Volume 8 Number 3 1980
Volume 8 Number 31980
Nucleic Acids Research
Nucleic Acids Research
Elongation of oligonucleotides in the 3'-direction with activated mononucleotides and their
analogs using RNA ligasel
E.Ohtsuka, T.Miyake, K.Nagao, H.Uemura, S.Nishikawa, M.Sugiura2 and M.Ikehara
Faculty of Pharmaceutical Sciences, Osaka University, 133-1 Yamadakami, Suita, Osaka 565, Japan
Received 5 December 1979
ABSTRACT
P -Adenosine 5'-P2-2',3' -ethoxymethylidene nucleosides
[A(5')ppN(Em)] from four common nucleosides have been prepared
and used for single addition of nucleotides to elongate oligonucleotide chains in the 3'-direction in RNA ligase reaction.
U-U-C, T-Y-C and A-C-C were used as acceptors. Structural
dependence in these acceptors was found to be smaller compared
to joining reactions between oligonucleotides. Adenosine analogs
including 8-bromo-, 2'-fluoro-, 2'-azido-,8,2'-0-cyclo-, 8,2'S-cyclo-adenosine, arabinosyladenine and 2'-deoxyadenosine were
added to the 3'-end of A-C-C by adenylation chemically followed
by joining with RNA ligase. Symmetrical 5'-pyrophosphates of
8-bromo-, 2'-fluoro- and 2'-azido-adenosine were not recognized
as donor substrates.
INTRODUCTION
RNA ligase has been shown to be a useful enzyme not only
for joining of oligonucleotides3 8 but also for addition of mono-
nucleotides by using either 3',5'-bisphosphorylated ribo- 9-11 and
deoxyribo-12 nucleosides or P1-adenosine 5'-P -nucleoside 5'pyrophosphate.13 Using 5'-labeled pCp a 3'-end labeling method
has been developed.14 The adenylated alkyl- or sugar phosphate
could be linked with the 3'-end of ribooligonucleotides.13 This
allowed alternative 3'-labeling by phosphorylation with a photolabile ester P2-[32 PIP -adenosine 5'-P -o-nitrobenzyl pyrophosphate. 5 When 5'-adenylated 5'-nucleotides were used for single
addition of nucleotides, multiple addition took place
Protection of 3'-ends was required if a
at the same time.
single nucleotide was to be incorporated. In joining of oligonucleotides, the protection of 3'-ends of 5'-phosphorylated
molecules (donors) was employed especially when they had chain
lengths longer than eight.16 3'-Phosphates5-9,15 or 2',3'-
© IRL Press Umited, 1 Falconberg Court, London W1V 5FG, U.K.
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Nucleic Acids Research
groups8 were used for oligonucleotide donors.
In this paper we report blocking of the 3'-hydroxyl group of
adenylated mononucleotides to allow a single addition to the 3'end of oligonucleotides. Addition of adenosine analogs, 2'-fluoro-,
ethoxymethylidene
2'-azido-, 8-bromo-, 8,2'-O-cyclo-, 8,2'-S-cyclo-adenosine,
arabinosyladenine and dA to a trimer (A-C-C) and recognition of
these analogs by RNA ligase are also reported.
MATERIALS AND METHODS
Oligonucleotides
A-C-C and T-g-C were synthesized by stepwise condensation
of mononucleotides in the 5'-direction using a similar method
described previouslyl7 and details for the synthesis of T-Y-C
will be published shortly. U-U-C was synthesized using protected
U-Up 8 as an intermediate and condensed with protected cytidine9
by means of triisopropylbenzenesulfonyl chloride as the reagent.
