosystems 392 DNA

synthesizer with a dimethoxy trityl protecting group on the

end (47). Oligonucleotides were puriÆed on a reversed-

phase Rainin Dynamax C

column on a Waters HPLC and

then deprotected by incubation in 80% acetic acid for 15 min.

After deprotection, the oligonucleotides were puriÆed again

by HPLC. Following puriÆcation, these oligonucleotides were

desalted on a Waters C

SepPak column and then converted

to a sodium salt using CM Sephadex C-25 (Sigma)

equilibrated in NaCl. The concentration of the oligonucleo-

tides was determined by UV-visible spectroscopy (Beckman

DU 7400) using the extinction coefÆcients estimated for

single-stranded DNA:

(260 nm, M

±1

) adenine (A) =

15 400; guanine (G) = 11 500; cytosine (C) = 7400 and

thymine (T) = 8700. Single strands were mixed with

equimolar amounts of complementary strand and annealed

using a Perkin Elmer Cetus Thermal Cycler by gradual

cooling from 90

∞

C to ambient temperature in 90 min. The

NMR samples were prepared by dissolving the oligonucleo-

tides in a buffer solution (5 mM Na

HPO

, 15 mM NaCl,

pH 7.0). The concentrations of the samples varied between 0.5

and 1.2 mM duplex.

Melting temperature experiments

The melting temperatures of the oligonucleotides were

determined from absorbance versus temperature curves

measured at 260 nm on a Beckman DU 7400 UV-visible

spectrophotometer. Ten micromolar duplex was used in a

buffer of 5 mM Na

HPO

, 15 mM NaCl, pH 7.0. The melting

proÆle of the duplexes was obtained by slowly lowering the

temperature (0.5

∞

C/min) from 75

∞

Cto10

∞

C and measuring

the absorbance at 260 nm at each temperature. The

values

represent the midpoint of the transition as obtained by Ætting

the melting proÆles with a sigmoidal expression in Origin.

NMR spectroscopy

One- and two-dimensional

H NMR spectra were taken in

both D

O and 90/10 H

O/D

O at 277 K on a Varian INOVA

600 MHz spectrometer. For the spectra taken in 90/10 H

O, WATERGATE gradient pulse water suppression (48)

was used. The conditions of acquiring the spectra were:

600 MHz, 20 p.p.m. sweep width, TPPI, 2048 complex points,

hypercomplex mode, 256 t

blocks, 64 scans per t

block, 1.3 s

relaxation delay, 120 ms mixing time. ROESY spectra (49,50)

were obtained at variable mixing times of 80, 150 and

300 ms keeping the other parameters the same as for the

WATERGATE NOESY experiments with the r.f. strength for

the spin-lock Æeld set at 3.5 kHz. Linear prediction was

employed in the indirect dimension to increase resolution

close to the diagonal. All two-dimensional processing was

carried out with VNMR 6.1b software (Varian) on a

SUN workstation. Chemical shifts were reported relative to

the internal standard, sodium 3-trimethylsilyl-[2,2,3,3-D

]

propionate (TMSP).

Imino proton exchange measurements

Imino proton exchange within the DNA duplex occurs through

the opening of the base pair followed by exchange from the

open state. The conformational features of this state are not yet

fully understood. To account for proton exchange data, it is

generally assumed that, in the open state, the imino groups are

fully accessible to solvent and the base-pairing hydrogen

bonds are broken such that the imino proton becomes

available for hydrogen bonding with water or catalyst

molecules (40). The chemical proton transfer step from the

open state is rate limiting, and a proton acceptor like OH

must

be added to accelerate the exchange close to opening-limited

conditions. The base-pair lifetimes are obtained by extra-

polation of the exchange times to inÆnite base catalyst

concentration.

