©
1996 Oxford University Press
2044-2053 Footnote
Non-hydrogen bonding `terminator' nucleosides increase the 3'-end homogeneity of enzymatic RNA and DNA synthesis
Non-hydrogen bonding `terminator' nucleosides increase the 3'-end homogeneity of enzymatic RNA and DNA synthesis
Sean
Moran
,
Rex X.-F.
Ren
,
Charles J.
Sheils
,
Squire
Rumney IV
and
Eric T.
Kool*
Department of Chemistry, University of Rochester,
Rochester
, NY 14627,
USA
Received February 29, 1996
;
Revised and Accepted April 18, 1996
ABSTRACT
We report the use of novel non-polar nucleoside analogues as terminators of enzymatic RNA and DNA
synthesis. Standard `runoff' RNA synthesis by T7 RNA polymerase gives RNA
products which have ragged ends as a result of transcription which often
extends beyond the end of the template DNA strand. Similarly, the Klenow
fragment of
Escherichia coli
DNA polymerase I tends to run past the end of the template strand during DNA
synthesis. We report here that certain non-hydrogen-bonding nucleoside analogues, when placed at the downstream 5
'
-end of a template DNA strand, cause the polymerases to stop more abruptly
at the last coding nucleotide. This results in a considerably more homogeneous
oligonucleotide being produced. Three novel nucleosides are tested as potential
terminators: 4-methylindole
[beta]
-deoxynucleoside (M), 1-naphthyl
[alpha]
-deoxynucleoside (N) and 1-pyrenyl
[alpha]
-deoxynucleoside (P). Comparison is made to an abasic nucleoside
([Phi]) and to unterminated synthesis. Of these, M is found to be the most efficient
at terminating transcription, and both P and M are highly effective at
terminating DNA synthesis. It is also found that the ability of a nucleoside to
stall synthesis when it is internally placed in the template strand is not
necessarily a good predictor of terminating ability at the end of a template.
Such terminator nucleosides may be useful in the preparative enzymatic
synthesis of RNA and DNA, rendering purification simpler and lowering the cost
of synthesis by preventing the uptake of potentially costly nucleotides into
unwanted products.
INTRODUCTION
In vitro
RNA transcription (
1
-
4
) is commonly used in the synthesis of milligram amounts of RNA oligonucleotides
(
5
-
6
) and in the preparation of longer RNA strands as well (
7
). The standard approach for the synthesis of short RNA oligonucleotides is the use of synthetic DNA template strands which contain an RNA polymerase
promoter at the 3'-end (
1
-
4
). The 5'-end of the RNA product is well-defined by the transcription start site immediately following
the promoter, but the 3'-end is less well-defined. When the polymerase reaches the end of the DNA template
strand, it is common for the enzyme to add one or more additional nucleotides
to the RNA chain (
1
-
4
).
This is undesirable for a number of reasons. First, the separation of an oligonucleotide only one base longer than the desired product can be
difficult and time consuming, and thus purification to a homogeneous product
can be problematic. Secondly, nucleotides are wasted in the production of a
considerable amount of unwanted longer RNA strands; this is more of a concern
when especially costly nucleotides (such as isotopically labeled ones for NMR
studies) are being used (
5
,
6
). Finally, ragged termini on the RNA transcripts can hamper further
manipulations, such as ligation reactions, which require a homogeneous 3'-end (
8
,
9
).
For the preparation of longer RNA strands using a DNA plasmid-derived template, one approach to generation of homogeneous ends is the
use of a ribozyme cassette which can be cloned into the template at or near the
desired 3'-end (
7
). Following transcription, the ribozyme motif self-cleaves to give the transcript a well-defined 3' terminus. For shorter RNA oligonucleotides it is common to
use a single-stranded synthetic DNA template which contains a double-stranded RNA polymerase promoter at the 3'-end (
1
-
4
). Ribozyme-mediated post-transcriptional cleavage is less practical to apply to shorter RNA
oligonucleotides since the synthetic DNA template would be increased in length
by dozens of nucleotides. Moreover, hammerhead-type ribozyme cleavage generates 2',3'-cyclic phosphate termini, whereas 3'-hydroxyl termini are needed for some
applications.
