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EL SEV I ER B i o c h i m i c a e t B i o p h y s i c a A c t a 1 2 4 4 ( 1 9 9 5 ) 2 5 9 - 2 6 8

Biochi~ic~ae t B io p h y s ic a ~ t a

S y n t h es i s a n d ch a ra c t er i s a t i o n o f f l u o res cen t o l i g o n u c l eo t i d es . E f f ec t o f

in terna l labe l l ing on prote in recogni t ion

P e r H a g m a r a, M i c h a e l B a i l e y a, G l e n n T o n g b, J i m H a r a l a m b i d i s b , W i l l ia m H . S a w y e r a,* ,

B a r r ie E . D a v i d s o n a

Russell Grimwade School of Biochemisto', Universi O, of Melbourne, Parkville, Victoria 3052, Australia

b Howard Florey Institute of Experimental Physiology and Medicine, Universi~' of Melbourne, Parkville, Victoria 3052, Australia

Re c e i v e d 2 2 Se p t e m b e r 1 9 9 4 ; r e v i s e d 1 2 D e c e m b e r 1 9 9 4 ; a c c e p t e d 1 0 J a n u a r y 1 9 95

A b s t r a c t

F l u o r e s c e n t l y l a b e ll e d 42 b a s e p a i r D N A d u p l e x e s w e r e s y n t h e s i se d t o e x a m i n e t h e i n t e r ac t i o n b e tw e e n t h e T y r R r e p r e s s o r p r o t e i n o f

E s c h e r i c h i a c o l i a n d i t s D N A r e c o g n i t io n s e q u e n c e. A n F m o c - p r o t e c t e d 5 - ( 3 - a m i n o p r o p - 1 - y n - 1 - y l ) - 2 ' - d e o x y u r i d i n ep h o s p h o r a m i d i t e w a s

s y n t h e s i s ed a n d i n c o r p o r a te d i n t o o l i g o n u c l e o t i d e s u s i n g s t a n d a r d / 3 - cy a n o e th y l p h o s p h o r a m i d i t e c h e m i s t r y . O l i g o n u c l e o t i d e s c o n t a in i n g

t h e 3 - a m i n o p r o p y n y l n u c l e o t i d e a t i n t er n a l p o s i t i o n s w e r e r e a c t e d w i t h f l u o r e s c e i n i s o th i o c y a n a t e t o g e n e r a t e f l u o r e s c e n t D N A m o l e c u l e s

u s e f u l f o r c h a r a c t e ri s i n g in t e r a c ti o n s b e t w e e n D N A a n d p r o t e i n s . S h o r t D N A d u p l e x e s w e r e i n v e s t i g a t e d w i th r e s p e c t t o t h e i r m e l t i n g

tempera tu res and the i r ab i l i ty to b ind TyrR . Ol ig onuc leo t ides con ta in ing a TyrR b ind ing s i t e were l abe l led in the cen t ra l r eg ion o f the

r e c o g n i t io n s e q u e n c e o r n e a r t h e 5 ' e d g e o f t h e r e c o g n i t i o n s e q ue n c e . F l u o r e s c e i n - l a b e l l e d o l i g o n u c l e o t i d e s c o u l d h y b r i d i s e t o f o r m

d u p l e x D N A , a n d g e l r e t a r d a t i o n e x p e r im e n t s s h o w e d t h a t t h e p r e s e n c e o f t h e d y e d i d n o t a l t e r t h e b i n d i n g a f f i n it y f o r t h e T y r R p r o t e i n

s ign i f i can t ly . F luore scence an i so t ropy mea sureme nts were used to exam ine the b ind ing equ i l ib r ium in low and h igh sa l t buf fe r s . A

d i s s o c i a ti o n co n s t a n t o f 2 0 0 - 5 0 0 n M w a s o b t a i n e d f o r t h e i n t e ra c t io n o f t h e T y r R d i m e r w i t h a 4 2 b p d u p l e x c o n t a i n i n g a c e n t ra l l y

l o c a t e d 2 2 b p T y r R b i n d i n g s i te .

Keywords: D N A - p r o t e i n i n te r a c ti o n ; F l u o r e s c e n c e o li g o n u c l e o ti d e ; R e p r e s s o r

1 . I n t r o d u c t i o n

P r o t e i n s w h i c h r e c o g n i z e a n d b i n d t o s p e c i f i c b a s e

s e q u e n c e s o n D N A c o n t r o l th e p r o c e s s e s o f r e p l i c a t i o n ,

t r a n s c r i p t io n , r e p a i r a n d g e n e r e g u l a t i o n ( f o r r e v i e w s , s e e

[ 1 - 3 ] ) . S t u d i e s o f t h e t h e r m o d y n a m i c s o f th e s e i n t e r a c t io n s

c o n t r i b u t e t o o u r u n d e r s t a n d i n g o f t he b a l a n c e o f fr e e a n d

b o u n d s p e c i e s t h a t i s l i k e l y t o e x i s t w i t h i n c e l l s . O f t h e

s e v e ra l m e t h o d s t h a t h a v e b e e n d e v i s e d t o m o n i t o r D N A -

p r o t e i n i n t e r a c t i o n s , g e l r e t a r d a t i o n a n d f i l t r a t i o n a s s a y s

h a v e g a i n e d w i d e a c c e p t a n c e , b u t t h e r e is c o n c e r n t h a t

s e p a r a t i o n o f b o u n d f r o m f r e e s p e c i e s i n h e r e n t in t h e s e

p r o c e d u r e s m a y p e r t u r b t h e e q u i l i b r i u m . I n t h i s r e s p e c t ,

s p e c t r o s c o p i c a s s a y s o f f e r t h e a d v a n t a g e t h a t th e c o n c e n -

t r a t io n o f f r ee a n d b o u n d s p e c i e s c an b e d e t e r m i n e d i n

s o l u t io n . T h e h i g h s e n s i t i v i ty o f f l u o r e s c e n c e d e t e c t i o n

a l l o w s a s s a y s t o b e c a r r i e d o u t a t n a n o m o l a r c o n c e n t r a -

* C o r r e s p o n d i ng a u t h or . E - m a i h S a w y e r @ b i o c h e m i s t ry . u n i m e l b .

e d u . a u . F a x : + 6 1 3 34 7 7 7 3 0 .

0 3 0 4 - 4 1 6 5 / 9 5 / $ 0 9 . 5 0 © 1 9 9 5 E l s e v i e r S c i e n c e B . V . A l l r i g h ts r e s e r v e d

SSD10304-4165(95)00015-1

t i o n s a n d t h e r e f o r e i n t h e v i c i n i t y o f t h e d i s s o c i a t i o n

c o n s t a n t s o f th e s e e q u i l ib r i a . C o m p l e x f o r m a t i o n m a y b e

f o l l o w e d b y ( i ) a c h a n g e i n t h e i n t r in s i c f l u o r e s c e n c e o f t h e

p r o t e i n , ( i i ) a c h a n g e i n t h e e x t r i n s i c f l u o r e s c e n c e o f a

l a b e l l e d p r o t e i n o r o l i g o n u c l e o t i d e , o r ( i ii ) a c h a n g e i n

f l u o r e s c e n c e a n i s o t r o p y d u e t o a d e c r e a s e i n th e r o t a t i o n a l

d i f f u s i o n o f t h e c o m p l e x [ 4] .

A c o m m o n l a b e ll i n g p r o c e d u r e u s e s s t a nd a r d p h o s p h o -

r a m i d i t e c h e m i s t r y [ 5 , 6] t o c o n j u g a t e a p r i m a r y a m i n e

l i n k e d v i a a s p a c e r g r o u p t o t he 5 ' - p h o s p h a t e g r o u p o f a n

o l i g o n u c l e o t i d e . A f l u o r e s c e n t d y e w i t h a n a m i n e - r e a c t i v e

g r o u p s u c h a s a s u l f o n y l c h l o r i d e o r a n i s o t h i o c y a n a t e m a y

t h e n b e c o n j u g a t e d t o t h e a m i n e u n d e r b a s i c c o n d i t i o n s .

T h e s e a m i n o l i n k e r s a r e c o m m e r c i a l l y a v a i l a b l e i n l e n g t h s

o f 2 to 1 2 c a r b o n a t o m s a n d l a b e l l i n g a t t h e 3 ' e n d i s a l s o

p o s s i b l e ( A p p l i e d B i o s y s t e m s , F o s t e r C i ty , C A ; C l o n t ec h ,

P a l o A l t o , C A ) . H o w e v e r , i f a n e n v i r o n m e n t - s e n s i t i v e

f l u o r o p h o r e i s to b e e m p l o y e d , i t m u s t b e p o s i t i o n e d c l o s e

t o t h e f o o t p r i n t f o r t h e i n t e r a c t i o n s o t h a t t h e p r o b e e n v i -

r o n m e n t i s a f f e c te d b y p r o t e i n b i n d i n g . I n t h e s e c a s e s, u s e

o f 3 ' o r 5 ' l a b e l l i n g re q u i r e s t r u n c a t i o n o f t h e o l i g o n u c l e o -



260 P. Hagmar et al. / Biochimica et Biophysica Acta 1244 (1995) 259 268

t i de se que nc e on a t l e a s t one s ide o f t he r e c ogn i t i on

se que nc e to a c h ie ve a pp rop r i a t e j ux t a pos i t i on o f t he p robe

a nd the bound p ro t e in . Suc h t runc a t ion ma y be ina pp rop r i -

a t e , s i nc e foo tp r in t ing a nd homology a na ly se s show tha t

the b ind ing o f some p ro t e in s i s se ns i t i ve t o t he l e ng th o f

f l a nk ing re g ions on e i t he r s ide o f a spe c i f i c s i t e . Fo r

e xa mple , t he e uka ryo t i c t r a nsc r ip t iona l a c t iva to r G C N 4

st rong ly in t e ra c t s w i th a t l e a s t 18 bp o f D N A , a s i t e muc h

la rge r t ha n the 9 bp c onse nsus se que nc e ide n t i f i e d by

homology w i th na tu ra l ly oc c u r r ing b ind ing s i t e s [7 ] . S imi -

la r ly , the Escherichia coli C A P p r o t e i n r e q u i r e s a 2 8 - 3 0

bp D N A mole c u le t o ob ta in fu l l b ind ing s t r e ng th [8 ] , a s

c ompa re d to t he 16 bp s t rong ly c onse rve d c onse nsus se -

que nc e [9 ] . In su f f i c i e n t l e ng th o f t he o l igonuc le o t ide ma y

inde e d e xp la in t he l ow a ssoc i a t ion c ons t a n t s found fo r

som e t r a nsc r ip t ion r e gu la to r s [10 ]. 5 ' -L a be l l i ng o f o l igo -

nuc le o t ide s a l so p re ve n t s rou t ine 32 P - l a be l l i ng a t t h i s e nd .

