volume 4 Numbers 1977
Nucleic Acids Research
Complementary addressed modification of yeast t R N A y a ' with alkylating derivative of
d(pC-G)-A. The positions of the alkylated nucleotides and the course of the alkylation in
the complex.*
N.I.Grineva, G.G.Karpova, L.M.Kuznetsova, T.V.Venkstern, A.A.Bayev
Inst. Org. Chem., Siberian Branch of the Academy of Sciences of the USSR, Novosibirsk, and
Inst. Mol. Biol., Academy of Sciences of the USSR, Moscow, USSR
Received 6 January 1977
ABSTRACT
Yeast tRNA*al alkylation with 2«. 3'-0-4-(N-2-chloroethyl-N-methylamino) benzylidene d(pC-G;-A proceeds at 20 -30 C
in the complementary complexes which are formed by d(pC-G)-A>RC1 binding to 3 sequences of tENA^81: \y-C-G58 i& *&« T
loop, C-G^Q at the 3'-side of the anticodon loop and C-G-jg in
the D loop. T^e reaction in the complexes results in A C T , l «
andvj^., alkylation to form B-/N-methyl-N-(formylphenyl7amixyyethyl^tRNAY91 with the relative rate constants of the alkylation that are 3 or 2 orders of magnitude higher than that
for the alkylation without a complex formation. It is the
third nucleotide from the 5'-terminus of the binding site of
the modifying agent that is subjected to alkylation in the
tRNA)f . The course of the alkylation does not depend on the
possible base pairing of the 3'-terminal nucleotide of the
reagent. The extent of the reagent binding and the relative
rate constants of the alkalytion in the complexes indicate
the following order of the complex stability: (V-C-Gcg)>
Cg-G 40 )«=(C-G 1g ) at 20° and (^-C-G 58 )>(C-G 40 )>(C-G 18 ) at
30 •
INTRODUCTION
Previous studies of alkylating derivatives of oligonucleotides (addressed reagents) have shown that these compounds
1—3
4
bind to complementary regions of rRNA
and DNA . Within a
complex highly efficient alkylation of A, G and C occurs if
they are located in a strictly definite position relatively
The abbreviations are used in the text: d(pC-G>A -deoxycytidylyl-(3'-5f)~deoxyguanylyl-(3l-5l)-riboadenosine-5'-phospha^
te; d(pC-G)-A>RCl - 2 f , 3'-0-4-(N-2-chloroethyl-N-methyl-amino)benzylidene d(pC-G)-A; R - B-/N-methyl-N-(4-formylphenyl)
amino/ethyl residue; Bz - benzoyl; CNEt - cyanoethyl; an anisoyl; ib - isobutyryl; Hy - hypoxanthine; D - dihydrouridine. Figure as an index at a nucleotide for example Asjineans
the position of the nucleotide in tRNA beginning from the
5'-end of the tRNA molecule.
© Information Retrieval Limited 1 Falconberg Court London W1V5FG England
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Nucleic Acids Research
to the binding sites'1 »>5. The efficiency of 2', 3»-0-4-(N-2-chloroethy1-N~methylamino)benzylidene oligonucleotides
/(Np)n_1N>ECl/ alkylation of rENA (competition factors) under
the condition of the complex formation is some orders of magnitude higher than that for the alkylation without complex
formation1»3*4.
Nucleotide N^ has been assumed to be conformationally
available for alkylation in a complex:
alkylation site
binding site
3
Indirect data '^ have shown that it is not the first hucleotide from the 5'-terminus of the reagent binding site. However the exact location of the alkylated nucleotide still
remained unknown.
I n order to prove the specificity of alkylation with
the complementarily addressed reagents unequivocally it would
be necessary to determine exactly what nucleotide of the polynucleotide chain is alkylated. For this purpose we have
studied the alkylation of a tENA with known primary structure, namely of yeast tENA^
'i A derivative of the trinucleotide d(pC-G)-A complementary to some tRNA^8 regions 2',3'-0-4-(N-2-chloroethyl-N-methylamino)benzylidene d(pC-G)-A:
d(pC-G)-A<^Q>CH-@-N^
2
2
/d(pC-G)-A>RCl/
OH-,
have been used as an alkylating agent.
Val
The structure of the alkylated regions of tENA^ , the
extent of the modification of each of them and the competition factors (the relative rate constants of the alkylation)
under various conditions have been estimated. Structural
analysis has allowed to locate the positions of modification
within the tENA molecule and to draw conclusions about some
features of the complementarily addressed alkylation mechanism.
1610
Nucleic Acids Research
Tha data obtained have permitted to answer some questions that are of considerable importance because they stipulate the possibility to use complementarily addressed alkylation for fragmentation' and sequencing of nucleic acids. These
questions are: does the alkylation really proceed in the vicinity of the ^'-terminus of the reagent binding site, if
the alkylating group is on the 3'-end of the reagent? How
far from the binding site is the modified nucleotide; What
is an accuracy of the binding and alkylation? What kind of
sequences do the addressed 'alkylating reagents bind to? Can
the reagent change the tertiary or secondary structure of
nucleic acids and interact with double-stranded regions of
the polynucleotide chain?