[B]

cl/

[B]

Nucleic Acids Research, 2002, Vol. 30 No. 21

4741

For a base pair with multiple open states, formed with rates

and closed with rates

, and provided that

, º.,

, the total imino proton exchange rate

equals

the sum of the exchange rates from each mode (40). To

simplify, where

is, as above, the intrinsic imino proton

transfer rate from the mononucleoside and

is a parameter

reØecting the different accessibility of the imino proton in the

open states and in the mononucleoside and [B] denotes the

concentration of the exchange catalyst, OH

in this case, then

for a base pair with a single opening mode (

= 1) and with

, this can be written as:

+1/

[B]

where

and

are the inverse exchange and opening rates,

respectively, and

is the base-pair dissociation

constant. A plot of

versus 1/[B] yields a straight line where

is obtained from the

-axis intercept and

from the

slope. The limit where equation

is valid is known as

Linderstr ̆m±Lang kinetics (51).

In this work, the oligonucleotides were titrated using

ammonia (4 M ammonia, 1.25 M ammonium chloride,

pH 10.0; Ricca Chemical Co.) as the exchange catalyst. The

oligonucleotide concentrations of the samples varied between

0.5 and 1.2 mM duplex, and the oligonucleotide sample was

buffered with 5 mM Na

HPO

, 15 mM NaCl, pH 7.0. The

imino proton exchange times (

) at different catalyst

concentrations were obtained from measurements of the

inversion recovery times in the presence (

rec

) and absence

(

aac

) of exchange catalyst according to the equation

=1/

rec

±1/

aac

Except for longitudinal dipolar relaxation, direct exchange to

water as well as exchange catalyzed by OH

ions and the

acceptor nitrogen of the opposite base contributes to the

recovery rate of the imino protons in the absence of added

catalyst 1/

aac

. However, these contributions remain constant

when the catalyst is added and will be canceled in equation

Consequently, the exchange time

represents exchange only

via the added catalyst. Thus, effectively, through measure-

ments of the longitudinal relaxation times,

, for various

imino protons as a function of catalyst concentration, we can

determine the imino exchange rates within the oligonucleotide

duplex.

The experiment was carried out by creating a designed

pulse sequence comprised of a 3.2 ms 180

∞

Gaussian pulse

(g3) for selective inversion, a variable delay, and a 5 ms 90

∞

Gaussian observe pulse (g4). Right shift and linear prediction

of the free induction decay are employed to correct for

magnetization evolution during the observed pulse. The

carrier frequency was centered on the imino proton region.

The spectral width was 7000 Hz and the experiments were

carried out at 600 MHz.

RESULTS AND DISCUSSION

Sequence design

The general 9mer oligonucleotide sequence d(GACA

T-

GTC)

; where

= G, T, A, C, was employed to determine the

base-pair lifetimes for DNA duplexes containing different

central single base mismatches (Table 1). A palindromic

sequence was utilized that is self-complementary except at the

mismatch site (X). This simpliÆes the

H NMR spectra and

facilitates the assignment of the protons. Table 1 also shows

the melting temperatures for the different duplexes and

demonstrates that duplexes containing mismatches are

thermodynamically destabilized as compared with the

matched pairs.

For the mismatched duplexes, only one strand was used,

resulting in a duplex with either AA, TT, CC or GG mismatch

at the central site (X); to obtain a complementary base pair at

site X, however, two different strands had to be used. This led

to the possibility of the formation of a mixture of matched and

mismatched duplexes as two separate strands were added to

form the fully matched GC and AT sequences. Given the

melting temperatures, that possibility was minimized by slow

cooling during hybridization of the duplexes and repeated

annealing of the sample to allow nucleation. In the case of the

AT-matched duplex, no resonances corresponding to the

imino protons of mismatched base pairs were observed. For

the GC matched duplex only a small fraction was observed

(see below), conÆrming the isolation of duplexes with

primarily Watson±Crick base pairs. The 9mer duplex was

also chosen in part to preserve the sequence used in guanine

oxidation studies reported elsewhere (38). The possibility

of hairpin formation is negligible with 9mer duplexes.