A similar problem of 3'-end heterogeneity also exists for enzymatic synthesis of DNA
sequences. Synthetic oligodeoxynucleotide templates are used in the production
of DNA oligonucleotides for NMR studies (
10
). For DNA synthesis of oligo- deoxynucleotides using Klenow fragment (KF) of
Escherichia coli
DNA polymerase I, a primer is either added to or is part of the template (
10
). Elongation of this primer to the end of the single-stranded template then gives the desired product. However, as with RNA
synthesis, undesired products one or two nucleotides longer are common (
10
,
11
).
Here we describe a new approach to making enzymatic synthesis of RNA and DNA
oligonucleotides with more homogeneous 3'-ends than was previously possible. We find that the addition of a
single non-coding nucleotide analogue to the 5' terminus of the template DNA strand can result in much more
efficient and specific termination at the desired site (3'-end of the product). The use of such `terminator' nucleosides
results in the production of much cleaner RNA and DNA oligonucleotide products, often in greater yields, and with more efficient use of nucleotides.
RESULTS
Our initial studies of non-polar DNA base analogues and their interactions with DNA and RNA
polymerases indicated that some of these unusual nucleosides were efficient at
stalling polymerase enzymes. This prompted us to study some of the most
efficient terminators for their potential utility in enzymatic DNA and RNA
synthesis; placement at the end of a template might stop polymerization at the
desired nucleotide (Fig.
1
). We therefore constructed several DNA template sequences which could be used
to test both DNA and RNA polymerases. We compared the oligonucleotide products of synthesis using normal DNA templates to those with templates containing our non-polar nucleosides at either internal or 5' terminal positions.
Figure 1
.
Illustration of poor termination which often occurs during enzymatic RNA and
DNA synthesis, yielding oligonucleotides one base longer than desired. On the
right is shown how end placement of a `terminator' nucleoside can cause a
polymerase to stop abruptly at the desired site.
Nucleoside design considerations
We have recently undertaken a study of non-polar isosteric analogues of the naturally occurring Watson-Crick bases (
12
,
13
). These have been designed to test the importance of hydrogen bonding and shape
complementarity in nucleic acid stability (
13
) and for information transfer via enzymatic polymerization. We observed that
when certain of these non-polar nucleoside analogues are included in a single-stranded DNA template, polymerases are very inefficient at inserting
normal Watson-Crick nucleoside triphosphates across from them or continuing
polymerization past them (see below). In general, nucleoside analogues which
are sterically or electronically incompatible with the active sites of
polymerases might be especially effective at stopping nucleic acid
polymerization at a specific site.
The 4-methylindole [beta]-nucleoside (
M
in Fig.
2
A) was designed to mimic the overall shape of deoxyadenosine, but without
hydrogen bonding groups. It and the related 4,6-dimethylindole nucleoside have been shown to pair very poorly with natural
Watson-Crick bases in duplex DNA (
13
). The [alpha]-naphthalene nucleoside (
N
) is slightly larger than a purine base; because of its lack of polar hydrogen bonding groups, its size, and its non-natural [alpha]-configuration, it is expected to pair poorly in duplex
nucleic acid and hence should be a very poor template for polymerases. The [alpha]-pyrene nucleoside (
P
) not only has the unnatural anomeric configuration, but it also possesses an
even larger aromatic system whose size approximates that of a full Watson-Crick base pair; these steric demands might be expected to exclude a
pairing nucleotide at the enzyme active site. This pyrene nucleoside also is of
considerable interest as a biophysical probe because of its fluorescence
properties (Paris,P. and Kool,E.T. manuscript in preparation). Finally, an
abasic nucleoside ([Phi]) (
14
) is included in these studies to test whether any effects observed are
specifically due the `base' moiety of the nucleoside rather than the sugar-phosphate backbone.
Figure 2
.
(
A
) Structures of non-polar nucleosides used as potential terminators of RNA transcription and
DNA elongation. Note that
N
and
P
nucleosides have alpha configurations about the C1' carbon. (
B
) Template DNA sequences used for RNA synthesis by transcription with T7 RNA
Polymerase and for DNA synthesis with the large fragment of
E.coli
DNA Polymerase I (Klenow fragment, KF). Upper case letters indicate the DNA
template and 18 nt T7 promoter for RNA transcription. The sequence of the
expected RNA product is shown in lower case letters; its length is indicated in
parentheses following the sequence designation. For DNA synthesis, elongation
proceeds from the 3'-end of the 18 nt T7 promoter which acts as a primer for DNA
polymerization. X indicates the terminator base which is
M
,
N
,
P
or [Phi].
