Guanine-uracil base-pairing

Vol. 46, No. 4, 1972

BIOCHEMICAL

AND BIOPHYSICAL

GUANINE-URACIL Sunney I. Chan,

Institute

BASE-PAIRING

Gregory

A. A. Noyes Laboratory

RESEARCH COMMUNICATIONS

C. Y. Lee,

Charles

F. Schmidt

of Chemical

Physics,*

California

California

91109

of Technology,

Pasadena, and

George ICN Nucleic 2727 Campus Received

January

P. Kreishman

Acid Research

Drive,

Irvine,

Institute

California

92664

10, 1972

The interaction of guanosine and 2’-deoxyuridine has been by high resolution pmr spectroscopy in DMSO-water mixtures. is presented for G-U base-pairing in solvent mixtures where the water content is sufficiently high. Downfield shifts were observed for the N(i)-H, NH, protons of G and the N t3)-H proton of U, suggesting that the complex formation involves three hydrogen-bonds and that the G base is pairing in the lactim-amino tautomeric structure. No evidence for G-U base-pairing in the “wobble” as well as other pairing schemes was obtained.

==-Y examine Evidence

The gnanine dominantly showed,

base in nucleic

in the lactam-amino however,

the abnormal

tautomeric

la&m-amino

species

erism.

be quite slow,

resonance

and the kinetics

The rate of interconversion and was dependent

In a recent paper, percentages

in aqueous solution

proton magnetic

both the equilibrium

is known to exist pre-

structure.

that it also exists in appreciable

Using high resolution termined

acid components

(10-X0/,)

at room

(pmr)

of this lactam-lactim

on the temperature

was found to

and pD of the solution.

exchange was shown to give rise to the guanine

broadening

observed

In its normal

lactam-amino

(C) to form the well-known

* Contribution Copyright

in the pmr spectra tautomer,

Watson-Crick

No. 4403.

0 1972, by Academic

Press, Inc.

1536

we detautom-

This slow tautomeric frequently

as

temperature.

spectroscopy,

between the two tautomers

we

of guanine

guanine

Hts)

derivatives.

(G) pairs with cytosine

G-C base-pair29

3 (Fig.

la).

This

’

Vol. 46, No. 4, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

C-G

U-G*

U-G

Fig.

1

G-C pairing of organic

(a) Normal

G-C base-pair.

(c) Wobble

G-U base-pair.

has the appropriate

posing thymine

(T) or uracil

of G is approximately

lactam-lactim

G*-U

pairing

lb).

G*-U

base-pair.

to pair with an op-

Although

in energy

the lactim

than the la&am

This consideration in solution side simply

suggests

1537

base’,

of hydrogen-bonds that it might

be

and to shift the la&am-lactim by flooding

the solution

base-pairing.

was found to be important

8 it is clear that evidence

tautomer

should be comparable

since the same number

here a pmr study of this G*-U

equilibrium

base is hydrated,

structure

or G*-U base-pair

G-C base-pair,

more to the lactim

We report

higher

G*-T

in each case.

to observe

equilibrium

(U) base (Fig.

of the abnormal

is formed

possible

(b) Abnormal

electronic

1 kcal/mole

to that of the normal (three)

Pa~rinq)

has been observed directly by nmr and ir techniques in a number solvents. 4-7 The lactim tautomer of G, henceforth denoted by

G*, however,

the stability

(Wobble

for G*-U

with U.

Since the

only when the guanine pairing

should be sought

Vol. 46, No. 4, 1972

BIOCHEMICAL

in aqueous solution. yield

results

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Unfortunately,

which are difficult

such experiments to interpret,

exchange between the water protons the base-pairing, the nucleic

and the protons

acid bases at the nucleoside

for the normal

base-pairing.

G-C base-pairing

have therefore

carried

choice of the water content, can be controlled,

No,-H

of G and the N(,) -H proton

The results

observation

we have summarized N(l)-H,

chemical

resonances

0.2 M dU at various

water concentrations

at this dU/rG

less large

and uracil

that proton resonance guanine base, creases

ratio

which is normally

and significantly,

studied

for the NH, and

between guanosine I and II.

In Table I,

Although

the shifts ob-

interaction

assigned

they are nevertheis occurring

protons

1538

between

shift was observed

to the N(,)-H

the induced shift observed -1.2 to -16.5

Hclt),

0.1 M rG and

are quite small,

downfield

for the

containing

proton

for of the

for this proton

in-

Hz over the range of water

(3 to 40 mole o/o). The increased

served for the NH, and the N(,)-H

sufficiently

of rG and the N(,)-H,

in DMSO.

The largest

with the water content from

concentrations

resonances

of dU for solutions

concentration

bases.

base-pairing

shifts which were observed

enough to infer that some pairing

the guanine

far enough toward

of the G*-U

in Tables

H(lr, and Hu,) proton resonances

NH,,

of

of U in the pmr spectrum.