Moreover, non-denaturing agarose gel electrophoresis with

the mismatch-containing duplexes indicated no formation of

hairpins in these duplexes (data not shown).

Imino proton resonance assignment

The imino proton resonances of the different oligonucleotides

were assigned primarily from the imino-imino crosspeaks in

the WATERGATE NOESY spectra in 90:10 H

O/D

solution (Table 2). The B-form structure of the DNA duplex

constrains the position of imino protons in the duplex to be

3.4 A

above or below one another; in the fully matched

Table 1.

The sequences and the melting temperatures (

) of the duplexes

containing a single base mismatch (XY) used in the NMR experiments

Duplexes

(

∞

5

-GACA

G

TGTC-3

34.9

3

-CTGT

G

ACAG-5

5

-GACA

C

TGTC-3

24.5

3

-CTGT

C

ACAG-5

5

-GACA

A

TGTC-3

32.1

3

-CTGT

A

ACAG-5

5

-GACA

T

TGTC-3

32.4

3

-CTGT

T

ACAG-5

5

-GACA

G

TGTC-3

47.8

3

-CTGT

C

ACAG-5

5

-GACA

A

TGTC-3

44.1

3

-CTGT

T

ACAG-5

These sequences represent the six oligonucleotides employed for the imino

exchange experiments. The single base mismatched or matched base pairs

are highlighted in bold.

Shown are the melting temperatures (

) of the duplexes determined as

described in Materials and Methods. Samples include 10

M duplex in a

buffer of 5 mM Na

HPO

, 15 mM NaCl, pH 7.0. The order of the melting

temperature varies as GC > AT > GG > TT ~ AA > CC.

4742

Nucleic Acids Research, 2002, Vol. 30 No. 21

duplex, each base pair contains one imino proton (N

Hin

thymine and N

H in guanine). These interactions between the

imino protons lead to crosspeaks in the imino-imino region of

the two-dimensional NOESY spectra of the DNA duplexes,

which can be exploited for the assignment of these protons.

Figure 1 shows the exchangeable imino region (9±

15 p.p.m.) of the one-dimensional

H NMR spectrum for

sequences containing GG, AA, TT, CC mismatches and for

the fully matched sequences containing AT and GC base pairs.

For all of the oligonucleotides under study, one observes three

main sets of imino protons: (i) thymine imino protons in the

range of 14.3±13.4 p.p.m., (ii) guanine imino protons mostly

in the range 12.8±12.4 p.p.m. and (iii) a set of imino protons

from the mismatched sites (GG and TT) in the range 10.2±

10.7 p.p.m.. These shifted resonances at the mismatch site are

more exposed and subject to exchange with solvent than those

of the stacked base pairs. Nonetheless, most of the imino

protons are quite well resolved. As is evident in Table 2, for all

of the duplexes, the chemical shifts of the outer imino protons

(G9N

H, T8N

H and G7N

H) remain quite close to each

other. This similarity in chemical shift is consistent with

previous observations that perturbations caused by the

mismatches are localized, limited essentially to the Øanking

bases (10±12).

The line-widths of the imino proton(s) for the different

duplexes containing mismatches clearly differ depending

upon the base composition of the mismatch. TT and CC

mismatch-containing duplexes exhibit larger line-widths as

compared with GG and AA duplexes. This variation in line-

width correlates with the relative stability of the duplexes;

such broad line-widths for duplexes containing TT and CC

mismatches have also been seen previously (24).

A representative imino-imino region in the NOESY spectra

of the duplex containing a GG mismatch is shown in Figure 2.

Since the imino protons are stacked one above another, the

interactions among them lead to a NOE walk of the imino-

imino cross peaks along the length of the oligonucleotide.