P1-Adenosine 5'-P2-nucleoside 5'-pyrophosphates
2',3'-Ethoxymethylidene nucleosides,20 8-bromoadenosine,21
2'-fluoroadenosine,22 2'-azidoadenosine,23 8,2'-0-cycloadenosine,
24
8,2'-S-cycloadenosine,25
were 5'and arabinosyladenine26
phosphorylated by treatment with phosphoryl chloride in triethylphosphate.27 A nucleotide (e.g. 2',3'-ethoxymethylideneadenosine
5'-phosphate, 175 A260, 0.011 mmol) and adenosine 5'-phosphoromorpholidate
(175 A260, 0.011 mmol) were rendered anhydrous by
evaporation of pyridine separately. The residues were dissolved
in DMF (1 ml), combined, concentrated to ca. 0.5 ml and kept at
30° for 2 days. The reaction was checked by paper
(Table I) and the mixture was subjected either to DEAE-cellulose
column equilibrated with 5 mM borate buffer (5 mM boric acid, 5
mM KC1, 0.027 N NaOH, pH 9.2) or to paper chromatography in
solvent D. After desalting either by adsorption to DEAE-cellulose
(bicarbonate) or by application to paper chromatography in solvent
B, nucleotides were coevaporated with methanol to remove a trace
of borate and further purified by paper electrophoresis at pH 7.5.
The yield of the pyrophosphate was 70 A260, 0.023 mmol, 21%.
Enzymes
T4 RNA ligase was purified as described previously.29 Polynucleotide kinase and other enzymes for characterization of the
electrophoreis
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Table I
Paper chromatography and paper electrophoresis
Compound
Rf
A
0.15
0.57
morphpA
0.52
pA(Em)
0.33
pU(Em)
0.30
pG(Em)
0.20
pC(Em)
0.35
pbr 8A(Em)
0.47
A(5')ppA
0.20
A(5')ppU
0.20
A(5')ppG
0.15
A(5')ppC
0.21
A(5')ppbr 8A
0.11
br8A(5')ppbr 8A 0.16
A(5')ppAf
0.11
A(5')ppAz
0.15
A(5')ppA5
0.08
0.08
A(5')ppA0
A(5')pparaA
0.08
A(5')ppdA
0.10
A(5' )ppA(Em)
0.37
A(5')ppU(Em)
0.36
A(5')ppG(Em)
0.40
A(5')ppC(Em)
0.37
A(5')ppbr 8A(Em)0.41
pA
A
B
0.35
0.64
0.64
0.58
0.59
0.47
0.57
0.61
0.41
0.43
0.29
0.35
0.37
0.42
0.42
0.50
0.26
0.28
0.21
0.34
0.58
0.44
0.51
0.58
0.62
C
0.22
0.65
0.57
0.44
0.54
D
0.16
0.50
Relative mobility
pH 7.5
1.0
0.0
0.50
0.58
0.65
0. 70
1.00
1.04
0;41
0.69
1.11
0. 39
0.52
0.19
0.24
0.17
0.18
0.28
0.63
0.64
0.90
0.28
0.28
0.33
0.26
0.26
0.28
0.32
0. 35
0.44
0.36
0.35
0.46
0.31
0.20
0.12
0.15
0.20
0.16
0.17
0.23
0.23
0.15
0.16
0.21
0.52
0.57
0.30
0.37
0.43
1.07
0.79
0.88
0.87
0. 80
0.85
0.62
0.84
0.79
0.79
0.76
0.88
0.88
0.79
0.88
0.80
0.80
0.65
products were obtained as described previously.4'18
RNA ligase reaction
A two fold excess of ATP with respect to donor molecules was
used unless otherwise specified, in the presence of 50 mM HEPESNaOH (pH 8.3), 10 mM DTT, 10 mM MgC12, 10 pg/ml BSA and RNA
ligase (88 units/ml) in total volume of 10 p1 at 250 for 1.5 hr.
Joined products were labeled by phosphorylation using polynucleotide kinase and [ -32PATP and counted by collection of DEAE-
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cellulose from homochromatography plates.
Other methods
Paper chromatography was performed by the descending technique
using solvent systems: A, isopopyl alcohol-concentrated ammoniawater (7:1:2, v/v); B, n-propyl alcohol-concentrated ammoniawater (55:10:35, v/v); C, ethanol-l M ammonium acetate, pH 7.5
(7:3, v/v); D, ethanol-l M ammonium acetate saturated with boric
acid, pH 7.0 (7:3, v/v). Paper electrophoresis was performed using
0.05 M triethylammonium bicarbonate (pH 7.5). Homochromatography30
was performed using Homomix I-V.31 Other methods for isolation
and characterization of products were also described in ref. 15.