(dU) are summarized the induced

hydration

But, by appropriate

of the interaction

Ht5) and Ht6) proton

served

to occur.

of individual

of our pmr studies

(rG) and 2’-deoxyuridine

complete

equilibrium

We

in DMSO-water

exchange rates can be rendered

direct

protons

experiments

the thermodynamics

slow to permit

necessary

at low temperatures.

in order to ensure

pairing

of

be that such interactions

except perhaps

G*-U

and the proton

concentrations

It might

the guanine base, and shift its la&m-lactim

the stacking

in

there has been no evidence

out the G-U base-pairing

side for significant

from

or nucleotide

in water.

The water is necessary

the lactim

which may be involved

arising

Moreover,

are quite weak in aqueous solution,

mixtures.

both because of the rapid proton

and because of complications

to detect the abnormal

in pure water (H,O)

induced shifts

ob-

of the guanine base with increasing

BIOCHEMICAL

Vol. 46, No. 4, 1972

TABLE

I.

Base-Pairing

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Shiftsa in DMSO-H,O

Mixtures

O.lMrG+0.2MdU

rG Protons

dU Protons

H,O Content Mole %

Htlr,

NH,

H(B) NW-H@(,)-W

3

-2.7

+1.2

-2.1

H(s)

- 1.2

H(1,)

H(e)

-1.6

-1.7

-1.3

+1.4 (6)

-2.5

-2.0

-1.0

-3.5

(5)

19

-2.6

-1.5

-3.0

- 8.5

NC,,-H

(10) 31

-2.5

-4.5

-3.5

(8)

-10.0

-3.4

-2.5

-3.0

-4.0 (16)

-5.0

-2.0

-3.0

-4.4 (20)

(20) 40

-2.5

-3.5

-3.5

-16.5 (30)

0.1 M rG + 0.1 M rC

H,O Content Mole %

H(,ft

-2

-2

aChemical

rG Protons NH, H(s)

NW-H

I-h,,)

H(s)

H(c)

NH,

-65.5

-144.0

-2.0

-14.0

-12.0

-59.0

shifts are given in Hz at 220 MHz.

denote linewidths

place (Table II).

DMSO mixture varied

Numbers

(in Hz) of the proton resonance

[dU ] / [rG ] concentration is taking

-14.0

rC Protons

ratio substantiates In these studies,

in parentheses

under consideration.

our contention the water content

that G-U pairing of the H,O-

was held fixed at N 10 mole % and the [dU ] / [rG ] ratio was

by varying

[dU]

at fixed

[rG j . 1539

Vol. 46, No. 4, 1972

TABLE

BIOCHEMICAL

II.

AND BIOPHYSICAL

G-U Base-Pairing Concentration

RESEARCH COMMUNICATIONS

Shiftsa as a Function Ratio in a 10 Mole

of [rG]/

o/c H,O-DMSO

[dU 1 Mixture.

dU Protons Hw )

-(10) 5.0

-11.5

H(s)

H(c)

NW-H -1.5 (10)

+0.5

-1.0

-3.0

-1.5

-3.0

-4.5

0

(25)

a Chemical

(in Hz) of the proton

The downfield

shifts

proton

the formation

of a hydrogen-bonded

H,O/DMSO

mixture

sides when the N(,)-H N(,, -site

shift of the amino

indicates

of G is blocked.

sine and 2’-deoxyuridine implicating

it is not possible

is pairing

with U.

a G-U base-pair proton

we surmise

The kinetics

ton of the lactam

species

group or when the

between these nucleosides,

involving

hydrogen-bonding

that it is the lactim equilibrium

so that the chemical a weighted

and the 0(,)-H

proton

1540

Since

of G with the guanine base in

of the overall

proton of G is in actuality

o/c

with 2-N, N-dimethyl-guano-

no interaction

to fast on the nmr time scale,

the “N(l)-H”

in a 10 mole

of the NH, group of G in the G-U pairing.

to construct

structure,

The

of 1-methyl-guanosine

by a methyl

experiments

also reveal

suggest

between the bases of these nucleo-

of G is replaced

of both the NH, group and the N(,,-H the lactam-amino

protons

of rG

these protons.

to 1-methyl-guanosine

Similar

the involvement

and NH, protons

involving

no interaction

proton

under consideration.

of these two nucleosides

complex

of 2’-deoxyuridine

in parentheses

for the N(,)-H

of dU upon the mixing

absence of a dU induced upon the addition

resonance

observed

and the N(,)-H

mediate

Numbers

shifts are given in Hz at 220 MHz.

denote linewidths

(13)

average

of the lactim

form of G which

is presumably

inter-

shift observed of the N(,rH species

for

proin both

Vol. 46, No. 4, 1972

the complexed interaction

BIOCHEMICAL

and the free states.

may be compared

of the G-C pairing is only present ture,

AND BIOPHYSICAL

The pairing

to the extent of 10-15s

we expect the base-pairing

magnitude

smaller

assuming

is clearly

of the following

in aqueous solution

and the G*-U

chemical

conditions, stability. in Table I.