Furthermore, the two imino protons from the mismatched

guanine (GG) base pair are positioned in close proximity to

each other and exhibit a strong NOE build up between them

(G5/G5

). The peak splitting for the G5 imino proton reØects

an asymmetry in conformation for the two Gs at the mismatch

site. One G of the GG mismatch has been seen to adopt the

syn

conformation with the alternate G in the

anti

form, with slow

exchange on the NMR time scale (17±19). This asymmetry at

the mismatch site extends out to other sites within the duplex;

peak splitting is observed not only for G5, but also for T6 and

G7 imino resonances. These peaks can easily be assigned from

the NOESY spectra.

The TT mismatch is known to be in intermediate exchange

between two asymmetric wobble structures involving two

imino to carbonyl hydrogen bonds (20±22), and we also

observe two peaks for T5 with a faster rate of exchange. Note

that one of the T5 peaks is much bigger than the other (T5

)

indicating that one of the conformations exchanges with water

at a faster rate relative to the other. For the TT mismatch-

containing duplex, the asymmetry can be seen to a small

extent on T6. Interestingly, for the CC mismatch-containing

duplex, a similar asymmetry is evident in the shifts for T6;

since the CC mismatch lacks imino protons, no asymmetry can

be detected directly at the mismatch site. This has been further

corroborated by the presence of a cross peak for the T6N

proton in the same phase as the diagonal peak in the two-

dimensional ROESY spectra of the TT and CC duplexes (data

Table 2.

The chemical shifts

(p.p.m.) of the imino proton resonances in the oligonucleotides containing the

intervening mismatches (XY)

Base pairs

X/Y5NH

(p.p.m.)

T6NH

(p.p.m.)

G7NH

(p.p.m.)

T8NH

(p.p.m.)

G9NH

(p.p.m.)

G¥G

10.65

13.76

12.70

13.92

12.71

10.22

13.46

12.47

13.89

T¥T

10.72

14.25

12.44

14.03

12.79

10.41(sm)

13.87(sm)

C¥C

14.20

12.54

14.04

12.80

13.87(sm)

A¥A

13.55

12.46

13.92

12.76

G¥C*

13.54

13.81

12.57

13.97

12.78

A¥T*

13.72

14.01

12.47

13.85

12.79

13.42

13.96

12.41

123456789

5

-GACA

X

TGTC-3

where

X, Y

=A,T,G,C

3

-CTGT

Y

ACAG-5

Samples for NMR studies are prepared as described in Materials and Methods and the proton chemical shifts

are relative to TMSP (0.00 p.p.m.). The samples were at a concentration of 0.5±1.2 mM duplex and taken in a

buffer solution of 5 mM Na

HPO

, 15 mM NaCl, pH 7.0 in 90:10 H

O/D

Designation of XY as shown in assembly.

The imino proton attached to the central mismatched base pair is denoted by X/Y5NH. The assignment of

the imino protons was accomplished from the imino-imino cross peaks of the WATERGATE NOESY spectra

of the oligonucleotides in 90:10 H

O/ D

O as described in Results and Discussion.

Because of the symmetry of the duplexes and the fact that each base pair can have only one imino proton

(except the mismatched pairs), imino protons at G1, T2, G3 and T4 sites are usually equivalent to that of G9,

T8, G7 and T6, respectively. The presence of the two values of the chemical shift reØects the loss of this

symmetry.

XY* corresponds to the Watson±Crick paired sequence.

Nucleic Acids Research, 2002, Vol. 30 No. 21

4743

not shown), which indicates chemical exchange involving that

imino proton. A combination of NOESY and ROESY spectra

can thus be used to distinguish between cross-relaxation and

chemical exchange (49). Note that our assignments of the

imino protons of the GG and TT pair are in close agreement

with those observed for related 9mers containing GG and TT

mispairs (24).

The one-dimensional spectrum for the AA mismatch-

containing duplex is relatively simple and is also easily

assigned from the imino-imino NOESY spectra. Only four

resonances from the four matched base pairs are evident. This

observation must reØect a primarily symmetric positioning of

the As within the mismatch site.