Nucleoside synthesis
The indole nucleoside
M
was prepared in a straightforward manner using the procedure previously
described for the 4,6-dimethylindole analogue (
12
). Naphthalene and pyrene analogues
N
and
P
were synthesized in only two steps from 1-bromonaphthalene and 1-bromopyrene, using a cadmium-mediated aromatic coupling procedure developed in our
laboratory (
15
). All three base-coupled compounds were deprotected, converted in good yield to the 5'-protected DMT ethers and then the 3' phosphoramidite derivatives using standard methods. The abasic
nucleoside and its phosphoramidite derivative were prepared by the published procedure (
14
). These four phosphoramidites were incorporated into DNA using standard
automated coupling cycles.
Template sequences for testing of terminators
Four different templates were constructed to test the ability of these
nucleotides to correctly terminate RNA and DNA polymerization at the end of the
template (Fig.
2
B). In the four sequences, two different bases (A and G) are situated at the
last template position to test nearest neighbor effects. The templates are
single-stranded and possess T7 promoters at the 3'-end. An 18 nucleotide (nt) top strand acts both as part of
the promoter for RNA synthesis and as a primer for DNA synthesis. The expected
RNA products of the four templates range from 16 to 34 nt in length. The RNA
product of the shortest template (RbSb) is a substrate for a hammerhead
ribozyme that is encoded by the longest template (Rib1). The other two
templates (U1 and U2) code for RNA hairpins that are recognized by the R17 coat
protein; they were used by Uhlenbeck and co-workers to optimize the
in vitro
T7 RNA polymerase system for the synthesis of short RNAs (
1
,
2
). The products of RNA synthesis were monitored by gel electrophoresis using 5'-end-labeling by initiating transcription with [[gamma]-
32
P]GTP or internal labeling with [[alpha]-
32
P]ATP or [[alpha]-
32
P]UTP. For DNA synthesis with Klenow fragment, the 5'-end of the primer strand is radiolabeled, and elongation products
are monitored by PAGE. Expected product lengths for DNA synthesis range from 33
to 51 nt.
Internal placement of terminators: effects on RNA and DNA synthesis
Since a number of modified DNA bases are known to act within template strands as
stops to polymerases, we decided to compare effects of the new nucleosides both
internally and at the terminus. We first tested the qualitative effects of
nucleosides
M
,
N
,
P
and [Phi] on RNA and DNA synthesis when they are internally located in a short
template strand (Fig.
3
). Templates with natural bases at the same position were examined for
comparison. For RNA synthesis with T7 RNA polymerase, all four non-natural nucleosides cause termination with high efficiency (Fig.
3
B). There is little or no incorporation of rNTPs across from the non-polar bases or the abasic site, and there are virtually no longer products
arising from read-through beyond the sites in question. With the natural nucleotides at this
position, synthesis proceeds as expected to the end of the template, and only
small amounts of products from normal abortive synthesis are visible at the
position where strong stops are seen with the non-natural nucleosides.
Figure 3
.
The efficiency of termination at sites within the template sequence. (
A
) Template sequence used to test efficiency of terminating bases at internal
position. X indicates the variable natural (
A
,
C
,
G
or
T
) or terminator base (
M
,
N
,
P
or [Phi]). (
B
) Autoradiogram of PAGE analysis of RNA synthesis products using T7 RNA
polymerase and template with internal non-polar nucleoside analogues. (
C
) Autoradiogram of PAGE analysis of DNA synthesis products using Klenow fragment
(exo
-
) DNA polymerase and template with internal non-polar nucleoside analogues.
B
C
For DNA synthesis with KF, the internally-placed non-natural nucleosides also act as strong terminators (Fig.
3
C). However, in contrast to the RNA results, all have nucleotides inserted
opposite them. The enzyme is apparently slow to elongate past the unusual base
pairs, since very small amounts of full length products are seen. Of the four
non-natural structures,
M
gives the most full length (and longer) products, although this is minor
relative to the amount of truncated product generated after addition of a base
across from
M
. Nucleosides
N
and
P
also have nucleotides inserted opposite them, but give very little full length
product. Overall, at internal positions, all four compounds act as efficient
terminators for both RNA and DNA synthesis, although with DNA synthesis they
seem to allow insertion opposite themselves prior to stalling.
Effects of end-placed terminators on RNA synthesis
We then tested the potential terminators at the 5'-end of the templates. T7 RNA polymerase reactions using normal
templates and ones with terminator nucleosides at the 5'-end reveal that the non-polar analogues can decrease the appearance of products
longer than expected (Fig.