MATERIALS AND METHODS
Bz &
d(pC-G)-A was prepared by treatment of pAfiz and
d[(CNEt)pCanpG ]° with triisopropylbenzene sulpfionyl chloride according to Khorana et al.
R . of d(pC-G)-A in solvent
(5) is 0.66. UV-spectra in water: X m a x 260 nm, ^- m i n 235 nm»
A
250^A260 °*93i A28O/'A260 0 # 6 ^» Venom phosphodiesterase digest of d(pC-G)-A contained dpC, dpG and pA in the ratio
1.1:1.0:0.8.
14
Reagent
C-d(pC-G)-A >RC1 was prepared by treatment of
d(pC-G)-A and
C-4-(N-2-chloroethyl-N-methylamino)benzaldehyde with 2,2-dimethoxypropane and CF^COOH in dry dimethylformamide according to . The sample contained 10% of
d(pC-G)-A. R^. in solvent (5) 1.35. W-spectra: X ^ 263 nm,.
£260 52.5-103.
Val
tRNAi
was isolated by V.D.Axelrod from unfractionated
'
•i'l
p
i'l 1
il
1p
ll
ll
H]
HHo]
>'<::.. The
Thepurity
purity of
of tRNAi
>'<
tRNA-i
Vali n
unit
£ tBNA
1 M
was not less than 90%. One -A260
°
'j
0»
tris HC1, 0.02 M MgCl o , pH 7.5 accepted 1.8-1.9 nmoles of
14
3
C-valineo The molar extinction coefficient 538*10^1'mole
—1
Val
13
tRNA^ al alkylation with d(pG-G)-A> JRC1 was performed in
cm
was used for tRNA^j
according to ,
buffer I (.O.OT M tris HC1, 0.2 U NaCl and 0.01 M UgCl 2 , pH
7.5). tRNA concentration in the experiments was 10.8 pit. Before incubation the solutions were treated with bentonite.
baker's yeast tRNA according to
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Nucleic Acids Research
The reagent was solved in buffer 2 (O.O2 M tris HC1, pH 7.5)
at 0° and the solution was mixed with an equal volume of cold
0.4 M NaCl, 0.02 M MgCl 2 in 0.02 M tris. This mixture and some drops of CHClo were added to the solution of tENA at 0°.
The mixture was incubated 7 days at 20° or 3 days at 30°. The
reagent concentration was 55 <uM at 30° and 208 jiM at 20°. During incubation the reagent was completely transformed into
the active alkylating intermediate that was consumed much
14
quicker than formed
. After incubation IM Na acetate, pH
3.8, was added to the reaction mixture up to 0.1 M and the
resulting mixture was incubated 3 hours at pH 3.8-4.0 at 40°
Val
in order to hydrolyze the acetal bond of alkylated tENA
,
d(pC-G)-A>B-t£NAlal (I), that yielded E~tBNAYal and d(pC-G)-A.
E-tENA)j
was isolated by gel filtration on Sephadex G-75
(column 1x43 cm, 45°, buffer 2, a flow rate 60 ml per hour;
fraction volume about 1 ml). The extent of tENA alkylation
was estimated from the radioactivity of the polymer fraction
(in dioxane scintillator using a Nuclear Chicago Mark 1 scintillation counter) and its absorbance at 260 nm (ET-4 spectrophotcmeter, Hitachi, Japan). The results were expressed
Val
in moles of the reagent bound per mole of tENA* .
Val
Acid-catalyzed hydrolysis of E-tENA^j
(20 £M) was carries out in 1 N HC1 at 100° for an hour. The products of hydrolysis were separated on Dowex 50W(H)x4 (column 1x16 cm, linear gradient from 0 to 6 N HC1, total volume of gradient was
200 ml, flow rate about 1 ml per minute). Badioactivity in
the peaks was measured after HC1 evaporation. The substances
in each peak were separated in the solvents (I), (2), (3)i
(7) and identified by comparison with authentic substances
according to .
Pyrimidyl-BNase (EC 2.7.7,16) digestion was performed
with an enzyme purified on CM-cellulose column according to
15
. 0.16 mM tENA^al or E-tRNA^al were incubated with pyrimidyl-RNase (20:1 w/w) in 0.01 M tris HC1, pH 7.5 and 0.001
M EDTA at 37° 18 hours. The enzyme was absorbed on bentonite
and the digest (1.1 ml) was passed through a BD-cellulose
1fi
(Cl"") column (1x10 cm. Plow rate 20 ml for an hour)
1612
Table 1. The mobilities of the alkylated oligonucleotides on a cellulose thin layer
relatively to Ap and to RA-G-Tp and UV-spectra of the compounds.