Also shown are the one-dimensional spectra for the fully

matched duplexes. For the GC duplex, the G5 imino is now

revealed at the expected shift for a matched base pair. In the

case of the GC duplex, there are also very small peaks evident

at 13.46, 10.65 and 10.22 p.p.m.; we attribute these to the

formation of a very small amount of GG mismatch-containing

duplex during the hybridization process. Interestingly, sharper

resonances are evident for the AT matched oligomer com-

pared with the GC matched duplex. In addition, in the case of

the AT matched duplex, peak doubling is evident for most of

the resonances, consistent with the symmetry breaking by the

TA base pair at the central site. Only one resonance at each

position is apparent for the matched GC oligomer; perhaps this

Figure 1.

One-dimensional

H NMR spectra of the exchangeable imino region (9±15 p.p.m.) of the oligonucleotides with intervening GG, TT, AA, CC mis-

matches and the fully matched AT and GC base pairs at 600 MHz. Samples contained 0.5±1.2 mM oligonucleotides, 5 mM Na

HPO

and 15 mM NaCl buffer

at pH 7.0 in 90:10 H

O/D

O at 277 K. The peaks are assigned from the respective imino-imino region of the two-dimensional NOESY spectra. Chemical

shifts are reported relative to TMSP (0.00 p.p.m.). The chemical shifts of the individual imino resonances in all the duplexes are given in Table 2.

4744

Nucleic Acids Research, 2002, Vol. 30 No. 21

results from a more central positioning of the G5N

H within

the duplex.

Imino proton exchange

The exchange times of the imino protons were obtained

through the measurement of the inversion recovery times of

the NMR. The addition of an exchange catalyst, NH

, yields,

in the limit of inÆnite catalyst concentration, the kinetic

parameters for the base-pair opening (equation

). Upon

titration with base, the general observation was broadening

and the gradual disappearance of the imino proton resonances

as the exchanges times became too fast to be observed on the

NMR timescale. Figure 3 shows the variation in the one-

dimensional spectrum for the GG mismatch-containing duplex

as a function of increased base concentration. Different imino

protons are seen to broaden out at differing levels of base

concentration. For example, G7N

H and T6N

H broaden out

at a much slower rate compared with the imino protons G9 and

G5. This is consistent with the fact that the outermost protons

(G9N

H and T8N

H), as well as the protons at the mismatch

site (G5N

H), are undergoing exchange with solvent at a faster

rate than the other internally positioned imino protons and,

hence, the fast relaxation is observed. The line-width of the

G9N

H imino proton is relatively broad compared with the

other imino protons even before the addition of the catalyst

due to fraying of the end base positions; that the imino

resonance for the end position is seen at all is somewhat

surprising.

In Figure 4, the exchange times at 277 K of the imino

protons of the base pairs of the oligonucleotide containing the

GG mismatch are displayed as a function of the inverse

ammonia concentration. The exchange times display the linear

dependence on the inverse base concentration, as expected

from equation

. Similar plots were drawn for the other

mismatches (Supplementary Material) and these plots were

found to be linear in all cases. Therefore, imino proton

exchange in these duplexes containing single base mismatches

does follow Linderstr ̆m±Lang kinetics (51). The base-pair

lifetimes,

, obtained from the linear Æts are given in Table 3.

Base-pair lifetimes

Fast exchange rates and therefore short base-pair lifetimes are

observed for the imino protons directly at the mismatch sites

as compared with the Watson±Crick base pair GC (

G5N

H=18

4 ms). This observation supports the fact that

the mismatches produce local destabilization in the helical

structure of the duplex leading to dynamics for the mismatches

that are different from those of the standard Watson±Crick

base pairs. Furthermore, the

of the GG mismatch site is

appreciably longer than that of the TT mismatch, 2 ms for

Figure 2.