La be l l i ng w i th in t he ba se se que nc e a vo ids t h i s p rob le m

a n d p r o v i d e s o p p o r t u n i t i e s f o r ' f l u o r e s c e n c e f o o t p r i n t i n g ' .

A numbe r o f l a be l l i ng s t r a t e g i e s a re a va i l a b l e ( fo r r e v i e w ,se e R e fs . [4 ] a nd [11 ] ) . La be l l i ng o f t he i n t e muc le o t ide

p h o s p h o r o u s b y s y n t h e s i s i n g a d e o x y n u c l e o s i d e 3 ' - p h o s -

p h o r o t h i o a m i d i t e h a s b e e n d e s c r i b e d b y C a r u t h e r s e t a l .

[12 ] . I t i s a l so poss ib l e t o r e p l a c e a nuc l e os ide w i th a

3 -c a rbon b r idge c a r ry ing a n a minobu ty l s ide a rm to w h ic h

a f l u o r e s c e n t p r o b e m a y b e c o n j u g a t e d ( C l o n t e c h , P a l o

A l to , C A ) . The 3 -c a rbon b r idge i s de s igne d to c onse rve the

in t e rphospha te d i s t a nc e bu t ha s t he d i sa dva n ta ge tha t a

nuc le os ide i s r e p l a c e d by a fo re ign b r idg ing g roup . O f

pa r t i c u l a r i n t e re s t i s l a be l l i ng a t ba se s w i th in t he se que nc e .

G i b s o n a n d B e n k o v i c d e s c r i b e d t h e s y n t h e s i s o f a p h o s -

p h o r a m i d i t e d e r i v a t i v e w i t h a p h t h a l i m i d e - p ro t e c t e d p r o p y -l a mine on the C 5 o f de oxyur id ine [13 ]. H o w e v e r , t he

ph tha l imide p ro t e c t ing g roup c a n be d i f f i c u l t t o r e move

c om ple t e ly unde r a lka l ine c ond i t i ons . U se o f t he B oc

p ro te c t ing g rou p [14] som e t ime s r e su l ts i n pa rt i a l o l i go -

nuc le o t ide de g ra da t ion du r ing t r i f l uo roa c e t i c a c id de p ro t e c -

t i on w i th c onse qu e n t l ow y ie ld s o f the fu l l le ng th p roduc t .

F m o c p r o t e c t i o n o f a n a l k y n y l a m i n o d e o x y u r i d i n e d e r i v a -

t i ve [15 ] a ll ow s e a s i e r r e mo va l o f t he g roup in a m m on ia

solut ion s a t 55 ° C an d w i l l be use d in the curren t s tudy .

T h e l e n g t h o f a n y s p a c e r o n a n a m i n o u r i d i n e d e r i v a t i v e

o r a t e r m i n a l l i n k e r b e c o m e s i m p o r t a n t w h e n f l u o r e s c e n c e

a n i so t ropy i s u se d to mon i to r t he D N A -pro te in a ssoc i a t ion .

Loc a l mo t ion o f t he dye a t i t s po in t o f a t t a c hme n t i s

p r o m o t e d b y l o n g s p a c e r s a n d c a n b e a m a j o r d e t e r m i n a n t

o f t he s t e a dy -s t a t e a n i so t ropy . W e now re po r t t he syn the s i s

o f a n a m i n o u r i d i n e p h o s p h o r a m i d i t e p o s s e s s i n g a s h o r t

a l k y n y l a m i n o s i d e a r m . I n c o r p o r a t i o n o f t h i s n u c l e o s i d e

in to a n o l igonuc le o t ide u s ing s t a nda rd so l id pha se phos-

p h o r a m i d i t e m e t h o d o l o g y e n a b l e s e f f i c i e n t s y n t h e s i s o f

o l igonuc le o t ide s w h ic h c a n re a c t w i th a va r i e ty o f a mine -

re a c t ive p robe s . W e i l l u s t r a t e t he u se o f t he o l igonuc le o -

t i d e c o n j u g a t e s a s p r o b e s f o r D N A - p r o t e i n i n t e r a c t i o n b y

in t roduc ing a f l uo re sc e n t l a be l a t spe c i f i c r e s idue s i n t he

r e c o g n i t i o n s e q u e n c e f o r a D N A b i n d i n g p r o t e i n , n a m e l y ,

t h e T y r R r e p r e s s o r p r o t e i n o f E. coli, a nd show tha t

s t e a dy -s t a t e f l uo re sc e nc e a n i so t ropy ma y be u se d to fo l low

t h e f o r m a t i o n o f t h e D N A - r e p r e s s o r c o m p l e x .

Ty rR re gu la t e s t he e xp re ss ion o f e igh t tr a nsc r ip t iona l

un i t s e sse n t i a l fo r a roma t i c a mino a c id b io syn the s i s a nd

t ra nspo r t [16,17 ]. The a c t iv i ty o f Ty rR i s modu la t e d by the

b i n d i n g o f A T P a n d t h e a r o m a t i c a m i n o a c id s . A t c o n c e n -

t r a t i ons a bove 100 nM a nd in the a bse nc e o f t y ro s ine ,

Ty rR e x i s t s p re domina n t ly a s a d ime r i n so lu t ion [18 ] . The

c onse nsus se que nc e fo r Ty rR b ind ing s i t e s i n D N A , so

c a l l e d T y r R b o x e s , i s T G T A A A N 6 T T T A C A . B o x e s t h a t

ha ve a s t rong homology w i th t he c onse nsus se que nc e b ind

TyrR in t he a bse nc e o f c o fa c to r s a nd a re r e fe r re d to a s

s t rong boxe s i n c on t ra s t t o w e a k boxe s t ha t ha ve w e a k

homology to t he c onse nsus se que nc e a nd re qu i re t he p re s -

e nc e o f A TP a nd ty ros ine fo r b ind ing o f t he p ro t e in . W e

ha ve syn the s i se d 42 bp o l igonuc le o t ide s c on ta in ing the

s t rong Tyr R box fou nd in t he r e gu la to ry r e g ion o f t he O'rR

ge ne w i th t he mod i f i e d de oxyur id ine in e i t he r o f tw o

pos i t i ons i n t he N 6 nonspe c i f i c spa c e r r e g ion . R e a c t ion o fthe se o l igonuc le o t ide s w i th f l uo re sc e in i so th ioc ya na te

y ie lde d f luo re sc e n t o l igonuc le o t ide s t ha t w e re a b l e t o hy -

b r i d i s e t o t h e i r u n m o d i f i e d c o m p l e m e n t a r y o l i g o n u c l e o -

t i de . A na lys i s i nd i c a t e d tha t t he r e su l t i ng doub le - s t r a nde d

m o l e c u l e s b o u n d T y r R t o t h e s a m e e x t e n t a s t h e u n m o d i -

f i e d T y r R b o x .

2 . M a t e r ia l s a n d m e t h o d s

2.1. Materials

3 - A m i n o p r o p y n e , 5 - i o d o - 2 ' - d e o x y u r i d in e , t e tr a ( tr i p h en -

y l p h o s p h i n e ) - p a l l ad i u m ( 0 ) a n d 2 - c y a n o e t h y l - N , N - d i i s o p r o -

p y l c h l o r o p h o s p h o r a m i d i t e w e r e p u r c h a s e d f r o m A l d r i c h .

3-tert-Butyloxycarbonylaminopropyne w a s f r o m S i g m a .

N - ( ( F l u o r e n - 9 -y l m e t h o x y c a r b o n y l )o x y ) s u c c i n im o d e a n d

d i m e t h y l f o r m a m i d e ( d i s ti l le d a n d s t o re d o v e r 3 A m o l e c u -

l a r s i e v e s ) w e r e o b t a i n e d f r o m A u s p e p , M e l b o u r n e . S t a n -

d a r d p h o s p h o r a m i d i t e s w e r e f r o m A p p l i e d B i o s y s t e m s .

D i c h l o r o m e t h a n e a n d m e t h a n o l w e r e d r ie d o v e r 3 A s i e v es .

A n a l y t i c a l t h i n - l a y e r c h r o m a t o g r a p h y ( T L C ) w a s p e r -

f o r m e d o n M e r c k S G - 6 0 p r e - c o a t e d p l as t i c p la t e s ( p a rt i c le

s i z e 5 - 40 / z m) a nd ra p id f i l t ra t i on c h ro ma to g ra ph y [19 ]

w a s p e r f o r m e d u s i n g t h i n - l a y e r c h r o m a t o g r a p h y g r a d e s i l -

i c a ge l (Me rc k SG -60 , pa r t i c l e s i z e 5 -40 /xm) . F luo re s -

c e i n - 5 - i s o t h i o c y a n a t e ( F I T C ) ( i s o m e r I ) w a s p u r c h a s e d

f r o m M o l e c u l a r P r o b e s .

2.2. Experimental procedures

Spectroscopy

A b s o r b a n c e s w e r e m e a s u r e d w i t h a C A R Y 5 s p e c t r o -

pho tome te r f i t t e d w i th t he rma l a c c e sso r i e s a nd in t e r fa c e d

to a c ompu te r fo r a c qu i s i t i on a nd a na ly s i s o f e xpe r ime n ta l

d a t a . A b s o r b a n c e v e r s u s t e m p e r a t u r e m e l t i n g c u r v e s w e r e



P. H agmar et al. / Biochimica et Biophysica Acta 1244 (1995) 259-268 261

m e a s u r e d a t 2 6 0 n m a t a h e a ti n g r a t e o f l ° C / m i n . T h e

t e m p e r a t u r e w a s m e a s u r e d w i t h a t h e r m i s t o r p r o b e i n s e r t e d

in bu f fe r i n a c uve t t e a d j a c e n t t o t he sa mp le i n t he

t h e r m o s t a t e d c e ll b l o c k . F l u o r e s c e n c e w a s m e a s u r e d w i t h a

P e r k i n - E l m e r L S - 5 l u m i n e s c e n c e s p e c t r o m e t e r . T h e f l u o -

re sc e nc e a n i so t ropy w a s de t e rmine d in t he u sua l w a y ,

a l l o w a n c e b e i n g m a d e f o r t h e g r a t i n g c o r r e c t i o n f a c t o r

[201.