Oligonuc-
Spot on
leotide
Fig. 2
RA-G-Tp
V Solvent
X.nm
E
(1) (6) (4) (5)
2
RA-G-Tp
I
231
312
232
311
0.81 0.75 L 8 7 1.28 7
257
349
255
348
256
350
258
350
250
348
13
253
350
2
253
350
235
310
234
312
7
253
348
13
253
352
0.53 0.71 1.0
1.0
7
13
2
R*-A-G-Dp
RI-A-Cp
2
CO
2
3
i
max min
1.08 0.77 2.65 1.51
239
319
232
311
231
308
230
307
230
306
3
max nun 2bO
34.0 20.8
28.3 5.2
29.6 20.5
27.6 3.8
29.0 23.7
30.6 4.4
39.2 25.0
30.2 4.5
31.8 29.1
31.2 7.4
26.8 24.5
30.2 5.0
34.0 20.4
29.8 6.2
A
25>
A
260
A
28>
A
260
33.2
0.96
0.59
1.03
0.60
28.0
1.00
0.50
39.2
0.90
0.61
28.0
1.14
0.64
25.7
1.03
0.61
31.9
1.06
0.72
1.06
0.78
1.05
0.66
28.0
28.8 16.8
26.5
30.0 7.3
25.8 23.5
30.8 5.8 24.2
Nucleic Acids Research
Unmodified oligonucleotides were eluted with 0.5 M NaCl in
0.05 M Na acetate, pE 6 (25 ml) and the alkylated derivatives
were eluted with the same solvent in aqueous ethanol (20%).
Becovery of radioactivity from the column was about 95%. The
peaks were twice desalted on Sephadex G-10 (30x1 cm) and were concentrated to 1 ml. The digestion products were separated by two-dimensional TLC on quartz plates with cellullose
layer according to
in the solvents (4) and (6) in the case of unmodified oligonucleotides or in solvents (I) and (5)
in the case of alkylated substances. The cellulose spots containing UV-absorbing substances were removed from the plate
as solid platelets using nitrocellulose (1-4%) solution in
17
ethanol-ether mixture according to
. The solid platelets
were used for counting the radioactivity in a toluene scintillator. After counting the plateles were washed with ether,
then eluted by 0.01 M tris HC1 to register the absorbance at
260 nm. The substances were repeatedly purified on cellulose
thin layers and their UV-spectra were recorded (table I ) . In
order to find alkylated compounds that are formed in minute
amounts and therefore can't be seen in UV-light screening of
17
the cellulose layer for radioactivity was performed
. Recovery of radioactivity from the plate was about 70%.
Nucleotide composition of alkylated oligonucleotides was
determined by digesting them with 1 N HC1 at 100° for an hour. The digest was fractionated by two-dimensional TLC on a
cellulose layer in solvents (2) and (7) (Fig.3, table 3 ) . After elution as mentioned above, the radioactivity and UV—
spectra of each spot was recorded; the microtechnique developed in the Institute of Organic Chemistry of Novosibirsk
18 1Q
was used for the latter purpose
• yQ Properties of authen4 20—24
tic compounds are listed in the papers •
.
Chromatography on Whatman FN-I and cellulose FND (Filtrak, DDE) were used for chromatography of tENA components in
the following solvents: (I) isopropanol - cone. NH,-H~O
(7:1:2); (2) isopropanol - H 2 0 (6:4); (3) isopropanol - cone.
HC1-H2O (1?O:41:39); (4) tert. butanol - 0.075 N HC00MH4,
pH 3.8 (1:1, pH 4 O 8); (5) n-propanol - cone. NH.,-H20 (55:10:
1614
Nucleic Acids Research
:35)i (6) isobutyric acid - 0.5 N HH 3 (5O)» pH 3.8; (7) ethanol - 1 M NH 4 acetate, pH 7.5 (7:3).
Sephadex was from Pharmacia, Sweden, and Dowex 50 - from
Serva, BED.
The relative rate constants of the alkylation (k^K" )
of the every site of the tENAJ
modification have been cal25 45
45
culated according to -" •* by the equation ^:
- [EN])
,,
[d(pC-G)-AECl]0 d - e - ^ * ) - [EN]
where k is the rate constant of the reaction of the individual alkyla ted residue in tENA with ethylene immonium cation
that is formed from the reagent in the rate determining step
25
of the reaction
. Is., is the rate constant of byreactions
of the intermediate with low molecular nucleophyles in the
solution, Nu i . [EN] is the concentration of the modified residue in tENA. This magnitude is determined from the experiment (table 2 and fig o 4). Initial concentration of every site of alkylation [ N ] is equal to [tENA] . k is the rate
constant of the rate determining step of the ethylene immonium cation formation; k = 6,3-10 s" at 20° and
k Q = 2,O9 # 1O~5S~' 1 at 30° 2 -\ The rate constants have been determined in the experiments performed under the same Nu. concentration. Under low extent of the alkylation k can be cal25
culated by the equation given in ^.
BESUI/rS AND DISCUSSION
It should be expected that in a complex alkylation will
proceed in the vicinity of the reagent binding sites. If it
Val
is really so the pattern of tENA^
alkylation in a complex
and without complex formation will be absolutely different.
Without complex formation, chloroethylmethylaminobenzylidene nucleosides, nucleotides and oligonucleotides are
shown to alkylate guanine mainly and cytosine a little in
tENA 1 4 > 2 4 » 2 5 . Any of the tENA^ 1 guanines exhibites similar
25
low reactivity J, tENA alkylation consumes negligible amounts of the reagent. The main part of it is used for the re14
action with water or nucleophiles
of the reaction mixture.