Expanded imino-imino region of the two-dimensional NOESY contours in 90:10 H

O/D

O for the duplex d(GACA

GTGTC)

containing an inter-

vening GG mismatch. Solution is 1 mM in DNA duplex, 5 mM Na

HPO

and 15 mM NaCl buffer at pH 7.0. Spectrum is acquired with a mixing time of

120 ms and at 277 K and collected at 600 MHz. The details of the NOESY experiment are outlined in Materials and Methods. Chemical shifts are reported

relative to TMSP (0.00 p.p.m.). The lines illustrate the NOE walk along the imino protons, which are stacked above or below one another (at a distance of

3.4 A

) along the oligonucleotide.

Nucleic Acids Research, 2002, Vol. 30 No. 21

4745

G5N

H in the GG pair, compared with 0.5 ms for T5N

Hin

the case of TT. The value of

obtained for the GG mismatch

is similar to values previously reported for a GT mismatch

(52).

The effect of the mismatches also propagates out to the base

pairs adjacent to it, T6N

H and G7N

H, such that each duplex

can be distinguished by its differential kinetics. As seen in the

chemical shift changes, the dynamic effects of the mismatches

are mostly localized in the vicinity of the mismatch site. This

Ænding is consistent with the crystallographic studies, which

show that the conformational differences between the normal

B-form and the mismatched DNA are small and affect solely

the local environment of the mismatched site (10±12). This

observation is also consistent with previous

H NMR studies

of DNA mismatches (16,28,52,53).

In fact, in the case of the duplexes containing CC and AA

mismatches, the base-pair lifetime at the mismatch site clearly

cannot be determined since there are no imino protons at the

mismatch site to measure the relaxation times. In those cases,

to compare the lifetimes with the other mismatches, the

neighboring imino protons (T6N

H) to the mismatch site were

considered.

The exchange times of T5

H of the TT mismatched

duplex and T6

H of the CC duplex could not be determined

due to very fast exchange and the rapid disappearance of the

peaks on the Ærst addition of the exchange catalyst. This

observation is consistent with the very short lifetime of the TT

mismatched base pair (

= 0.5

0.3 ms) obtained

independently from the other imino proton (T5N

H) at the

mismatch site. Unusually fast exchange is also observed at

= 0.5

0.2 ms) of TT duplex, providing evidence

of fast exchange occurring around the vicinity of the

mismatch.

In terms of overall kinetics, the duplex containing the GG

mismatch displays the longest base-pair lifetime, followed by

AA, CC and then TT with the shortest lifetime. We believe

that the short base-pair lifetimes reØect an increased lateral

motion of the base pair, leading to an increased disruption of

the

stacked array. This disruption affects not only the imino

protons at the mismatch site, but also the imino protons up to

two neighboring base pairs away from the mismatch. In all of

the sequences under study, the outermost GC and AT base

pairs exhibit similar base-pair lifetimes.

Matched sequence

The base-pair lifetimes in the fully matched GC-containing

oligonucleotide are comparable to the values reported pre-

viously for fully matched duplexes (42). However, quite

contrary to our expectation, the base-pair lifetime for the AT

base pair in the sequence context of 5

-A

T-3

was found to

be much shorter than AT base pairs in other sequence contexts

(42,43) and even shorter than many mismatches (

of T5N

0.4 ms). Interestingly, the Øanking base pairs of the

central AT base pair also show unusually rapid lifetimes as

compared with the fully matched GC sequence. Note the very

fast lifetime of the G7 base pair (

of G7N

H=3

1 ms) in

this sequence. Also note that, in this given AT duplex,

different AT base pairs show different lifetimes. The one

Øanked by GC base pairs on both sides exhibits the longest

lifetime (

of T8N

H=12

3 ms) which can be rationalized

based on the fact that the GC base pairs can provide better

stacking for the AT base pair. It is apparent that the value of

the base-pair lifetimes depends upon the sequence context, as

the sequence context imposes different local conformational

Øexibilities on the duplex DNA. These structural variations

are reØected in the base-pair opening dynamics.