G e l r e ta r da t ion a s s ay

D o u b l e - s t r a n d e d 4 2 m e r s i n t h e e x p e r i m e n t a l b u f f e r w e r e

mix e d w i th a l i quo t s o f a Ty rR s toc k so lu t ion , bu f fe r a nd

g lyc e ro l t o g ive the r e l e va n t c ons t i t ue n t c onc e n t ra t i ons i n

10% g lyc e ro l . A f t e r 20 min inc uba t ion , 10 /z l sa mp le s

w e r e l o a d e d o n a 7 . 5 % p o l y a c r y l a m i d e g e l a n d e l e c -

t r o p h o r e s e d a t 1 5 m A c o n s t a n t c u r r e n t i n 9 0 m M T r i s -

b o r a t e, 2 m M E D T A b u f f e r ( p H 8 . 3 ) (1 x T B E ) . G e l s w e r e

s t a i n e d w i t h e t h i d i u m b r o m i d e a n d p l a c e d o n a U V - t r a n s -

i l l u m i n a t o r f o r p h o t o g r a p h y o r s c a n n i n g w i t h a C C D c a m -

e r a. I n t h e l a tt e r ca s e , B I O - P R O F I L s o f t w a r e ( V i l b e r L o u r -

ma t , Ma rne l a V a l l~e , F ra nc e ) w a s u se d to qua n t i t a t e t he

ima ge s . Fo r fu l l ge l r e t a rda t ion t i t r a t i ons , t he i n t e g ra t e d

in t e ns i t i e s fo r t he f r e e o l igonuc le o t ide s w e re f i t t e d to a

s ing le - s i t e b ind ing mode l u s ing the non - l ine a r r e g re ss ion

op t ion o f S igm a Plo t 5 .0 ( Ja nde l Sc i e n t i f i c , Sa n R a fa e l ,

C A ) .

N M R

S p e c t r a w e r e r e c o r d e d a t 3 9 9 . 9 M H z ( ~ H ) a n d a t 9 9 . 9 8

M H z ( 1 3 C ) o n a J E O L G X 4 0 0 i n s t r u m e n t . T h e i n t e r n a l

r e f e r e n c e w a s r e s i d u a l s o l v e n t i n d 6 - D M S O . ~ 3 C - N M R

a s s i g n m e n t s w e r e c o n f i r m e d b y D E P T e x p e r i m e n t s w i t h a135 ° ~H se l e c tion pu l se . The 3~p spe c t rum w a s ob ta ine d

o n a V a r i a n 3 0 0 s p e c t r o m e t e r a t 1 2 1 . 4 2 M H z u s i n g 8 5 %

H 3 P O 4 / H 2 0 a s e x t e rn a l r e f e r e n ce . T h e n u m b e r i n g s y s t e m

f o r c o m p o u n d s 3 a n d 4 i s s h o w n i n S c h e m e 1.

t h e w o r k - u p p r o c e d u r e . T h e r e a c t i o n , p e r f o r m e d o n a 7 . 9

m m o l s c a l e, w a s c o m p l e t e a f te r 2 h a s j u d g e d b y T L C

u s i n g e t h y l a c e t a t e a s t h e s o l v e n t. A G I X 8 ( H C O 3 ) i o n

e x c h a n g e r e s i n ( 3 - f o l d e x c e s s ) w a s a d d e d w i t h 5 0 %

M e O H / d i c h l o r o m e t h a n e ( 10 m l / m m o l o f nu c le o si de ).

A f t e r 0 .5 h t he r e s in w a s r e move d by f i l t r a t i on a nd the

so lve n t r e move d in va c uo . Pu r i f i c a t ion w a s by ra p id f i l t r a -

t i o n c h r o m a t o g r a p h y . T h e c o l u m n w a s p r e - w a s h e d w i t h

9 0 % d i c h l o r o m e t h a n e / t r i e t h y l a m i n e ( 1 c o l u m n v o l u m e )

f o l l o w e d b y d i c h l o r o m e t h a n e ( 2 c o l u m n v o l u m e s ) b e f o r e

a pp l i c a t ion o f t he sa mp le . The c rude r e s idue w a s the n

a pp l i e d to a s i l i c a ge l c o lum n (10 g o f TLC s i l i c a ge l pe r

m m o l o f n u c l e o s i d e ) ; t h e i n it i al e l u a n t w a s

d i c h l o r o m e t h a n e , f o l l o w e d b y 5 0 % e t h y l a c e t a t e / d i c h l o r o -

me tha ne , a nd the n f ina l ly e thy l a c e t a t e . The de s i r e d nuc le -

os ide 3 w a s ob ta ine d a s a f a w n so l id w i th t yp i c a l y i e ld s

b e t w e en 3 5 - 6 0 % . 1 H - N M R (D M S O - d 6 ) : ~ 2 .2 5 (m , 2H ,

2 × H 2 ') , 2 .96 (dd, 1H, H5 ' , J = 10.3 , 4 .4 Hz) , 3 .08 (dd,

1H, H5" , J = 10.4 , 2 .7 Hz); 3 .29 (s , 2H, H9), 3 .73 ( t , 1H,

N 9 H , J = 5 . 3 H z ) , 3 . 9 0 ( m , I H , H 4 ' ) , 4 . 1 4 ( m , 1 H , H 3 ' ) ,

4 . 2 0 ( t , I H , F m o c H 9 J = 6 . 8 H z ) , 4 . 2 8 ( d , 2 H , F m o c

C H 2 , J = 7 .0 H z ) , 5 .2 9 ( d, 1 H, 3 ' O H , J = 4 . 4 H z ) ,

7 . 1 0 - 7 . 5 0 ( m , 1 7 H , F m o c + P x H ) , 7 . 6 9 ( d , 2H , F m o c H I

a n d H 8 , J = 7 .3 H z ) , 7 .89 (d , 2H , Fmoc H 4 + H 5 , J = 7.3

H z ) , 7 .98 (s , IH , H 6) . 13C -N MR (D M SO - d6) : 6 30 .4

( C 9 ) , 4 0 . 3 ( C 2 ' ) , 4 6 . 6 ( F m o c C 9 ) , 6 3 . 7 ( C 5 ' ) , 6 5 . 7 ( F m o c

C H z ) , 70 .8 (C 3 ' ) , 74 .2 (C 8) , 75 .5 (Px C 9) , 85 .3 (C I ' ) , 85 .9

(C 4 ' ) , 89 .7 (C 7) , 98 .2 (C 5) , 116 .3 (Px C H ) , 120 .1 (Fmoc

C3 and C6) , 122.3 , 123.9 , 124.0 (Px CH), 125.2 (Fm oc C2

and C7) , 125.8 , 126.7 (Px CH ), 127.1 , 127.6 (Fm oc C1

and C8) , 128.0 , 129.0, 129.1 , 129.7 (Px CH ), 140.7 (F mo c

C 4a a nd C 4b) , 143 .1 (C 6) , 143 .7 (Fmoc C 8a a nd C 9a ) ,148.3 (C2) , 149.3 (Px CI ' ) , 150.53, 150.67 (Px C4 a and

C l 0 a ) , 1 5 5. 8 ( F m o c C O ) , 1 6 1 .6 (H R - F A B M S : e x a c t m a s s

found 760 .2659 (M + H ) , c a l c u l a t e d fo r ( C 4 6 H 3 7 N 3 0 8 ) q-

H 760 .2659. R F 0 .21 (e thyl ace ta te ) .

M a s s s p e c t r o m e t r y

S a m p l e s f o r F A B - M S m e a s u r e m e n t s w e r e s u s p e nd e d i n

a p o l y e th y le n e g l y c o l ( 6 0 0 ) / t h i o g l y c e r o l / g l y c e r o l / D M S O

ma t r ix . In t he c a se o f t he nuc l e os ide p hosph ora m id i t e , i t

w a s ne c e ssa ry to i nc lude 3 -n i t robe nz y la l c oho l a nd t r i e th -

y l a mine in t he ma t r ix t o ob ta in a s t rong mo le c u la r i on . The

i o n i s a t i o n g a s w a s X e . I o n - s p r a y m a s s s p e c t r o m e t r y m e a -

s u r e m e n t s w e r e p e r f o r m e d o n a P e r k i n E l m e r P E S C I E X

L C / M S , i n bo t h th e p o s i t iv e a n d n e g a ti v e i o n is a t io n

m o d e s . T h e s a m p l e s w e r e p r e p a r e d a c c o r d i n g t o t he m e t h o d

of R e ddy a nd Ide n [21 ] .

2 .3 . Sy n the s i s o f 5 -p r opy n y lam ino nuc le os ide phos pho-

r am id i t e

5 - ( 3 - ( F l u o r e n - 9 - y l m e t h o x y c a r b o n y l ) a m i n o ) p r o p - l- y n - 1 -

y l ) -5 ' -O - ( 9 -phe ny lx an the n - 9 - y l ) -2 ' -de ox y ur id ine 3

The re a c t ion c ond i t i ons u se d w e re i de n t i c a l t o t hose

p re v ious ly de sc r ibe d [15 ] bu t mod i f i c a t ions w e re ma de to

S y n t h e s is o f n u c l eo s i d e p h o s p h o r a m i d i t e 4

T h e n u c l e o s i d e p h o s p h o r a m i d i t e 4 w a s s y n t h e s i s e d o n a

2 .2 mmol sc a l e by the me thod o f S inha [6 ] e xc e p t t ha t a

3 - f o l d e x c e s s o f p h o s p h i t y l a t i o n r e a g e n t a n d d r y

d i c h l o r o m e t h a n e w e r e u s e d . T h e r e a c t i o n w a s m o n i t o r e d

b y T L C ( 9 9 % e t h y l a c e t a t e / t r i e t h y l a m i n e ) a n d w a s c o m -

p le t e a f t e r 80 min . The c rude r e s idue w a s pu r i f i e d by ra p id

f i l t r a t i on c h roma tog ra phy (20 g s i l i c a ge l fo r 2 .2 mmol

s c a l e r e a c t i o n ) . T h e c o l u m n w a s p r e - w a s h e d w i t h e t h y l

a c e t a t e / d i c h l o r o m e t h a n e / t r i e t h y l a m i n e ( 4 5 :4 5 : 1 0, 1 X

c o l u m n v o l u m e ) f o l l o w e d b y e t h y l a c e t a t e / d i c h l o r o -

m e t h a n e ( 5 0 :5 0 , 2 X c o l u m n v o l u m e ) . T h e s a m p l e w a s

l o a d e d o n t h e c o l u m n i n e t h y l a c e t a t e / d i c h l o r o -

m e t h a n e / t r i e t h y l a m i n e ( 5 0 : 4 9 : 1 ) . T h e c o l u m n w a s t h e n

e l u t e d w i t h e t h y l a c e t a t e / d i c h l o r o m e t h a n e / t r i e t h y l a m i n e

o f the fo l low ing c omp os i t i ons : 50 :49 :1 , 75 :24 :1 , a nd

98:1:1 .