semi-quantitatively

KT * = G

(1)

G*-U

(2)

constants

for the lactam-lactim

respectively.

at sufficiently

of dU, so that the observed

of

equilibria:

base-pairing

D [G*-U]

of G

at room tempera-

are of comparable

in Table II can be analyzed

Here KT and K are the equilibrium

[dU],

tautomer

borne out by the data summarized

G* + U Z

we expect

study

shifts in the G-U case to be an order

G

erism

for the G-U

in a parallel

Since the lactim

and G-C base-pairs

The data summarized in terms

I).

observed

than those for the G-C case under similar

that the G*-U

This expectation

shifts

with those observed

in pure DMSO (Table

RESEARCH COMMUNICATIONS

For weak G-U interaction,

high stoichiometric

base-pairing

tautom-

concentrations

shift can be adequately

approximated

by 6 obsd EA where A is the chemical relative

librium

shift of a proton

of G is roughly

i. e., these protons

and G-C pairing, constant

induced

complex

If we can now assume that A for

the same in the G*-U are shifted

for the pairing

complex

as in the G-C

by about the same amount

of magnitude

as that previously

in DMSO.

for the N cl)-H (0(,)-H) 1541

by G*-U

then the data suggest that the equi-

between G* and U is of the order

of the G-C complex

shifts observed

(3)

in the hydrogen-bonded

state.

and KT * 0.05 to 0.1,

which is about the same order the formation

bul,

(1 + KT) + K KT [dU j,

to that in the uncomplexed

the NH, protons complex,

K%

of 1 M-l,

reported

for

Using this K value and the dU proton

of G, we estimate

Vol. 46, No. 4, 1972

that this proton

BIOCHEMICAL

is shifted

the uncomplexed ppm downfield

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

N 2-3 ppm downfield

state upon G-U pairing. from

from its spectral

Since this proton appears

TMS in the absence of complex

shift is approximately

13.0 ppm downfield

from

formation,

which is what one would expect for the chemical

hydrogen

in a relatively

0-H.

. +0 hydrogen-bond.

We have also noted broadening U upon mixing

ing was found to be more water content, observed

lmebroadening

might

concentrations.

at higher

be due to enhanced

these resonances

we exclude

in the rG-dU

and we suspect that it arises of the overall Finally,

proton

since we observe

of G and U in comparative

equilibria

chemical

our contention

(Fig.

either

of

and reflects

the dy-

by Crick.

equilibrium

This

is not im-

that the la&am-lactim The lack of

in dry DMSO also enables us to

of other base-pairing

lc) proposed

rG or

the broadening

of the guanine base.

of G-U base-pairing

the guanine base is in the la&am-amino pairing

water

of the N-H

induced shifts for any of the G or U resonances.

is induced by the hydration

rule out the importance

of G was also

of dU to rG in dry DMSO does not

would seem to suggest that the lactam-lactim

for any form

At a given

denoted by (1) and (2).

result

evidence

involving

exchange,

in significant

tautomerism

This linebroaden-

must be due to the G*-U base-pairing,

we note that the addition

in DMSO and supports

of G and

This water-dependent

broadening

Thus,

result

portant

resonances

resonance

ratio.

little

this possibility.

from

’

exchange at the higher

experiments

system

its chemical

water contents.

[dU ]/ [rG]

- 10.5

shift of the bridge

I and II).

of the NC,)-H (0(,)-H)

with increasing

However,

dU alone in solution,

namics

(see Tables

pronounced

the broadening

to increase

resonances

of the N-H proton

of the two nucleosides

in

TMS in the hydrogen-bonded

complex,

strong

position

form,

schemes

between G and U when

most notably

the “wobble7’

lo

Acknowledgment. This work was supported in part by Grant GM 14523-05 from the National Institute of General Medical Sciences, U. S. Public Health Service, and by Grant GP-8540 from the National Science Foundation.

1542

Vol. 46, No. 4, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

References Lee, G. C. Y., and Chan, S. I., J. Amer. Chem. Sot., in press. Watson, J. D., and Crick, F. H., Nature, 1’71 737 (1953). Watson, J. D., and Crick, F. H., Nature, l-7-f 964 (1953). Kyogoku, Y., Lord, R. C., and Rich, A., J%er. Chem. Sot., 89, 496 (1967). Kyogoku, Y., Lord, R. C., and Rich, A., Science, 154 518 (1966). :* and Penman, S., J. Mol. Biol., -15 220 (rss’s). 7: E%?na?k, R. A ., and Cantor, C. R., J. Amer. Chem. Sot., 90- 5010 (1968). Lee, G. C. Y., and Chan, S. I., manuscript in preparation. 9”: Pimentel, G. C. , and McClellan, A. L., “The Hydrogen Bond, ” Freeman, ;;w;o;k (;960). 10. 5 548 (1966). , . . , J. Mol. Biol., ;:

1543