This observation is particularly interesting because it shows

that the dynamics of a fully matched base pair in a given

sequence context can be comparable to a mismatched site even

though this may not be reØected in the melting temperatures.

Thus, an individual Watson±Crick base pair within a DNA

helix may not necessarily be the most stable and well stacked.

The AT base pair in general has a longer base-pair lifetime

(>1 ms but <5±6 ms) under other sequence contexts (44). In

Figure 3.

The titration of 1 mM d(GACA

GTGTC)

with 4 M ammonia

buffer in 90:10 H

O/D

O followed by

H NMR. Shown is the imino proton

region of the spectra taken at 600 MHz and 277 K. Conditions are 1 mM

DNA duplex, 5 mM Na

HPO

and 15 mM NaCl buffer and 4 M in the

titrant ammonia buffer. Chemical shifts are reported relative to TMSP

(0.00 p.p.m.). [B] denotes the concentration of the ammonia buffer in the

sample solution at different points of the titration.

4746

Nucleic Acids Research, 2002, Vol. 30 No. 21

contrast, long tracts of AT base pairs show very long base-pair

lifetimes (>100 ms) (43). The alternating sequence 5

-TATA-

is also known to be particularly Øexible (54). Thus, the base-

pair lifetime for a given base pair depends sensitively upon the

sequence context and composition. A systematic study of this

effect is in progress. It is interesting to note that we have also

Figure 4.

Exchange of the imino protons of the duplex d(GACA

GTGTC)

containing the intervening GG mismatch versus the inverse of the concentration of

ammonia catalyst. The exchange times (

) of (A) G7N

H, (B) G9N

H, (C) G5/G5

H, (D) T6N

H and (E) T8N

H imino protons are individually displayed

as a function of inverse ammonia concentration (1/[B]). The data points used in the drawing of these lines are normalized based on at least three trials

. The

straight lines are obtained by Ætting to equation

, with the exchange times weighted according to their errors. The corresponding base-pair lifetimes (

)

thereby obtained from extrapolation are displayed in Table 3. Two independent line Ættings are accomplished for the imino proton at the mismatch site

(G5/G5

H).

Nucleic Acids Research, 2002, Vol. 30 No. 21

4747

seen evidence of the local Øexibility of this sequence in studies

of long-range DNA-mediated charge transport (38).

Internal dynamics of mismatched and matched base

pairs

One expects that the imino proton of a Watson±Crick base pair

(e.g. a GC pair) within a double helix cannot exchange on a

rapid timescale, since neither solvent nor catalyst has access

to the proton. Exchange, therefore, proceeds via three

consecutive steps: (i) base-pair opening, whose rate is 1/

followed by (ii) proton exchange from the open state and

(iii) closing the base pair (55):

GH*C

GH* + C

(i)

(ii)

(iii)

The base-pair lifetimes of the mismatches are found in general

to be shorter than the Watson±Crick base pairs. Indeed in

many cases, no `opening' of the mismatched base pair is

required. This means that the lifetime of the mismatches in the

closed state is greatly reduced relative to that of the normal

Watson±Crick base pairs. It has been shown (52) that ~0.1% of

the GT mismatches are in the open solvent-accessible state

and GT is one of the more thermodynamically stable

mismatches. The mismatches, in general, are more accessible

to solvent and other extraneous molecules than the

Watson±Crick bases. This may have relevance to how the

mismatches are recognized by the mismatch repairing proteins

and how the mismatches may act as `hot spots' in reactivity

studies (56).

The imino exchange experiments measure primarily the

lateral motions of the base pairs. These opening motions may

occur by different pathways and may be coupled to bending

(57±60), which in turn affects the stacking of the base pairs.