The f r a c t ions c on ta in ing the de s i r e d p roduc t w e re

poo le d , t he so lve n t w a s r e move d in va c uo , a nd the r e su l t -



262 P. Hag mar et al. / Biochimica et Biophysica Acta 1244 (1995) 259 268

i ng th i c k sy rup w a s r e d i sso lve d in t o lue ne (4 ml ) a nd

p re c ip i t a t e d f rom ra p id ly s t i r r i ng he xa ne (30 ml ) a t room

te mpe ra tu re . The f ine c o lou r l e ss p re c ip i t a t e w a s c o l l e c t e d

by g ra v i ty f i l tr a t i on , a i r -d r i e d a nd d ri e d in va c uo - (16 h )

( 1 . 1 4 g , 5 5 % ) . T L C ( 9 9 % e t h y l a c e t a t e / t r i e t h y l a m i n e )

s h o w e d t w o c l o s e s p o t s ( R F 0 .3 7 a n d 0 . 4 4 ) c o r r e s p o n d i n g

to the tw o d i a s t e re o i som e rs o f t he p roduc t . Pa r t i a l ~H - N M R

(C D 2C 12) mix tu re o f d i a s t e re o i som e rs : 6 3 .05 , 3 .07 (dd ,

1H , H 5 ' , J = 7 .6 , 2 .9 H z ) , 4 .55 (m , 1H , H 3 ' ) , 6 .26 , 6 .28

( d d , I H , H I ' , J = 6 . 0, 1 .4 H z ) , 7 . 0 0 - 7 . 8 0 ( m , 2 1 H , F m o c

a nd Px a rom a t i c (H ) , 8 .31 , 8 .34 ( s , 1H , H 6) . 31P-N M R

(CD2C12): 6 148.8 and 149.1 . FABMS: m / z 959.4 .

H R - F A B M S : e x a c t m a s s f o u n d 9 6 0 .3 7 2 9 ( M + H ) , c a l c u -

la ted form (C55H54N,509P) + H 960 .3737 .

Synthesis o f oligonucleotidesT w o 4 2 b p D N A f r a g m e nt s ( 4 2 A / 4 2 B a n d N S I / N S 2 ,

se e F ig . 1 ) w i th se que nc e s t a ke n f rom the 5 ' r e gu la to ry

r e g i o n o f t h e ~rR ge ne w e re syn the s i se d . The se que nc e s

c o r r e s p o n d t o t h e f o l l o w i n g r e g i o n s o f ~,rR: 4 2 A / 4 2 B , b p2 1 8 - 2 5 9 ; N S 1 / N S 2 , b p 2 8 2 - 3 2 3 [ 22 ]. O n e o f t h e o l ig o -

nuc le o t ide s , 42A , w a s a l so syn the s i se d w i th t he mod i f i e d

de oxyur id ine r e p l a c ing the t hymid ine a t pos i t i on 7 , 19 o r

22 f rom the 5 ' - e nd ( (7 )42A , (19 )42A a nd (22 )42A ) . T he 42

b p o l i g o n u c l e o t i d e s w e r e c h o s e n b e c a u s e 1 0 b p f l a n k i n g

re g ions on e i t he r s ide o f t he 22 bp c onse nsus se que nc e

w e re found to i nc re a se t he b ind ing a f f in i ty fo r Ty rR .

O l i g o n u c l e o t i d e s w e r e s y n t h e s i s e d o n a n A p p l i e d B i o s y s -

t e rn s Mode l 381A syn the s i z e r , u s ing s t a nda rd /3 -c ya noe thy l

p h o s p h o r a m i d i t e c h e m i s t r y . T h e m o d i f i e d p h o s p h o r a m i d i t e

w a s u s e d i n p l a c e o f t h e n o r m a l t h y m i d i n e p h o s p h o -

ra mid i t e a t t he spe c i f i e d pos i t i ons i n t he 42A se que nc e .Fo r t h i s c oup l ing s t e p , t he mod i f i e d phosphora mid i t e so lu -

t i on w a s p re pa re d a t tw ic e the c onc e n t ra t i on (0 .2 M in

a n h y d r o u s a r g o n - d e g a s s e d a c e to n i t ri l e ) o f t h e n o r m a l p h o s -

pho ra mid i t e so lu t ions , a nd the c oup l ing t ime w a s e x t e nde d

to 15 ra in (no rma l ly 30 s ) . The o l igonuc le o t ide s w e re

c l e a v e d o f f t h e s u p p o r t w it h 2 5 % a m m o n i u m h y d r o x i d e a s

t r i t y l -on de r iva t ive s , a nd subse que n t ly de p ro t e c t e d in c on -

c e n t r a t e d a m m o n i u m h y d r o x i d e ( 3 5 % ) a t 5 5 ° C f o r 1 8 h .

The o l igonuc le o t ide s w e re t he n d r i e d in a va c uum c e n -

t r i fuge . A d rop o f t r i e thy la mine w a s a dde d e ve ry 30 ra in

du r ing d ry ing to ke e p the so lu t ion ba s i c i n o rde r t o p re ve n t

de t r i t y l a ti on . Th e y i e ld s fo r 1 / xmo l syn the se s o f o l igo -

nuc le o t ide s c on ta in ing the mod i f i e d u r id ine ba se w e re

a b o u t 5 0 % a s j u d g e d f r o m t h e D N A b a s e a b s o r p ti o n a t 2 6 0

nm. Th i s i s c ompa ra b le t o t he y i e ld s ob ta ine d u s ing s t a n -

d a r d p h o s p h o r a m i d i t e s .

T h e o l i g o n u c l e o t i d e s w e r e c h a r a c t e r i s e d b y P A G E , r e -

v e r s e - p h a s e H P L C a n d U V s p e c t r o p h o t o m e t r y . F o r t h e

P A G E a n a l y s i s , 5 - 1 0 / z g o f ea c h o l i g on u c l e o t id e w a s

loa de d on a 1 mm th i c k 20% a c r y l a m ide gel a nd e le c -

t r o p h o r e s ed a t 1 . 70 W / g e l c o n s t a nt p o w e r i n 1 × T B E f o r

17 h . G e l s w e re s t a ine d w i th 0 .02% me thy le ne b lue so lu -

t i on fo r 30 min , de s t a ine d fo r a f e w hou rs a nd pho to -

g r a p h e d . R e v e r s e - p h a s e H P L C w a s c a r r ie d o u t o n a C - 1 8

c o l u m n ( f l o w r a t e 1 m l / m i n ) w i t h a g r a d i en t f r o m 1 0%

a c e ton i t r i le in 100 mM a que ous t r i e th y la m mo nium a c e t a t e

(pH 7 .0 ) ( so lve n t A ) t o 100% a c e ton i t r i l e ( so lve n t B ) a t

1% inc re a se i n a c e ton i t r i l e /min fo l low ing a 4 min i so -

c ra t i c s t e p o f so lve n t A (G ra d ie n t 1 ) . Mo la r a bso rp t iv i t i e s

fo r t he o l igonuc le o t ide 42me rs c a l c u l a t e d f rom the se -

quen ces [23] were as fo l lows : 42A, E260 = 4 .41 • 105 M l

c m -1" , 42B , ~260=5.01 " 105 M I cm 1; NS 1, e260 =4 . 4 4 . 1 05 M i c m 1 ; N S 2 , e 2 6 0 = 4 . 4 4 . 1 0 5 M i c m - 1 .

N o c o r re c t ion due to t he p re se nc e o f t he mod i f i e d de -

oxyu r id ine in s t e a d o f t hym id ine w a s m a de due to t he sma l l

c ha nge in ove ra l l a b so rp t iv i ty t h i s subs t i t u t i on c a use s .

FITC-labellingThe 42me r c on ta in ing the mod i f i e d u r id ine ba se (40

nmol ) w a s d r i e d in a va c uum c e n t r i fuge . The sa mple w a s

the n d i sso lve d in 50% a c e t i c a c id in w a te r (200 /~1 ) a nd

i n c u b a t e d a t r o o m t e m p e r a t u r e f o r 2 0 m i n t o r e m o v e t h e

d ime thoxy t r i t y l g roup . The sa mple w a s d r i e d a ga in a nd

re s idua l d ime thoxy t r i t y l a l c oho l w a s r e move d by e tha no lp re c ip i t a t i on . The d r i e d o l igonuc le o t ide w a s r e d i sso lve d in

200 m M sod ium c a rbona te bu f fe r (40 /~1 ) (pH 10 ) t o g ive

a c o n c e n tr a t io n o f a b o u t 1 m M . A 2 5 0 m M s o l u ti o n o f

F I T C in d i m e t h y l f o r m a m i d e ( 8 / x l ) w a s a d d e d t o g i v e a

50 - fo ld mo la r e xc e ss o f dye to o l igonuc le o t ide . A f t e r 4 h

inc uba t ion a t room t e mpe ra tu re i n t he da rk , a dd i t i ona l

F ITC (250 mM, 8 / .d ) w a s a dde d a nd the r e a c t ion a l l ow e d

to p roc e e d fo r a fu r the r 18 h . U nre a c t e d F ITC w a s r e -

mo ve d by ge l f i l t ra t i on th rough Se p ha de x G -25 in 100 mM

t r i e t h y l a m m o n i u m a c e t a t e . T h e e l u a t e w a s c o l l e c t e d a n d

c ha ra c t e r i se d by a bso rp t ion a t 260 nm a nd 495 nm. The

f l u o r e s c e i n - l a b e l l e d 4 2 m e r w a s p u r i f i e d b y r e v e r s e - p h a s e

TyrR BO X

T G T G T C A A T G A T T G T T G A C A G A*' - T T T C C G T C T T A A C C T T C C T G - 3 ' 4 2 A

3 ' - A A A G G C A G A A A C A C A G T T A C T A A C A A C T G T C T T T G G A A G G A C - 5 ' 4 2 B

5 ' - N N T G T A A A N N N N N N T T T A C A N N , - 3 ' C o n s e n s u s

5 ' - T C A T A T T A A T T G T T C T T T T T T C A G G T G A A G G T T C C C A T G C G T - 3 ' N S I

3 ' - A G T A T A A T T A A C A A G A A A A A A G T C C A C T T C C A A G G G T A C G C A - 5 ' N S 2

F i g . 1 . S e que nc e s o f syn t he s i s e d o l i gonuc l e o t i de s . T he a s t e r i sks i nd i c a t e t hym i d i ne nuc l e o t i de s t ha t w e r e r e p l a c e d by t he m od i f i e d de oxyur i d i ne ( one

r e p l a c e me n t i n e a c h o l i gonuc l e o t i de ) t o p r ov i de t he f l uo r e sc e n t l y l a be l l e d o l i gonuc l e o t i de s .