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Nucleic Acids Research
60
G G G U C
C
U
m1A
55V
Fig e 1. Scheme of possible d(pC-G)-A>ECl binding to yeast
ai
tE
. The reagent is denoted by a line; the arrows indicate the direction of alkylation observed in the complexes
and the position of the alkylated nucleotides (circled). The
points denote tRNAJJal nucleotides which could participate in
the reagent binding. The hatched line denotes unrealized
binding (4,5).
Fig.1 shows a plausible scheme of d(pC-G)-A>ECl compleVal
mentary binding with appropriate sequences of tENA^ - • The
reagent can be expected to form a perfect complex with three
Val
base pair in two sequences of tENA^ , namely: *cc-C-Gcg and
Val
Uc-C-Go. Three sites of tENAi
can bind the reagent due
d
to
CG base pairing and 4 sites - to GA base pairing to yield
partially base paired complexes. Hence, theoretically the alkylation of about 9 sites of tENA^
should be expected to
occur in the complexes. In order to decrease the number of
possible points of d(pC—G)-A>EC1 alkylation in the complexes
with tRNA^al, we have studied tENA^al alkylation at 20° and
30° to avoid the formation of partially base-paired complexes, stability of which is naturally diminished.
The acetal bond of the reagent in the first product of
alkylation (I)* is subjected to hydrolysis at pH 4 1 4 . This
The formation.,of analogous alkylated ENA has been proved
previously3t~4.
°
^
1616
Nucleic Acids Research
Val
r e s u l t s in B-[N-methyl-N-(4-formylphenyl)amino]ethyl-tENA,(
al
(B-tRNA^ ) and d(pC-G)-A.
d(pC-G)-A(
0
Val
Two reagent residues are attached to tENA^
under 19 times
excess of the reagent and at 20° and only 0.67 B residue under 5 times reagent excess and at 30° (table 2 ) . Both species, Ro-tENA and uE n co-tBNA have been studied.
Val » ° /
E-tENA/i
was digested with ENase A. Alkylated and unmodified oligonucleotides of tENA)Ja were separated on a ED-cellulose column. The fingerprint of the unmodified oligonucleotides on a cellulose thin layer did not differ from that of
the native tENA-[
digest
. But the content of some of the
oligonucleotides was lower than in the case of the native
tENA. These oligonucleotides were expected to be A-G-Tp,
I-A-Cp and A-G-Dp. However, they were not completely resolved on the fingerprints. The alkylated oligonucleotides were
separated in the same way (fig.2). The homogeneity of each
of them was checked on a cellulose thin layer in 2 additional
solvent systems. Mobilities and UV-spectra of the alkylated
oligonucleotides were recorded (Table I ) .
Table 2 shows that there are three main alkylated oligonucleotides, which amount to 93% of all modified substances.
The nucleotide composition of the main modification products
was estimated after acid-catalyzed hydrolysis of each of
them. TLC separation of the acid hydrolyzates is given in
fig.3» Table 3 and 4 show some characteristics of the products of acid-rcatalyzed hydrolysis of the alkylated oligonucleotides. One can see that oligonucleotide I (fig.2) contains
equimolar amounts of Gua, Tp and the products of EAde transformation in acid . The presence of Tp allows unequivocally
to ascribe to this oligonucleotide the structure of RA-G-Tp.
The latter can be formed only by Ac, alkylation in A-G-Tp of
tENA;,
1617
Nucleic Acids Research
Figure 2. Two-dimensional separation of the alkylated oligonucleotides of a pyrimidyl-ENAse d i gest of E2-tENAi
on a cellulose
thin layer. Alkylation was performed at 20 . I - solvent ( 4 ) .
I I - solvent ( 5 ) .
1 _ EA-G-Tp, 2 - B¥-A-G-Dp.
3 - EI-A-Cp. Shaded spots are
revealed both by OV-absorption
and radioactivity. Unshaded
spots are revealed only by r a dioactivity.
Table 2 . Alkylated oligonucleotides
of Eyrimidyl-RNase d i gest ofSEtENAia± modified with n^C-d(pC-G)-A>ECl
a t 20°C
and 30°C **. [tBHA] 1 0 . 8 uM
Alkylated oligo- Spot No.,
C-Product content Modification
nucleotide com - F i g . 2
extent of
i n True U l g H B i , Jo
position
oligonucleo.1 _
EA-G-Tp
E<P-A-G-Dp
EI-A-Cp
Not i d e n t i f i e d
1
2
3
4
5
6
4-VtA
ert
30° •*
47.5
20.8
24.3
64.5
4.1
1.2
_
0.2
1.0
2.8
1.2
0.4
1.2
0.3
0.5
9
0.5
10
11
A-
20° *
7
8
-3-1—
1.0
0.1
-
6.4
22.2
0.9
tide, E/oligonucleotide
20° * 30°**
0.94 0.43
0.41 0.04
0.48 0.15
0.081
0.023
0.006
0.010
0.020
0.010
0.002
-
al
*The extent of the intact tRNA;T modification i s 1.97;
[reagent]/[tENA] = 1 9 .
The extent of the denatured tRNAY81 modification i s 0.67;
[reagent]/[tENA] = 5 .