4
). For the standard RbSb template (without a terminator) there is a substantial amount of transcription product with one extra base; quantitation of radioactivity in these bands shows
the amount of
n
+1 (17mer) product is 45% that of the desired full-length 16 nt oligomer (Table
1
). In addition, there are at least four bands of yet greater length present.
These bands are due to self-priming of the RNA product (results not shown and ref.
16
) where nascent transcripts with self-complementary 3'-ends form a duplex which is elongated by the polymerase. The
actual full-length product was identified by isolating various bands labeled
internally with an [[alpha]-
32
P]rNTP, removing the 5' triphosphate with calf intestinal phosphatase, 5'-end-labeling with ATP and T4 polynucleotide kinase, and
comparing their gel mobility to that of a chemically synthesized, 5'-end-labeled RNA marker with the same sequence as the expected
product (results not shown).
Figure 4
.
Effect of terminators at the end of the template on RNA synthesis products.
Autoradiogram of PAGE analysis of RNA synthesis products using T7 RNA
polymerase and templates with non-polar nucleoside analogues (X) at their 5' termini. Lengths of the expected full-length products are indicated on the side of the gel.
Table 1
.
Effects of terminators on RNA synthesis
Nucleotides
M
,
N
and
P
are placed at the 5'-end of the template strand, and transcription is carried out with
T7 RNA polymerase. For comparison are the results with [Phi] (an abasic nucleoside) and with no terminator present. `
n
' is full-length product;
n
+1 is the product resulting from addition of an extra nucleotide.
a Quantitation of 32P labeled products by phosphorimaging. Error limits are standard deviations for
multiple experiments.
b Efficiency is defined as the (
n
/
n
+1) ratio for terminated case divided by the ratio with no terminator.
c Error is estimated at +-30% in single experiments.
Addition of 4-methylindole [beta]-deoxynucleoside (
M
) to the 5'-end of the RbSb template significantly decreases the amount of
product >16 nt. The 17mer and longer bands appear quite significantly lighter
on the autoradiogram (Fig.
4
); quantitation shows that the n+1 band drops from 45% (when unterminated) to 20% of the full-length product (Table
1
). The terminator efficiency, defined as the
n
/
n
+1 ratio for terminated template divided by the ratio for unterminated template,
averaged 2.3 for multiple experiments, meaning that the terminator cuts the
relative
n
+1mer product amount by 2.3-fold.
The decrease in
n
+1mer was found to be less when the RbSb template is terminated with the
naphthalene [alpha]-nucleoside (
N
); the terminator efficiency in this case is 1.4, corresponding to a 15% drop in this
unwanted product. Results for the pyrene [alpha]-nucleoside (
P
) were virtually the same, with a terminator efficiency of 1.7 (Table
1
). The nucleoside lacking a base altogether ([Phi]) gave no termination at all, with slightly greater amounts of
n
+1mer being observed than with the unterminated template; this establishes that
the bases (not the backbones) of
M
,
N
and
P
are responsible for their termination properties.
To test the generality of termination, we then examined three other templates
for RNA synthesis; we compared the most successful terminator (
M
) from the previous experiments with results with the abasic nucleoside [Phi] and the unterminated template (Fig.
4
). For the U1 and U2 templates, termination with
M
also decreases the percentage of
n
+1mer products present. Quantitation of the bands shows that
M
improves the
n
/
n
+1mer ratio by 1.7-fold. The abasic nucleoside slightly increases the
n
/
n
+1 ratio for the U1 template (10% relative decrease in
n
+1mer), but gives termination roughly equal to that of
M
for the U2 template (Table
1
). When the longest template (Rib1) is terminated with the
M
base, an apparent ~2-fold increase in the relative amount of the undesired
n
+1mer product is observed, and results are similar with the abasic nucleoside [Phi]. Comparison of the results for templates RbSb and U1, which have
different end bases, shows that the terminator
M
gives the same efficiency.
Examination of the overall results shows that four unterminated templates give
n
+1mer amounts which range from 38 to 83% of that for the desired full-length product. The most efficient terminator nucleoside is the 4-methylindole nucleoside
M
, which is effective in three of four cases, giving a ~2-fold reduction in longer products. The abasic nucleoside has little
or no effect on product lengths. Transcription reactions both with 500 [mu]M and with 1 mM NTP concentrations gave identical results. In addition,
examination of efficiency at different time points showed that relative
efficiency of
M
termination stayed approximately constant; however, we did observe slower RNA
synthesis overall with the terminated template (data not shown).