RESULTS AND DISCUSSION
Synthesis and purification of P1-adenosine 5'-P -nucleoside 5'-
pyrophosphates (3) and symmetrical pyrophosphates (4)
Nucleosides were 5'-phosphorylated as described in Materials
and methods. 2',3'-Ethoxymethylidenenucleoside 5'-phosphates were
prepared also from corresponding 5'-nuclotides as shown in Chart
1 and desalted by adsorbing to DEAE-cellulose.20 1 was allowed to
react with adenosine 5'-phosphormorpholidate (2)28 and the pyroChart 1
B
B
-OH
Nucleoside
+
HC(OEt)3
POCl3
(Ot3
H0404J
HO-POJ
0t
O
LOvCc,H
(1)
NpA (2)
A(5')ppN(Em)
N(5')ppN
DMF
(3)
(4)
phosphate (3) was isolated by ion-exchange chromatography on
DEAE-Sephadex to separate 2',3'-protected compounds from cisdiol compounds. A typical elution profile is shown in Fig. 1.
Rf values and relative mobilities of pyrophosphates (3) are
shown in Table I. Symmetrical pyrophosphates such as P 'P2_21azidoadenosine 5'-pyrophosphate were obtained by treatment of
with DCC followed
N,3'-dibenzoyl-2'-azidoadenosine
by ammoniacal treatment. Pyrophosphates were characterized by
hydrolysis with venom phosphodiesterase.
5'-phosphate33
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10
I
z
0.3
5
I PAX0I'
0.2
0.1
A(5)ppA
/\IV
200
300
400
fr. No.
Fig. 1 Chromatography of the reaction mixture in the synthesis
of A(5')ppA(Em) on a column (1.1 x 52 cm) of DEAE-Sephadex A-25
(chloride form). Elution was performed with a linear gradient of
sodium chloride (0-0.3 M, total 500 ml) in 5 mM borate buffer.
Addition of 2',3'-ethoxymethylidenenucleoside 5'-phosphates to
the 3'-end of oligonucleotides
2',3'-Ethoxymethylidenenucleoside 5'-phosphates from four
major nucleosides were added to the 3'-end of trimers, A-C-C,
U-C-C, U-U-C and T-Y-C by using adenylated pyrophosphates (3)
and RNA ligase. The results are summarized in Fig. 2. pG(Em) was
incorporated at the fastest rate in every case and the presence
of 15% DMS&4increased the yield in the case of U-U-C. There were
differences on rates depending upon donors. However, relatitvely
less sequence preferences of acceptors were observed compared to
ligase reactions between oligonucleotides. Therefore the present
method for addition of mononucleotides may be useful to elongate
the chain in 3'-direction especially when the chain is a poor
acceptor e.g. oligouridylates.4
Addition of adenosine analog 5'-phosphates to the 3'-end of
A-C-C
Incorporation of adenosine analogs to the 3'-terminus of
tRNA has been performed by the use of tRNA nucleotidyl trans605
Nucleic Acids Research
XPYPZ + A(PPNEM) XPXPZpN(EM)
N= A -A-Ats
CQ
ACC
UCC
U -0-oG -o-oC -x x-
5~~~~0
50
1.
40~~io
20
2
~~~~00w)
(hr.)
.)
2
20
40h0w.)'
20
Fig.
2 Addition of nucleotides to the 3'-ends of trimers. 1 mM
acceptors and 2 mM donors were used in the standard conditions.