Hydrolysis of the most rapidly moving oligonucleotide 3 lias
revealed Ada, Cp and an alkylated base the mobility of which
i s similar to that of EGua (Table 3 ) t however the shape of
the UV-spectra of the altolated monomer derived from oligonucleotide 3 i s most likely to be close to that of B.7 tty27
(Table 4 ) . Hence, the structure of the parent oligonucleotide
1618
CO
R Cp
3
Gua
0
Cp
UP
Gua
Ade
•
e
Gua
.V'
R7Gua
CP O
Cp
R Cp
Ade
o
8o
3
R 7 Gua
Cp
Ade
Ade
14,C-RHy
Figure 3. Fingerprints of the products of acid-catalyzed hydrolysis
of RA-G-Tp (a), R*U -A-G-Dp (b) and RI-A-Cp (c) on a cellulose
thin layer. T - solvent (2); Tl - solvent C7)7 Shaded spots
denote the products of hydrolysis; authentic substances in the
direction I are denoted by a continuous line, in the direction II
by a dashed one; o - start points.
II
Cp ^'J R'Gua
Gua
7
R'Gua
3
CD
V)
CD
0)
u>
J3
a
Z
o_
w
o'
Nucleic Acids Research
Table 3. Components of acid hydrolysate of the main alkylated
oligonucleotides and their mobility on a cellulose
thin layer relatively to Gua.
Oligonuc- Components Molar ratio
leotide of the di- of the comgest
ponents
BA-G-Tp
Gua
Tp
'l.O
EAde*
0.9
Gua
BV-A-G-Dp
Ade
E*p
BI-A-Cp
Dp
Ade
Cp
EHy
Authentic
compounds:
Gua
Ade
Cp
Tp
7
1.1
1.0
Efi
(3)
(7)
1.0
1.0
1.0
2.2
6.4
0.4
2,6; 2 .9 3.4; 6.6 0.8; 0.9
1.4
1.9
1.0
'1.0
0.8
0.8
1.0
2.1
4.9
1.1
1.1
2.4
5.2
0.3
1.0
'I.4
2.0
1.1
1.1
1.1
2.1
3.8
0.4
E Gua 20
EHy 1
yj* von p a p e r
1.0
1.7
1.0
1.4
2.1
2.4
2.6
2.0
'l.O
2.0
4.0
1.3
1.0
6.5
1.9
1.2
0.4
0.4
1.1
3.3
0.9
2.1
3.2-3.6;5.0
E Ade J
E 3 Cp
in solvent
uua
(2)
2.2
3 is RI-A-Cp that is due to alkylation of the tENA;,
don loop.
antico-
The digest of oligonucleotide 2 contained Ade, Gua and
EWp. The latter was identified by comparison of the UV-spectra (Table 4) with that of me1*p 2 8 » 2 9. Tlle uv-spectra of this
compound in alkali indicate the presence of R-typ as admixtu*
re. There is no sequence in tBNA^| nwhich can form a trinucleotide with a 5'-terminal Vp. The latter can arise only in
the case when the phosphodiester bond on the 3'-site of «¥p
loses the ability to be digested by pyrimidyl-ENase. The onVal
ly sequence of tENA^
which is composed of A, G, ^ is
^
j
*
This sequence must have a 3l-terminal Dp if it
was due to pyrimidyl-ENase digestion. The latter was indeed
revealed by the method of Janion and Shugar ^ in an acid
digest of the oligonucleotide 2. The mobility of the substance giving transcient UV-absorbance after alkali addition,
corresponded exactly to that of Dp, and its quantity was
1620
Nucleic Acids Research
Table 4. DV-spectra of alkylated components of acid hydroly'
zates of alkylated oligonucleotides
Component
pH
I
EHy
7
13
I
^P
7
13
A iim
min
max
292
352
255
352
246
305
29.5
232
292
232
292
232
292
16.4 16.4
27.8
17.2
17
28.0
17.2 16.8
28.0
299
16.8 13.5
12.2 13.6
355
262
355
262
355
RAde*
7
20.4 20.2
30.6
20.6 20.2
35.6
13.5 13.4
255
352
262
258
235
328
295
max 260
275 inflexion
^So
""^260
1.49
0.93
0.64
1.74
0.88
0.57
2.18
0.93
0.77
1.58
0.91
0.43
1.56
0.83
0.49
1.55
0.83
0.79
0.15
0.17
1.09
0.84
1.09
9.8
equimolar to the three other compounds of the oligonucleotide. Thus we can draw the conclusion that the structure of
oligonucleotide 2 is E*^^-jl-G-D16p. E*pA is digested much
slower than^pA, that is in agreement
with the data concerng
ing with poly-me<l> digestion 3°»3
It should be mentioned that the composition and sequence of unmodified nucleotides in the alkalyted oligonucleotides give us enough information to reconstruct the whole
structure of the oligonucleotide. Really, if the trinucleotide from E-tENA/j
pyrimidyl-ENase digest contains G and T
their sequence has to be G-T and the third nucleotide, namely
RA, can be only on its 5*-end. Therefore the structure of
oligonucleotide 1 is EA-G-Tp. Further, if the alkylated oligonucleotide 2 consists of A, G, D and an alkylated nonomer,
Vfil
their sequence should be E*-A-G-Dp in accordance with tENA^
primary structure ' . A and C identified in the oligonucleo1621
Nucleic Acids Research
tide 3, can be only in the sequence A-Cp. The A-Cp sequence
is present in 3 sites of tRNAJj , namely in 36-37, 38-39 or
74-75 positions. Oligonucleotide 3 can't be A ^ - R ^ - C ^ or
•A^-BA-C^g because the acid-catalyzed hydrolysis of the main
alkylated oligonucleotides revealed three different alkylated
monomers (table 3) in them. RVp and RA were already observed
to be in the oligonucleotides 1 and 2. Therefore it is likely
that alkylated monomer of the third oligonucleotide is due
to I,,, Crj-x or C~rj alkylation. Clear difference between the
mobility of the alkylated monomer and that of RCp
(table 3)
enables us to identify it as Hy derivatives (RHy) in the
RI^c-A—Cp-jo sequence.