Stacking is mostly a `vertical' effect along the axis of the

duplex while the opening motions are `horizontal' effects.

However, these two are clearly related, possibly one motion

reinforcing the other. Molecular dynamics (MD) simulations

have been performed on the lateral motion of duplexes

containing mismatches (61). Consistent with the short lifetime

of mismatches, opening Øuctuations in MD runs are larger at

mismatched base pairs 5±7

∞

, than fully matched CG, 4

∞

.It

should, however, be emphasized that, by measuring the base-

pair lifetime, we are essentially quantifying only one type of

dynamic motion occurring in the DNA duplex. Mismatches

can bring about changes in a variety of other internal motions

in DNA like helical twisting, propeller twisting, base tilting,

base rolling, etc. and these internal motions are not reØected in

the study of base-pair lifetimes, although they may be related.

CONCLUSIONS

A systematic study of the base-pair dynamics of mismatch-

containing oligonucleotides has been carried out using imino

exchange measurements. We have observed very fast base-

pair opening rates in the mismatch site, indicative of the local

distortion generated around the mismatch. Some mismatches

generate more distortion than others, and these differences are

captured in the faster base-pair lifetimes. A relative ordering

of the mismatches with respect to the base-pair lifetime has

been achieved. In general, the mismatches are found to be

kinetically destabilized relative to the Watson±Crick base

pairs. However, certain Watson±Crick base pairs, under a

given sequence context, may behave very much like a

Table 3.

The base-pair lifetimes

(

) of the oligonucleotides containing intervening mismatch (XY)

(ms)

Base pair

T5NH

G5NH

T6NH

G7NH

T8NH

G9NH

G¥G

124

515

0.9

T¥T

0.5

0.3

110

314

0.5

0.2

C¥C

XX10

312

A¥A

XX15

312

310

210

G¥C*

X18

412

318

511

215

A¥T*

0.4

112

312

123456789

5

-GACA

XTGTC-3

where

Y=A,T,G,C

3

-CTGT

YACAG-5

Samples for the determining the base-pair lifetime were prepared and the experiments based on imino proton

exchange were performed as described in Materials and Methods. The samples were at a concentration of

0.5±1.2 mM duplex and taken in a buffer solution of 5 mM Na

HPO

, 15 mM NaCl, pH 7.0 in 90:10 H

Shown are the mean and standard deviation of the base-pair lifetime values (

) of the mismatches/base

pairs (T5/G5NH) and their Øanking base pairs based on at least three trials.

Designation of XY as shown in assembly.

Two independent values of the base-pair lifetimes at the mismatch site are obtained for the duplexes

containing GG mismatch for each trial.

In the case of TT mismatch, two different lifetimes are obtained for the T6NH proton (Results and

Discussion).

Since only guanine and thymine have imino protons, no base-pair lifetimes are obtained for CC and AA

duplexes directly at the corresponding mismatch sites.

The base-pair lifetime corresponding to the T6

NH of the CC mismatch could not be determined because of

very fast relaxation on the addition of base.

XY* corresponds to the Watson±Crick paired sequence.

4748

Nucleic Acids Research, 2002, Vol. 30 No. 21

mismatch in terms of base-pair opening dynamics. The faster

dynamics associated with the mismatches and with some

Watson±Crick base pairs requires consideration in how these

sites may be recognized and repaired by proteins.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at NAR Online. Figures

depicting the exchange of the imino protons of the duplexes

containing the intervening AA, CC, TT, mismatch and the

Watson±Crick bases GC and AT base pairs versus the inverse

of the concentration of ammonia catalyst are given in Figures

S1±S5.

ACKNOWLEDGEMENTS

We thank Dr Scott Ross and Helen Chuang, a summer

undergraduate research fellow, for technical assistance. We

are also grateful to the National Institutes of Health

(GM33309) for their Ænancial support of this work.

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