An aliquot (1 ul) was taken at different time intervals, labeled
by 5'-phosphorylation and applied to homochromatography. Dotted
lines indicate the reactions in the presence of 15% DMSO.
ferase32 and it was concluded that nucloeside 5'-triphosphates
were not recognized by the enzyme when a nucloside had syn conformation. Recognition of syn-conformers by RNA ligase was tested
in reaction using 5'-adenylated 2',3'-ethoxymethylidene 8-bromoadenosine 5-phosphate (3) as a donor and A-C-C as an acceptor. As
shown in Fig. 3,pbr8A was incorporated into the oligomer at the
similar rate that was found for pA. This suggests that a synconformer can be recognized by RNA ligase and pbr8A may well be
incorporated to the 3'-terminus of tRNAs which lack the terminal
pA using the present derivative or the 3',5'-bisphosphorylated
compound. Addition of 5'-phosphates of 2'-fluoroadenosine (Af),
2'-azidoadenosine (Az), 8,2'-0-cycloadenosine (A0), 8,2'-S-cycloadenosine (AS), arabinosyladenine (araA) and deoxyadenosine (dA)
were tested using the method similar to above. As shown in Table
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Nucleic Acids Research
(.)
100
ACC + A(s)ppA(Em)
9-4J.,-
50
CCAe.o
A/A
-*o--AC-ACCbFANM-*--*-
20
40(hr)
Fig. 3 Incorporation of 8-bromoadenosine 5'-phoshate. Reaction
conditions were the same as described for Fig. 2 except for 10%
DMSO in the solution.
II the analogs were joined to A-C-C. Since these analogs possesed
the 3'-hydroxyl group, multiple additions were observed in the
cases of A, br8A, Af and Az. The relative yields of products with
longer chain length are also shown in Table II. The yields were
estimated by homochromatography after labeling at the 5'-end with
[T- P]ATP and polynucleotide kinase. The tetramers containing A°,
AS, araA or dA where no multiple additions were detected or
multiple additions were not clear were isolated by paper chromatography and then tested for their ability as an acceptor by incubating *pA-C-C-N with *pAp and,RNA ligase. The tetramers containing AO or As at the 3'-end did not react with *pAp, while
the tetramer *pA-C-C-araA accepted *pAp. *pA-C-C-araA*pAp was
recovered from homochromatography plates and characterized by
digesting with RNase T2 plus phosphatase to yield araA*pA, which
was detected by paper chromatography at pH 7.5. *pA-C-C-dA was
found to be hydrolyzed to *pA-C-C presumably due to the presence
of DNase activity.
Structural requirement for the cofactor ATP
All adenylated nucleoside 5'-phosphates so far tested reactee
as an activated donor molecule as shown above. On the other hand
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Table II
Acceptor
A-C-C
(n mol)
5.0
5.0
1.0
1.0
5.0
5.0
4.0
4.0
1.0
1.2
1.2
Addition of adenosine analog 5'-phosphates by RNA ligase
Donor
n mol
A(5' )ppA
A(55 )ppbr 8A
A (5' ) ppAf
A(5' )ppAz
A(5' )ppA0
A (5') ppAS
A(5' )pparaA
A(5' )ppdA
br8A(5' )ppbr8A
Af (5 ' ) ppAf
Az (5' ) ppAz
20
20
4. 8
4.0
20
20
20
20
20
2. 4
2. 4
DMSO En
% u/ml
Product
A-C-C-N
63
10
125
10
10
10
15
15
15
15
125
125
125
88
88
88
95
10
15
15
88
88
0
88
0
88
63
58
65
90
A-C-C20
26
15
14
0
0
a
a
92
85
0
(N)nn
0
0
0
Slower traveling spots were detected by homochromatography
but they were not measured.
a,
symmetrical 5'-pyrophosphates of adenosine analogs (br 8A, Af and
Az) did not react as a donor molecule in the conditions shown in
Table II. This indicates that triphosphates from these nucleside
analogs may not be recognized by RNA ligase in the first stage of
This
the reaction where adenylation of the enzyme occurs.
adenylic acid is then transfered to the 5'-phosphate of donor
molecules to give activated intermediates.4 Presumably adenosine
analog 5'-phosphate linked oligonucleotide 5'-phosphates also
can not be recognized by RNA ligase. It seems that the structural
requirement of adenylates as a cofactor is quite strict.
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Nucleic Acids Research
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