According to the analysis of the alkylated oligonucleotides, tRNA^al alkylation with d(pC-G)-A> RC1 results in the
modification of the sites: Jl,,, I.,,- and S^-j both at 20° and
at 3O0 (Table 2 ) . The modification extent of these three sites amounts to 93-94% of the whole reaction. Incorporation
of 2 moles of the reagent per 1 mole tRNA^ al (R2~tRNA) modifies Acoi I35 and W^ to 94, 48 and 41% respectively. Therefore R 2 -tRNA^ al is a mixture of (RAc-,, RI-jc)-tRNA^a:L and
(RAc-,1 R^O-tRNA,, probably with little admixture of
(RI 3 5 , Kf^3)-tRNA)j . The alkylation at 30 incorporation
0.67 mole of the reagent per mole of tRNA (RQ 6r,-tRNA^al);
the analysis shows that 43% R is covalently attached to A r y
4% R - to V,,3 and 1 % R - to I__ (Table 2 ) .
The positions of the alkylated nucleotides in
indicate that the d(pC-G)-A >RC1 modification affects the
nucleotides which are in the vicinity of the ^'-terminus of
the tRNA sequences complementary to the reagent. The latter
are *cg-C-G,-o in the T loop, C-G^Q in the anticodon loop and
^,Q in the D loop (the sites 1, 2 and 3 on Fig.1). At the
same time the pattern of tRNA},
alkylation with 2 1 , 3»-0-4-(N-2-chloroethyl-N-methylamino)benzylidene uridine-5'-inethylphosphate (mepU>RCl) that does not form complexes with
tRNA is completely different; this reagent has been shown to
alkylate any of all 18 G, I and 5'-terminal phosphate but
Aco and vi)^, don't react in the case.
1622
Nucleic Aeids Research
An active intermediate of (Np) N > R C 1 , ethylene immonium
derivative, that is formed in the rate determining step of
the alkylation, was shown to react with HNA completely for
their formation in the complexes '* and in a negligible extent without complex formation *»''4,25# <pna reactivity of the
alkylating reagents is used to estimate by competition factors (relative rate constants of alkylation). Table 5 shows
the relative rate constants of the tHNAY al alkylation with
d(pC-G)-A>HCl for every site of modification that have been
determined according to 2^»^5 f r o m data o f the table 2 and
fig.4.
Table 5. The
the alkylation of
k-v. (competition
6.01 M tris
1-1
fact ._,
HC1, pH 7.4.
Alkylation site
Vnl
1
Reagent
intact tRNA
20°
A C ,GTV|CG,-Q
1.4 *10 4
I,,-ACACG40
3.3 *103
*13 A G D C G 18
•^AGDCG^g + spot 4
Spot 4*
Spots 5-10* an average
Any of 18 G
average
d(pC-G)-A>RCl
2.6 *10 3
denatured
tENA
30°
2.4 *10 4
6.6 *10 3
1.8 *10 3
3
3-4 *10
4.2 *10 2
5.9 •10 1
1.3 •10'1
an
60 (40°)**
mepU> RC1
2.1 •io1
J
35
Three main sites of d(pC-G)-A>RCl
alkylation an ave- +10 fold
rage
d(pC-G-A)
+10 fold
d(pC-G)
See table 2 and fig.2;
1.3 •10 4
1.5 *10 3
2.1 *10 3
Without Ms 2*
The reactivity of Ac,, ly- and <V^, that are in the vicinity of the sequences complementary to d(pC-G)-A>RCl is 3
1623
Nucleic Acids Research
Figure 4. The plots of the
extent of tHWA][al alkylation
with d(pC-G)-A>RCl in the
presence of the varying amounts of d(pC-G-A) (1),
d(pC-G) (2) and (Ap)?A (J).
(3)
(tHNA) 8.9'ICT6
(dCpCM^-A > RCl) Q
0.2 M NaCl, 0.01
0.01 M tris HC1,
0
4
8
M;
4.54- 1O"5M;
M MgCl 2 ,
pH 7.4; 20°.
12
[oiigomer]0/ [ d(pC-G)-A>RCl]o
or 2 orders of magnitude higher than that of mepU>R01 g 5 and
that of other points of alkylation with d(pC-G)-A> RC1. The
number of these products of the alkylation, their negligible
content and low reactivity that are similar to that under
mepURCl alkylation indicate that they are due to alkylation
by the non-bound free reagent. Considerable difference in the
direction and in the relative rate constants for the alkylation with mepTJ>RCl and for the alkylation of the peculiar
sitea with d(pC-G)-A >RC1 indicate that the latter proceeds
in the complex.