P . H agm a r e t al , / B i oc h i m i c a e t B i ophy s i c a A c t a 1244 ( 1995) 2 59- 268 2 6 3

C l 8 H P L C u s i n g t he f o l l o w i n g e l u t io n s e q u e n c e ( G r a d i e n t

2 ) : (1 ) 4 r a in i s oc ra t i c e lu t ion w i th s o lve n t A , (2 ) s o lve n t B

i n c r e a se d b y l % / m i n f o r 8 m i n , ( 3 ) s o l v e n t B i n c r e as e d

b y 0 . 5 % / r a i n f o r 2 4 m i n ( t h e r e g i o n w h e r e t h e 4 2 m e r

e lu t e s ) , (4 ) s o lve n t B inc re a s e d to 100% in 4 r a in fo l low e d

by (5 ) a 10 min i s oc ra t i c s t e p a t 100% s o lve n t B . The f low

r a te w a s 1 m l / m i n . T h e f l u o r e s ce i n - l a b e ll e d 4 2 m e r w a s

the n d r i e d in a va c uum c e n t r i fuge . The l a be l l i ng ra t io

d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l ly w a s i n t h e r an g e 0 . 9 -

1.1.

Annealing o f DNA

C o m p l e m e n t a r y 4 2 m e r s w e r e d i s s o l v e d in d i s ti ll e d w a -

t e r a nd p re c ip i t a t e d w i th e tha no l t o r e move pos s ib l e f luo -

r e s c e n t c o n t a m i n a n t s f r o m t h e H P L C s o l v e n t s a n d a n y

r e m a i n i n g t r i e t h y l a m m o n i u m a c e t a t e s a l t . T h e d r y p e l l e t s

w e r e d i s s o l v e d i n th e e x p e r i m e n t a l b u f f e r ( 1 0 0 m M K C 1,

2 5 m M K H 2 P O a - K 2 H P O 4 , 1 m M E D T A , 0 .1 m M D T T ,

0 . 0 2 % ( w / v ) N a N 3 ( p H 7 . 5 ) ) a n d t h e i r c o n c e n t ra t i o n s

d e t e r m i n e d b y a b s o r p t i o n s p e c t r o s c o p y . N o c o r r e c t i o n w a s

ma d e fo r t he a bs o rp t ion o f f luo re s c e in a t 260 nm s inc e the

c o r r e c ti o n w o u l d b e l e s s t h a n 3 % o f t h e D N A a b s o r p ti o n

[24 ]. Equ imo la r a mou n t s o f the tw o s t r a nds w e re mixe d

a nd inc uba te d a t 90°C fo r 5 min a nd the n c oo le d a t a r a t e

o f 0 . 5 % ° C / m i n . T h e p u r i t y o f t he d o u b l e s t r a n d ed o l i g o -

n u c l e o t i d e w a s m o n i t o r e d b y n a t i v e P A G E a n d b y D N A

me l t ing a na ly s i s .

TyrR preparation

TyrR w a s p re pa re d a s p re v ious ly de s c r ibe d [25 ] a nd

t ra ns fe r re d to the e xpe r ime n ta l bu f fe r by C e n t r i c on c e n t r i f -

uga t ion . To d e te rmin e the mo la r a bs o rp t iv i ty o f Ty rR [26] ,

a s a mp le w a s de na tu re d in 6 M gua n id ine hyd roc h lo r ide

fo r 2 h a t 0 ° C , a n d a bs o rb a nc e s m e a s u re d fo r t h re e na t ive

a nd de na tu re d p ro te in d i lu t ions . The mo la r a bs o rp t iv i ty o f

T y r R m o n o m e r s (EZS0) w a s found to be 3447 0 M t c m - ~.

3 . R e s u l t s

3.1. Synthesis of the modified phosphoramidite

W e h a v e p r e v i o u s l y r e p o r t e d t h e p r e p a r a t i o n o f t h e

Y -p ro te c t e d C 5-a lk yny l nuc le o s ide 3 (Sc h e me . 1 ) [15 ]. In

0

H N I

1 2

I I

2

N H 4 0 . ,~

o ~-._ o. 92',, J ~ N~-~ a ,. ~ ~ "rF "~.?~

, o

o .

6 7

l(b)

N _ _ _ _ _ _ _ f

S c h em e 1 . ( a) ( P h 3 P ) 4 P d ° / C u I / E t 3 N / D M F . ( b ) 2-Cyan•ethy•-N•N-diis•pr•py•ch••r•ph•sph•ramidite/diis•pr•py•ethy•amine/dichl•r•methane.



26 4 P. Hagmar et al. / Biochimica et Biophysica Ac ta 1244 (I995) 2 59- 268

t ha t s tudy , c oe lu t ion o f t he s t a r ti ng ma te r i a l Px ldU 1 w i th

t h e p r o d u c t 3 u n d e r a v a r i e t y o f T L C c o n d i t i o n s m a d e

mon i to r ing o f t he c oup l ing re a c t ion a nd the subse que n t

pu r i f i c a t ion d i f f i c u l t . W e ha ve now found tha t 100% e thy l

a c e t a t e i s a n e xc e l l e n t TLC so lve n t t ha t r e so lve s t he

s t a r t i ng ma te r i a l f rom the p roduc t . I t s u se a l l ow s the

re a c t ion to be mon i to re d a nd the p roduc t t o be e f fe c t ive ly

p u r if i ed . P a l l a d i u m ( 0 ) - c a t a ly s e d c o u p l i n g o f t h e F m o c - a l -

kyne 2 w i th 5 ' -p ixy l - IdU 1 ga ve the de s i r e d a lkyny l nuc l e -

o s ide 3 i n y i e ld s r a ng ing be tw e e n 35 a nd 60%. U n l ike the

a n a l o g o u s r e a c t i o n s b e t w e e n 5 ' - p i x y l - I d U 1 a n d p r o p a r g y -

l a mine s p ro t e c t e d w i th a c id - l a b i l e B oc a nd P ixy l g roups , a

ma jo r f l uo re sc e n t s ide -p roduc t w i th a s ign i f i c a n t ly l ow e r

R F w a s fo rm e d du r ing the w ork -u p . In i t i a l a na ly s i s o f t h i s

s i d e - p r o d u c t b y F A B m a s s s p e c t r o m e t r y w a s i n c o n c l u s i v e ,

w i th on ly low mole c u la r f r a gme n t i ons de t e c t e d . Subse -

q u e n t a n a l y s i s b y i o n - s p r a y m a s s s p e c t r o m e t r y g a v e p s e u -

d o m o l e c u l a r i o n s o f 53 8 ( M + H ) a n d 5 3 6 ( M - H ) , u s i n g

pos i t i ve a nd ne ga t ive i on sou rc e s , r e spe c t ive ly . B y a na logy

w i th the w ork o f R ob ins a nd B a r r [27 ], t he f l uo re sc e n ts ide -p rodu c t w a s a ss igne d the s t ruc tu re o f 5 . H ow e v e r ,

e f f i c i e n t pu r i f i c a t ion p roc e du re s a l l ow the de s i r e d nuc le o -

s ide 3 t o be i so l a t e d in h igh pu r i t y . Phosph i ty l a t i on a t t he

3 ' - h y d r o x y l o f 3 g a v e t h e d e s i r e d m o d i f i e d p h o s p h o -

ra mid i t e 4 . The ~H - a nd ~3C -N MR spe c t ra o f nuc l e os ide

Y - p h o s p h o r a m i d i t e s a r e c o m p l e x d u e t o t h e e x i s t e n c e o f

the R p a nd S p d i a s t e re o i some rs a nd IH-31p a nd 13C-31pc oup l ing . W e the re fo re c ha ra c t e r i se d the t a rge t a mid i t e 4

by a c om bina t ion o f 3~ p_ a nd I H -N M R spe c t ro sc o py , a nd

h i g h - r e s o l u t i o n F A B m a s s s p e c t r o m e t r y . T h e 3 ~ p - N M R

spe c t rum sh ow e d tw o re sona nc e s a t 8 148.8 a nd 149.1,

i n d i ca t iv e o f t w o p h o s p h o r a m i d i t e d i a s t er e o m e r s . I n c o n -t r a s t t o a n e a r l i e r r e po r t [28 ] , w e f ind FA B ma ss spe c t rom-

e t ry i n t he pos i t i ve i on i sa t ion mode to be a n e xc e l l e n t

m e t h o d f o r c h a r a c te r i s in g n u c l e o s i d e p h o s p h o r a m i d i t e s . B y

e m p l o y i n g a m a t r i x w h i c h c o n s is t s o f a ' c o c k t a i l ' o f

po lye th y le ne g lyc o l (M r 600 ) , t h iog lyc e ro l , g lyc e ro l ,

D MSO , t r i e thy la mine , a nd 3 -n i t robe nz y l a l c oho l , i t i s pos -

s ib l e t o ob ta in s t rong mo le c u la r i ons fo r t he mod i f i e d

phos phora mid i t e 4 a nd a l so a ser i e s o f 5 ' nuc l e os ide

p h o s p h o r a m i d i t e s ( d a t a t o b e p u b l i s h e d e l s e w h e r e ) . T h e

a b i l i t y t o ob ta in a h igh re so lu t ion p se udomole c u la r i on

( M + H ) o f n u c l e o s id e p h o s p h o r a m i d i t e s p r o v i d e s a n a d d i-

t i ona l w a y o f c ha ra c t e r i s ing the se c omple x a nd l a b i l e

m o l e c u l e s . T h e p s e u d o - m o l e c u l a r i o n o f 9 60 . 3 7 2 9 i s c o n -

s i st e n t w i t h t h e m o l e c u l a r f o r m u l a o f 4 .