Previously we established that (Ap)cA>RCl binding with
rHKA was directed by the oligonucleotide part of the reagent,
the latter and (Ap)^A competed for the binding to oligo-U sequences of rRNA, whereas pC 6 did not effect. Now, Fig.4 shows
that d(pC-G-A) and d(pC-G) inhibit the alkylation of tHNA^ al
with d(pG-G)-A> RC1 efficiently, but (Ap),A. The competition
effect is increased under rising excess of d(pC-G-A) and
d(O-G). The latter inhibits the alkylation slightly weaker as
one could expect. d(pC-G-A) and d(pC-G) as well as
d(pC-G)-A >RG1 are self-complementary oligomers; hence, the
competition observed can be due to the oligomer binding to
the alkylation sites in tRNA^al and to the reagent. But the
main effect of the inhibition is connected with the first
cause as constants of the oligomer-oligomer binding are assum1624
Nucleic Acids Research
ed to be lower than that of oligomer-polymer binding. On the
other hand the relative rate constants for the alkylation of
the 3 main sites in tHNA do not change when increasing the
reagent excess from 1 to 10 (average k p 1,5*10* M ~ 1 ) , whereas
k p is diminished twice when d(pC-G-A) is added to the mixture
up to the ratio of d(pC-G-A):d(pC-G)-A >HC1 1:1 under the sum
ten fold excess of them in relation to tRNAY . The weaker
competition of d(pC-G) than d(pC-G-A) supports this consideration because both of them have the same sequence C-G complementary to the reagent.
All these data together allow us to draw the following
conclusions: (i) tRNA alkylation with d(pC-G)-A> RC1 really
proceeds in the complexes of the oligonucleotide part of the
alkylating reagent with complementary sequences of tRNAl i
y-C-G,-8, C-G 18 and C-G 40 ; (ii) It is the third nucleotide
from 5'-end of the binding site of the reagent that is alkylated in the complexes formed by pairing of the entire oligonucleotide part of the reagent as well as in the complexes
that are formed by two 5'-terminal bases only. The pairing of
the 3'-terminal base of the reagent seems to be not essential
for the direction of the alkylation in the complementary
complexes. This seems to be analogous to the phenomenon of
wobble binding in the codon-anticodon interaction.
Among the not identified alkylated products (table 2)
the product 4 is formed at 20° with rather high relative rate
constant (table 5) to be alkylated with the free reagent. It
can be due to alkylation within a complex that is present in
low concentration or to be the result of partial pyrimidylENase digestion of R^-A-G-Dp. To solve this question, the
content of the alkylated oligonucleotides (table 2) have been
compared with the content of the alkylated monomers, that
have been estimated by analysing products of the acid-catalyzed hydrolysis of R-tHNA* . The products obtained were
isolated after Dowex 50 H+ column chromatography and separation on paper. RAde* was found to be 47.5%, RHy - 24.5% and
R4»p - 24.0% of all alkylated products. Rade and RHy content
is in good coincidence with
1625
Nucleic Acids Research
that of RA-G-Tp and RI-A-Cp. But EH>p content is 3.2% higher
than that of RV-A-G-Dp. So far as \J> can't be aIkylated with
the free reagent J the content difference is most likely due
to partial enzyme digestion of RVpA phosphodiester bond. The
data above permit us to evaluate the extent of RV-A-G—Dp digestion in the range of 13-17%. Taking into account the digestion extent of R*-A-G-Dp, the extent of 4 ^ alkylation can
be considered to be about 49% instead of 41% under alkylation
at 20° and to be 5% instead of 4% under alkylation at 30°.
The difference between the extent of modification (table 2) and the relative rate constants for the alkylation of
Val
deverse sequences of tENA" complementary to d(pC-G)-A BC1
(table 5) mean that the concentrations and stabilities of the
complexes with tRNA sequences: D-C-G^8; A - C - G ^ Q and >*-C-G,-Q
increase in the series: 1 , 1 , 4.5 for the intact tRNA alkylation at 20° and 1.0, 3.7, 14 for the denatured tRNA alkylation at 30°. The difference observed can be interpreted as a
difference in the binding constants. According to Lewis et
al
13 times difference is observed between the binding
constants of the complexes formed by two O G pairing and by
two C»G and one A*U. Therefore, the sequence y-C-Gc8 in the
denatured tRNA^8 can be considered to bind at 30° the entire
reagent whereas D-C-G^g binds only C-G part of the reagent.
The latter way of pairing is most likely to occur under the
reagent binding with A-C-G^. At 20° the reagent binding with
Val
and V-C-Gcg in the intact tRNA,]
is perhaps restricted somehow as C - G ^ binds the reagent as well as C-G^g and
the difference between the reagent binding with >J>-C-G,-g and
C-G/io is less than that at 30°. The consideration is consistent with some data concerning *V • A pairing-' and D inability
to pair with A ^ as well as with the data of a restriction
of the complementary oligomer binding to the T-*f-C-G sequence
of some tRNAs 32,37,47,48^
Val
The sequence Uc-C-Gr; in the acceptor stem of tENA/j
(fig.1) could in principle participate in complementary base
pairing of d(pC-G)-A>RCl to form a complex with 3 pairs if
the reagent would be able to compete with the sequence
1626
Nucleic Acids Research
that is paired in the native tENA a with Uc-C-G^.