3.2. Synthesis an d labelling o f oligonucleosides

T h e m o d i f i e d a n d n o r m a l o l i g o n u c l e o t i d e s u s e d i n t h i s

s tudy w e re syn the s i se d u s ing s t a nda rd so l id -pha se /3 -

c y a n o e t h y l p h o s p h o r a m i d i t e m e t h o d o l o g y [ 5 , 6 ] . I t w a s

though t de s i r a b l e t o l e ng the n the s t a nda rd c oup l ing t ime o f

30 s t o 15 min a nd a l so to i nc re a se t he c onc e n t ra t i on o f t he

so lu t ion f rom the u sua l 0 .1 M to 0 .2 M to e nsu re ma x i -

m u m c o u p l i n g e f f i c ie n c y o f th e m o d i f i e d p h o s p h o r a m i d i t e .

<: .<

C~ Or?

O,2 <~ U'~

C',2 0, 2 ~_~

... .. . . . . . . .......... ........

a b c d e f g h

F i g . 2 . E l e c t r ophor e s i s (7 M u r e a , 20% p o l ya c r y l a m i de ge l ) o f t he 42 bp

o l i gonuc l e o t i de w i t h t he mod i f i e d de oxyur i d i ne a t pos i t i on 22 f r om t he

Y - e nd ( ( 22 ) 42A ) : ( a ) c r ude t r i t y l -O N r e a c t i on mi x t u r e , ( b ) H P L C- pur i f i e dt r i t y l - O F F sa mpl e , ( c ) c r ude r e a c t i on mi x t u r e a f t e r F I T C c on j uga t i on , ( d )

H P L C- pur i f i e d f l uo r e sc e i n - l a be l l e d 42me r , ( e ) unm odi f i e d t r i ty l - O F F 42A .

O l i gonuc l e o t i de mod i f i e d a t pos i t i on 19 f r om t he 5 '~e nd ( ( 19 ) 42A ) : ( f )

c r ude t r i t y l - O N r e a c t i on mi x t u r e , ( g ) c r ude r e a c t i on mi x t u r e a f t e r F I T C

c on j uga t i on , ( h ) H P L C - pur i f i e d f l uo r e sc e i n - l a be l l e d 42me r .

T h e c o u p l i n g e f f i c i e n c y o f t h e m o d i f i e d p h o s p h o r a m i d i t e ,

a s a sse sse d by t r i t y l a ssa ys be fo re a nd a f t e r i t s a dd i t i on ,

w a s c o m p a r a b l e t o t h a t o f n o r m a l p h o s p h o r a m i d i t e s ( b e -

tw e e n 98 to 100%) . A na lys i s o f t he c rude 5 ' - t r i t y l -O N

ol igonuc le o t ide s by 20% de na tu r ing PA G E (F ig . 2 , l a ne s a

a nd f ) i nd i c a t e d a h igh de g re e o f pu r i t y w i th no de t e c t a b l ea m o u n t s o f f a il u r e s e qu e n c e s . R e v e r s e - p h a s e H P L C a n a l y -

s i s o f one o f t he se c rude m od i f i e d o l igonuc le o t ide s (o l igo -

nuc le o t ide 42A w i th t he mod i f i e d u r id ine in pos i t i on 22

f r o m t h e Y - e n d ) s h o w e d t w o m a j o r p e a k s w i t h r e te n t io n

t ime s o f 17 .3 min a nd 24 .8 min (F ig . 3A ) . The pe a k a t

24 .8 min w a s c o l l e c t e d , de t r i t y l a t e d in t he u sua l ma nne r

a n d t h e n r e - a n a l y s e d b y H P L C ( F i g . 3 B ) a n d P A G E ( F i g .

2 , l a ne b ). B o th a n a ly se s i nd i c a t e d the p re se nc e o f on ly

one spe c i e s . I t i s no t e w or thy tha t no t on ly i s t he re t he

e xpe c te d s ign i f i c a n t d i f f e re nc e in r e t e n t ion t ime s be tw e e n

t r i t y l -O N a nd t r i t y l -O FF o l igonuc le o t ide s on re ve r se -pha se

H PLC , bu t t he re i s a l so a no t i c e a b le r e duc t ion in t he

e l e c t ropho re t i c mob i l i t y o f t r i t y l -O N o l igonuc le o t ide s r e l a -

t i v e t o t r i t y l - O F F s a m p l e s i n P A G E a n a l y s i s . C o n s e -

que n t ly , pu r i f i c a t ion by e i t he r me thod i s s t r a igh t fo rw a rd .

In ou r c a se , t he pu r i t y o f t he c rude o l igonuc le o t ide s w a s

su f f i c i e n t fo r u se i n l a be l l i ng s tud ie s w i thou t p r io r pu r i f i c a -

t ion .

Y i e l d s o f t h e D N A - f l u o r e s c e i n c o n j u g a te s a l w a y s e x -

c e e de d 60%. F ig . 2 ( l a ne s c a nd g ) show s the e l e c -

t ropho re t i c pa t t e rn s o f t he c rude r e a c t ion mix tu re s . The

dye - l a be l l e d o l igonuc le o t ide ha s r e duc e d e l e c t ropho re t i c

mob i l i t y c ompa re d w i th t he un la be l l e d spe c i e s a nd c ou ld

be pu r i f i e d e a s i ly f rom a ge l . Pu r i f i c a t ion by re ve r se -pha se



P. Hagmar et al. / Biochimica et Biophysica Acta 1244 (1995) 259 -26 8 26 5

0r.DO,2

A

B

o

r~,.Q

o5o

,.o

I I0 1 0 2 0 3 0

E l u t i o n t i m e { m i n u t e s )

F i g . 3 . H P L C c h r o m a t o g r a m s o f ( 2 2 ) 4 2 A . ( A ) C r u d e t r it y l -O n r e a c t i o n

mixture (Gradient 1). (B) Re-chromatographed detritylated 42mer (Gradi-

ent 1). (C) Crude reaction mixture after FITC conjugation (Gradient 2).

(D) H PLC-purified fluorescein-labelled 42m er (Gradient 1). See text for

details of gradients.

H P L C w a s p r e f e r r e d s i n c e l a r g e r a m o u n t s o f m a t e r ia l

c o u l d b e p r o c e s s e d a t a n y o n e t i m e . A c h r o m a t o g r a m o f a

p u r i f i c a t i o n o f c ru d e l a b e l l e d 4 2 m e r i s s h o w n i n F i g . 3 C .

F l u o r e s c e i n - l a b e l l e d 4 2 m e r e l u t e d a t 2 2 m i n ( t h e l a s t m a j o r

p e a k ) a n d w a s s h o w n t o b e p u r e b y P A G E ( F i g . 2 , l a n e s d

a n d h ) a n d H P L C ( F i g . 3 D ) .

3 . 3 . A n n e a l i n g a n d t h e r m a l a n a l y s i s

A n n e a l e d o l i g o n u c l e o t i d e s , w h e t h e r c o n t a i n i n g t h e

m o d i f i e d d e o x y u r i d i n e o r n ot , m i g r a t e a s s in g l e b a n d s o f

t h e s a m e m o b i l i t y o n n a t i v e p o l y a c r y l a m i d e g e l s ( F ig . 4 ).

T h e s m a l l a m o u n t s o f s i n g l e - s t r a n d e d m a t e r i a l ( F i g . 4,

l a n e s a , d ) a r e d u e t o a s l i g h t e x c e s s o f t h e u n l a b e l l e d

s t r a n d a d d e d d u r i n g s a m p l e p r e p a r a t i o n . T h i s e x c e s s e n -

s u r e s t h a t n o s i n g l e - s t r a n d l a b e l l e d m a t e r i a l i s p r e s e n t

w h i c h w o u l d o t h e r w i s e i n t e r f e r e w i t h f l u o r e s c e n c e a n i s o t -

r o p y m e a s u r e m e n t s . T h e s t e a d y - s t a t e f l u o r e s c e n c e a n i s o t -

r o p y o f f l u o r e s c e i n - l a b e l l e d s i n g l e - s t r a n d e d 4 2 m e r ( e x c i t a -

t i o n a t 4 9 5 r i m , s li t w i d t h 2 . 5 r i m , a n d e m i s s i o n a t 5 2 0 n m ,

s l it w i d t h 2 0 n m ) i s 0 . 0 9 . I n t h e d o u b l e - s t r a n d e d c o m p l e x ,

t h e a n i s o t r o p y i n c r e a s e s s l i g h t l y to 0 . 1 0 .

D N A m e l t i n g c u rv e s f o r t h e u n m o d i f i e d 4 2 m e r

O 2 O 2

•~ . , ~ o 2

O 2 0 2 0 ,2 U ]

o~ o 2

• ~ 02 09

~ ~ Z

- -+ - - + - + - +

I >~ o 2 o 2

a b c d e f g h i j k

Fig. 4. The effect of TyrR on DN A 42m ers in PA GE . Labelled and

unlabelled oligonucleotides (2.5 pmol) in the absence (- ) and presence

(+ ) of 5-fold molar excess TyrR: unmodified 42A /42B (a,b); f luores-

cein-labelled in position 19 (c,d) and position 22 (e,f); unlabelled nonspe-

cific control sequence NS I/N S2 (g,h). 25 pm ol TyrR dimer ( i) , 14 pmol

single-stranded 42A (j), and 42B (k).

4 2 A / 4 2 B a n d tw o f l u o r e s c e i n -l a b e l le d d u p l e x s a m p l e s in

t h e e x p e r i m e n t a l b u f f e r a r e s h o w n i n F ig . 5 . A s a n a l y s e d

b y t h e t w o - s t a t e m i d p o i n t m e t h o d o f B r e s l a u e r e t a l . [ 2 9 ] ,

u n m o d i f i e d 4 2 A / 4 2 B h a s a m e l t i n g p o i n t o f 6 5 .9 ° C . F o rt h e f l u o r e s c e n t o l i g o n u c l e o t i d e s l a b e l l e d i n t h e 1 9 a n d 2 2

p o s it io n ( F ( 1 9 ) 4 2 A / 4 2 B a n d F ( 2 2 ) 4 2 A / 4 2 B ) , t he m e lt in g

t e m p e r a t u r e s a r e 6 3 . 2 ° C a n d 6 3 . 8 ° C , r e s p e c t i v e l y . T h e

s l i g h t d e s t a b i l i s a t i o n o f t h e c o m p l e x m a y r e s u l t f r o m t h e

i n t r o d u c t i o n o f t he n e g a t i v e l y c h a r g e d d y e i n t o a n e g a -

t i v e l y c h a r g e d p o l y e l e c t r o l y t e . F o r c o m p a r i s o n , t h e r e -

I I I I I [

1 . o

~ 0 . 9 o J ' 10 . 8 -

I I I I I I

8 0 4 0 5 0 6 0 7 0 8 0

T e m p e r a t u r e ( °C )

F i g. 5 . M e l t i n g c u r v e s o f 4 2 m e r s . N o r m a l i s e d a b s o r b a n c e ( 2 6 0 n m ) a s a

f u n c t i o n o f t e m p e r a tu r e f o r t h e u n la b e l l e d 4 2 A / 4 2 B ( b r o k e n l i n e) a n d

f l u o r e s c e in - l a b e l le d F ( 1 9 ) 4 2 A / 4 2 B a n d F ( 2 2 ) 4 2 A / 4 2 B ( s o li d li n e s) .



266 P. Hagmar et al. / Biochimica et Biophysica Ac ta 1244 (1995) 259 -268

placement of one G-C base pair for an A-T base pair (and

thus the removal of one hydrogen bond) in a 42met is

expected to give a l°C reduction in melting temperature

[30,31]. The increase in absorbance at 260 nm of the

unmodified 42A/42B from room temperature to the com-

pletion of chain melting is 30%. The corresponding value

for F(19)42A/42B and F(22)42A/42N is 29% indicating

that the dye label does not cause any significant decrease

in the base pairing.

3.4. Gel re tardation assay

The fragments 42A/42B and NSl/NS2 have identical

sequences to segments of the 5' regulatory region of the E.

co l i gene tyrR (Fig. 1). 42A/42B contains a strong TyrR

box, whereas NSI/NS2 does not. We have performed

DNase I footprinting experiments with large DNA frag-

ments containing the entire 5' tyrR regulatory region,

including the segments in 42A/42B and NSI/NS2 (P.

Maroudas and B.E. Davidson, unpublished data). Theseexperiments showed that the TyrR box equivalent to that

in 42A/42B bound TyrR in the presence or absence of

tyrosine, whereas NSI/NS2 DNA did not bind TyrR

under any conditions. We therefore carried out gel retarda-

tion assays to determine if the TyrR box was still able to

bind TyrR when present in the smaller fragment

(42A/42B) and the effect, if any, of the fluorescein moi-

ety on the TyrR-binding properties.

The electrophoretic mobility of 42A/42B is markedly

decreased by the presence of TyrR (Fig. 4, lanes a and b),

indicating binding of the protein to the fragment. The

presence of TyrR does not affect the mobility of NS 1/N S2(Fig. 4, lanes g and h). We conclude that non-specific

binding of TyrR to DNA does not occur under the condi-

tions of the experiment and that retardation o f 42A /4 2B is

due to sequence-specific binding of TyrR to the TyrR box.

The mobilities of derivatives labelled with fluorescein at

positions 19 and 22 also decreased in the presence of TyrR

(Fig. 4, lanes c-f). The extent of the decrease is the same

as that seen with unlabelled 42A/42B. This result shows

that the fluorescein group at positions 19 and 22 does not

prevent the binding of TyrR to the TyrR box in these

fragments.

The assays shown in Fig. 4 were carried out with a

5-fold molar excess of TyrR dimer over DNA fragment.

Since this excess could have obscured small differences in

the protein binding properties of the fragments, we carried

out gel retardation assays at a series of increasing concen-

trations of TyrR and a fixed concentration of either unla-

belled or fluorescein-labelled 42A/42B (Fig. 6A, results

for F(22)42A/42B not shown). Densitometric analysis

revealed no significant differences between the resulting

gel patterns, indicating identical affinities of the duplexes

for TyrR within the error limits of the experiment (Fig.

6B). Assuming a stoichiometry of one protein dimer boun d

per molecule of DNA fragment, curve fitting procedures

A 4 2 A / 4 2 B F ( 1 9 ) 4 2 A / 4 2 B

0 0 ----~ ' , '--~ t"g . O0 < O 0 C~ ",, '--~ -," ~ [ 'X2 CO LO

B 20

15

10

I I I I I I

v ~ -

V . . . . . . . . V

0 I I I I I I

0 1 2 3 4 5

[TyrR] t ( f fM)

Fig. 6. The effect of increasing TyrR concentration on the mobility ofDNA 42reefs in PAGE. A, lanes contained 1 /,tM of unlabelled(42A/42B) and fluorescein-labelled (F(I9)42A/42B) duplexes. Thenumbers above the lanes indicate the TyrR dimer/42mer ratio. B,

scanned intensities of the free DNA band for 42A/42B (circles) andF(19)42A/42B (triangles). The solid and broken lines are the fittedcurves representing dissociation constants of 200 nM and 500 riM,respectively.

gave a dissociation constant of 200-500 nM for the inter-

action of TyrR with the three DNA fragments under the

conditions used.

3 .5 . S tea dy - s ta te f luoresce nce an i so t ropy t i t ra t ions

The binding of TyrR to the labelled 42mers reported

here was not accompanied by any change in the intrinsic

fluorescence of the TyrR or in the fluorescein fluorescence

of the conjugates. Consequently, fluorescence anisotropy

becomes the most appropriate fluorescence method for

following complex formation. Fig. 7 shows the fluores-

cence anisotropy titration of a 42met labelled at position 7

(F(7)42A/42B) with TyrR under low salt (buffer plus 18

mM KCI) and high salt (buffer plus I00 mM KCI) condi-

tions; values of K d were 335 nM and 380 nM, respec-

tively. The specificity of TyrR binding is demonstrated by

the fact that no anisotropy increase is observed when

NSI/NS2 is used in the titration (Fig. 7). Although the

binding energy is only marginally greater under the low



P. Hagmar et al. / Biochimica et Biophysica Acta 1244 (1995) 259 -26 8 267

~>~ 0.14

e

03

0 " 1 2 ~

0u )Q )L.

0

0 . 1 0l a . .

0 0

0 0 0 0

I I I0 . 0 0 . 5 1 . 0 1 . 5

[TyrR] #MFig. 7. Fluorescence anisotropy titrations of fluorescein-labe lled duplexes

(F(7)42A/42B) (510 nM) with TyrR in a buffer containing 4.5 mM

KH 2P Oa /K 2H P Q , 0.1 mM ATP, 5 mM MgC12, pH 7.5, under low salt

(buf fer + 18 mM KCI) (~ ) or high salt (buf fer + 100 mM KCI) ( n)

conditions. The abscissa represents the total TyrR added. Excitation and

emission wavelengths were 495 nm and 520 nm, respectively.

Anisot ropies were mea sured 5 rain after the addition of each aliquot of

TyrR and are the average o f 3 readings. Solid lines are the nonlinear leas!

square s fit for the low salt (K d = 335 n M) and h igh salt ( K d = 380 nM)

titrations. Hollow circles are for the titration of NS 1/N S2.

sa l t c ond i t i on , a n i so t rop ie s i n t he p re se nc e a nd a bse nc e o f

TyrR w e re c ons i s t e n t ly h ighe r i n t he l ow sa l t bu f fe r .

4 . D i s c u s s i o n

T h e e x p e r i m e n t s d e s c r i b e d i n t h i s p a p e r s h o w t h a t

o l igonuc le o t ide s c a n be in t e rna l ly l a be l l e d by f luo re sc e n t

p robe s a t t he pos i t i on o f a t hymine ba se i n t he se que nc e .

T h e p r i m a r y a m i n o g r o u p u s e d f o r t h e c o n j u g a t i o n i s

a t t a c he d to C 5 o f t he de oxyur id ine v i a a ve ry sho r t a nd

r ig id a lkyne l i nka ge . Mode l l i ng s tud ie s show tha t t he

a l k y n e l i n k e r w i l l p r o j e c t o u t o f t h e m a j o r g r o o v e o f

d o u b l e - s t r a n d e d D N A . I n c o n t r a s t t o t h e p h t h a l i m i d e p r o -

t e c t ion g roup w h ic h c a n be d i f f i c u l t t o r e move [13 ] , t he

F m o c g r o u p i s r e a d i l y c l e a v e d u n d e r n o r m a l o l i g o n u c l e o -

t i de de p ro t e c t ion c ond i t i ons . B e ing ba se d on a s t a nda rd

/3 -c ya noe thy l phosphora mid i t e , t h i s de r iva t ive c a n be in -

c o rpo ra t e d e a s i ly i n to a n o l igonuc le o t ide u s ing e s t a b l i she d

a u t o m a t e d t e c h n iq u e s . S e v e r a l h u n d r e d m i c r o g r a m s t o a

f e w m i l l i g r a m s o f t h e m o d i f i e d o l i g o n u c l e o t i d e c a n b e

syn the s i se d a s r e qu i re d . The pa l l a d ium(0 ) -c a t a ly se d c ou -

p l i n g o f th e F m o c - p r o t e c t e d p r o p y n y l a m i n e 2 a n d t he

5 ' - p i x y l - 5 - i o d o d e o x y u r i d i n e 1 w a s h a m p e r e d b y t h e f o r m a -

t i o n o f s i g n i f i c a n t a m o u n t s o f a f u r a n o p y r i m i d i n o n e s i d e -

p roduc t a s a r e su l t o f c yc l i sa t i on w i th c onc omi ta n t l o ss o f

the Fmoc g roup . Th i s s ide r e a c t ion i s no t obse rve d in t he

c o u p l i n g o f b o x - o r p i x y l - p r o t e c t e d p r o p a r g y l a m i n e s t o

5 ' - p i x y l - 5 - i o d o d e o x y u r i d i n e u n d e r s i m i l a r c o n d it i o n s a n d

i s h e n c e m o s t p r o b a b l y d u e t o t h e r e m o v a l o f t h e b a s e - l a -

b i l e a m i n o p r o t e c t i n g g r o u p . H o w e v e r , m o d e r a t e t o g o o d

y ie ld s o f t he de s i r e d a lkyny l nuc l e os ide a re s t i l l ob t a ina b le

d u e t o e f f i c i en t c h r o m a t o g r a p h i c w o r k - u p .