Within this complex G 2 alkylation could be expected and after pyrimidyl-ENase digestion pG-EG-Up should appear. However no such alkylated oligonucleotide was found in the digest,
moreover there was only 1.6% E-Gua among the alkylated monoVal
mers after acid hydrolysis of tENA-j . The whole amount of
E-Gua is considered to be due to alkylation with the free
reagent. Hence the reagent can't bind to Uc-C-G™ neither at
20° nor at 30°. Preliminary denaturation of tENA by heating
at 80° in the absence of Mg + does not change the result of
the alkylation. The result is consistent with some data of
Uhlenbeck et al. 32,33^ p O ngs et al. 35.37 a n d H Oge nauer et
al.
who have shown that two-stranded regions of tENA don't
bind complementary oligonucleotides (under great excess of
tENA).
Oligonucleotides were shown by many workers to bind specifically to complementary single-stranded antisequences of
HNA and polynucleotides. According to Lewis et al.
dimers
can bind if they revealed two C«G pairs with constant binding 5,8*10^ at 0° even in the Mg 2 + absence. But Pongs et all''
and Uhlenbeck-1 did not observe C-G-A and C-G-A-A binding
with tENA Phe , tENA fMet and tENA171" at 0° whereas U-C-G,
U-C-G-A, G-A-A and G-A-A-C bound to these tENAs. From this
it was concluded that sequence G-T-W-C-G in the T-loops was
accessible to complementary oligomers but in a restricted
manner-^. Later C-G-A-A, U-C-G-A and U-C-G binding to the
T-loops of tENA^911 4 7 and tENA f M e t ^ was found but not as
the entire oligomers. One of the possible reason of an apparent restriction of the binding of G and G-C containing oligomers may be their ability to self-aggregation ". The aggregation of the oligomers at 0° is more efficient than that
at elevated temperature. We have performed our experiments
at 20-30° under rather great excess of the oligomer nevertheless our results don't contradict the data mentioned above. So the relative rate constants of the alkylation (table
5) shows that the alkylation of the T and A loops of the deVal
natured tENA/)
is facilitated in comparison with the alkyla-
1627
Nucleic Acids Research
tion at 20° of the native one whereas the D loop alkylation
is diminished as it should be expected due to decrease of the
binding constant and the complex concentration with temperature elevation. The alkylation at 20° is more drastic condition of course than the binding at 0° and the spatial structure of tENA may be changed easier.
JI/I
According to Privalov et al.
the tertiary structure
of tBNA^ al is stable in 0.15 M NaCl and 1 mM MgCl 2 up to 40°.
Hence we can conclude that the three-dimensional structure
Val
of tBNA/]
undergoes no essential changes under alkylation
conditions used in the absence of the reagent. But the reaVal
gent does change the tertiary structure of tENA*
anyhow as
Val
it binds to the sequences of tRNA^
some nucleotides of
which participate in hydrogen bonding from the point of view
of the tertiary structure which presumably is similar to all
tENAs 38,41,42^ I n t n Q f a c t d ( pC _G)-A >BC1 binds to the T, D
and A loops where 1 or 2 base are paired in the double or
three-dimensional structure of tENA and it alkylates A,--,
which is involved in the T stem. But it can't bind to U-C-Gr,
in the acceptor stem. Analysis of d(pC-G)-A>RCl binding to
Val
tHNAsj
allows us to emphasize the following: 1) Sequences
localized in long double-stranded regions of tENAs don't react with alkylating trinucleotide reagents at least if the
interaction leads to formation of the same number of base pairs in a new forming complex as in the initial double-stranded region of SNA. 2) The reagent can interact with a nucleotide of terminal base pair of the anticodon stem. Such binding increases the number of base pairing in comparison with
the state before binding (2 new bonds are formed and 1 is
eliminated). 3) The reagent competes for the nucleotides of
the T and D loops that form base pairs with one another in
the tertiary structure. The binding results in 3 new base
pairs whereas 2 previously existed pairs are broken in the
T loop. In the T and D loops we observe the formation of 5
new pairs and elimination of 2-4 initial.
The data obtained allow us to make the conclusion that
the alkylating derivatives of the oligonucleotides can bind
1628
Nucleic Acids Research
to complementary single-stranded regions or to sequences with
only single paired bases and enable highly specific modification at definite sites of nucleic acids, e.g. the; realize in
the fact complementarily addressed modifications. The latter
can be used for structural analysis of nucleic acids.
ACKNOWLEDGEMENT
The authors are very thankful to Dr. V.D.Axelrod for the
Vnl
generous gift of tENA^
and Drs V.V.Vlasov, L.Z.Khutorjanskaya, L.F.Shershneva, T.N.Shubina for a help in the work.
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Dedicated to the memory of Jerome Vinograd.
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Nucleic Acids Research
1632