O l igonuc le o t ide s w i th spe c i f i c de oxy thymid ine s r e -

p l a c e d b y t h e m o d i f i e d d e o x y u r i d i n e w e r e s y n t h e s i s e d i n

h igh y i e ld s a nd w e re found to l a be l w i th h igh e f f i c i e nc y .

T h e s e o l i g o n u c l e o t i d e s c a n b e p u r i f i e d b y p o l y a c r y l a m i d e

e l e c tr o p h o r e s i s o r b y t r i t y l - O N r e v e r s e - p h a s e H P L C . A l t e r-

na t ive ly , pu r i f i c a t ion ma y be in i t i a t e d a f t e r c on juga t ion o f

a p robe , r e su l t i ng in t o t a l pu r i f i e d y i e ld s o f a bou t 20%.

The 5 ' - e nd o f t he o l igonuc le o t ide i s s t i l l a va i l a b l e fo r

subse q ue n t 32p_ la be ll i ng i f r e qu i re d . Th e l a be l l e d o l igo -

n u c l e o t i d e s h y b r i d i s e w i t h c o m p l e m e n t a r y s t r a n d s t o f o r m

d o u b l e - s t r a n d e d D N A w i t h m e l t i n g c h a r a c t e r i s t i c s s i m i l a r

to those o f un la be l l e d D N A . G e l r e t a rda t ion a ssa ys i nd i -

c a t e d th a t th e b i n d in g a f f i n it y o f 4 2 A / 4 2 B f o r T y r R w a s

no t a f fe c t e d by the p re se nc e o f t he f l uo re sc e in l a be l (K d =

2 0 0 - 5 0 0 n M ) . G i v e n t h e u n c e r t a i n t y i n d e t e r m i n i n g t h e

a m o u n t o f o l i g o n u c l eo t i d e s b y t h e g e l s c a n n i n g p r o c e d u r ea nd the poss ib l e d i s to r t i on o f t he e qu i l i b r ium by se pa ra t ion

o f t h e fr e e a n d c o m p l e x e d d u p l e x e s o n t h e g e l , w e c h o s e

no t t o pu r sue the de t e rmina t ion o f t he b ind ing c ons t a n t t o

g re a t e r a c c u ra c y by ge l r e t a rda t ion a na ly se s .

S e v e r a l t h r e e - d i m e n s i o n a l s t r u c t u r e s o f D N A - p r o t e i n

c o m p l e x e s s h o w t w o r e c o g n i t i o n h a l f - s i t e s i n t h e D N A

s e p a r a te d b y o n e t u rn o f th e D N A h e l ix [ 3 2 - 3 6 ] . D N A -

pro te in i n t e ra c t ions oc c u r p re domina n t ly on one s ide o f t he

he l i c a l ba rre l , w i th t he ma jo r g roov e o f t he spa c e r D N A

be tw e e n the ha l f - s i t e s be ing tu rne d a w a y f rom the p ro t e in .

A p r o b e p o i n t i n g o u t f r o m t h e m a j o r g r o o v e o f t h e s p a c e r

re g ion shou ld thus i n t e r fe re min ima l ly w i th p ro t e in b ind -ing . The f luo re sc e in c on juga te s o f 42A w e re l a be l l e d a t

posi t ions 19 and 22 in the N 6 r e g i o n o f th e T y r R b o x . T h e

a b s e n c e o f a n y e f f e c t o f t h i s b u l k y g r o u p o n t h e T y r R

b ind ing a f f in i ty i nd i c a t e s t ha t Ty rR doe s no t i n t e ra c t w i th

t h e m a j o r g r o o v e i n t h i s r e g i o n o f t h e T y r R b o x . T h e

a na lys i s o f t he b ind ing p rope r t i e s o f o the r de r iva t ive s o f

42A in w h ic h the l a be l i s a t t a c he d to d i f f e re n t pos i t i ons

w i l l he lp t o de l ine a t e t he se i n t e ra c t ions more p re c i se ly .

T h e f l u o r e s c e i n - D N A c o n j u g a t e s r e p o r t e d h e r e h a v e

s t e a d y - s t a t e a n i s o t r o p i e s ( 0 , 1 0 - 0 . 1 3 ) c o n s i d e r a b l y l o w e r

tha n the l im i t ing a n i so t ropy fo r f l uo re sc e in (0 .39 ) [37 ] . A

42 bp dup le x a c t ing a s a r i g id rod w ou ld ha ve d i f fu s ion

c oe f f i c i e n t s fo r ro t a t i on a bou t i t s l ong a nd sho r t a x i s o f

DII = 1 .3 . 106 s ~ and D , = 1 .6 . 107 S -] , a nd the re fo re

c o r re l a t i on t ime s o f a bou t 770 n s a nd 62 n s , r e spe c t ive ly .

C ons id e r ing the sho r t l if e - t ime o f f l uo re sc e in (2 -4 n s ) , the

low a n i so t rop ie s sugge s t t ha t e ve n w i th a r e l a t i ve ly sho r t

l i nke r suc h a s t he a minopropyny l g roup the p robe unde r -

goe s subs t a n t i a l l oc a l mo t ion . Inde e d , t ime - re so lve d f luo -

r e s c e n c e s t u d i e s t o b e r e p o r t e d e l s e w h e r e s h o w t h a t 4 7 -

52% o f t he a n i so t ropy de c a y i s due to f a s t mo t ion o f t he

p robe w i th a c o r re l a t i on t ime o f 16 -340 ps (B a i l e y e t a l . ,

unpub l i she d da t a ) . I t is poss ib l e t ha t t he h ighe r a n i so t rop ie s

obse rve d a t l ow ion ic s t r e ng th a re due to a more r e s t r i c t e d



26 8 P . H a g m a r e t al . / B i o c h i m i c a e t B io p h y s i c a A c ta 1 2 4 4 (1 9 9 5 ) 2 5 9 - 2 6 8

m o t i o n o f t h e p ro b e a t it s p o i n t o f a t t a c h m e n t t o t h e D N A

b r o u g h t a b o u t b y e l e c t r o s t a t i c r e p u l s i o n b e t w e e n t h e a n -

i o n i c f l u o r e s c e i n m o i e t y a n d t h e c h a r g e d p h o s p h a t e b a c k -

b o n e o f t h e D N A . F o r t he F ( 7 ) 4 2 A / 4 2 B c o n j u g at e , b in d -

i n g o f T y r R c a u s e d l i tt l e c h a n g e i n t h e f a st c o r r e l a t i o n t i m e

i n d i c a t i n g t h a t th e f a s t m o t i o n o f t h e p r o b e a t i ts p o i n t o f

a t ta c h m e n t w a s p r e s e r v e d i n t h e D N A - T y r R c o m p l e x .

I n t e r n a l l y f l u o r o p h o r e - l a b e l l e d o l i g o n u c l e o t i d e s s h o u l d

p r o v e p a r t i c u l a r l y u s e f u l f o r s t u d y i n g t h e b i n d i n g o f re -

p r e s s o r p r o t e in s t o m u l t i p l e o p e ra t o r s o n D N A . F o r e x a m -

p l e , f o u r o f t h e e i g h t t r a n s c r i p t i o n a l u n i t s r e g u l a t e d b y

T y r R c o n t a i n a s t ro n g a n d a w e a k T y r R b o x i n c l o s e

p r o x i m i t y i n t h e s e q u e n c e [ 1 8 ] . B y c o n j u g a t i n g t h e d y e t o

t h e d i f f e r e n t s i t e s , t h e b i n d i n g a f f i n i t i e s m a y b e s t u d i e d

i n d e p e n d e n t l y . A n i n v e s t i g a t i o n o f t h e D N A b i n d i n g p r o p -

e r t i e s o f T y r R a l o n g t h e s e l i n e s i s c u r r e n t l y u n d e r w a y i n

o u r l a b o r a t o r y .

Acknowledgements

W e w o u l d l i k e to th a n k M r . P e t e r M a r o u d a s ( R u s s e l l

G r i m w a d e S c h o o l o f B i o c h e m i s tr y ) f o r p ro v i d i n g t h e T y r R

p r o t e in , D r . A l u n J o n e s ( 3 D C e n t r e , U n i v e r s i t y o f Q u e e n s -

l a n d ) f o r p e r f o r m i n g t h e i o n - s p r a y m a s s s p e c t r o m e t r y m e a -

s u r e m e n t s a n d M r . S t e w a r t T h o m p s o n ( V i c t o r i a n C o l l e g e

o f P h a r m a c y , M o n a s h U n i v e r s i t y ) w h o k i n d l y p r o v i d e d t h e

F A B m a s s s p e c tr a . T h i s re s e a rc h w a s s u p p o r t e d b y A u s -

t r al i an R e s e a r c h C o u n c i l g r a n t s t o W . H . S . a n d B . E . D . a n d

b y t h e C o m m o n w e a l t h A I D S R e s e a rc h G r a n ts C o m m i t t e e

o f A u s t r a l i a b y a p r o j e c t g r a n t a n d a p o s t g r a d u a t e s c h o l a r -

s h i p to G . T . T h e H o w a r d F l o r e y I n s ti t u te i s s u p p o r t e d b y a

b l o c k g r a n t f r o m t h e N a t i o n a l H e a l t h a n d M e d i c a l R e -

s e a r c h C o u n c i l o f A u s t r a l i a . P . H . i s t h e r e c i p i e n t o f a

S w e d i s h N a t u r a l S c i e n c e R e s e a r c h F e l l o w s h i p . M . B . i s t h e

r e c i p i e n t o f a n A u s t r a l i a n P o s t g r a d u a t e R e s e a r c h s c h o l a r-

s h i p .

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internal labelling fmoc

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