Effects of terminators on DNA synthesis
The same templates were then used to study nucleosides
M
,
N
and
P
as potential terminators for DNA synthesis. Comparison was again made with
abasic nucleoside [Phi] and the unterminated templates. Experiments were carried out with the
Klenow fragment of DNA polymerase I, using both a mutant enzyme lacking the 3'-5' exonuclease activity (designated exo
-
) and the wild type (exo
+
).
The results show that the non-natural nucleosides act as very efficient terminators of DNA synthesis.
Experiments with KF (exo
-
) using the RbSb template show (Fig.
5
; Table
2
) that the unterminated template gives fully five times as much
n
+1 product as the desired full-length product. The addition of the terminators reverses this unfavorable
ratio, giving the desired product as the major band. The terminator
M
gives 2.3 times as much full-length product as
n
+1mer, improving the relative full-length amount by almost 10-fold (its terminator efficiency). The naphthalene terminator
N
also worked very well (with a terminator efficiency of ~10) but less so than 4-methylindole
M
. Finally, the pyrene terminator
P
was quite effective, giving 4.4-fold more full-length product than
n
+1mer (an efficiency of >30). By comparison, the template ending with the abasic
nucleoside [Phi] gives results identical to those with no terminator at all (Table
2
).
Figure 5
.
Effect of terminators at the end of the template on DNA synthesis.
Autoradiogram of PAGE analysis of DNA synthesis products using the Klenow
fragment of DNA polymerase I. Templates have non-polar nucleoside analogues (
X
) at their 5' termini. Lengths of the expected full-length products (and
n
+1 products) are indicated on the side of the gel. The reactions with the
longest template (Rib1) are loaded before the other samples in order to obtain
better resolution between the
n
and
n
+1 products of primer elongation.
Table 2
.
Effects of terminators on DNA synthesis
Nucleosides
M
,
N
and
P
are placed at the 5'-end of template strands, and synthesis is carried out with KF DNA polymerase (exo
-
or exo
+
). For comparison are the results with [Phi] (an abasic nucleoside) and with no terminator present. `
n
' is full-length product; `
n
+1' is the product resulting from addition of an extra nucleotide.
a
Quantitation of
32
P-labeled products by phosphorimaging. Error limits are standard deviations
for multiple experiments.
b
Efficiency is defined as the (
n
/
n
+1) ratio for terminated case divided by the ratio with no terminator.
c
Error is estimated at +-30% in single experiments.
Experiments with same RbSb templates but with the wild-type KF (exo
+
) enzyme also show substantial increases in the desired product when the three
non-natural nucleosides are used as terminators (Fig.
5
; Table
2
). With the exonuclease activity, the undesired
n
+1mer product in the unterminated case is lowered significantly, although the
band is still 79% the intensity of the full-length product. With the terminators
M
,
N
and
P
the amount of undesired product becomes very small; the percentages of
n
+1 product relative to the desired product range from 19% for
M
to 9% for
P
. With this enzyme, the abasic nucleoside is seen to increase the amount of
undesired product, with an efficiency less than one.
Using the (exo
-
) KF mutant, we then tested the generality of termination with the three other
templates using nucleoside
M
, compared to the abasic nucleoside and the unterminated case. Results show that
DNA synthesis on the unterminated templates results in greater amounts of
n
+1mer than the actual desired product, with the latter making up only 10-50% of the two bands (Fig.
5
). In all three cases, addition of the terminator greatly increases both the
absolute and relative amounts of desired full-length product, with terminator efficiencies ranging from 2.7 to 6.4
(Table
2
). The abasic nucleoside again has either no effect or a negative effect on
desired product.
DISCUSSION
The present studies show clearly that a single non-hydrogen-bonding nucleoside added to the end of a template strand can significantly
improve the ratios of desired oligonucleotide products to longer undesired
ones. Interestingly, it is also clear that termination ability of nucleosides
at positions internally situated in a template is not necessarily a good
predictor of their properties when placed at the end of a template. For
example, the three analogues
M
,
N
and
P
are only moderate terminators of RNA synthesis at the template end, but are
strong terminators internally. It is possible that the strong tendency for
termination in this case arises in part from the proximity of the site in
question to the promoter, since RNA synthesis is aborted more easily in the
first few nucleotides (
17
). However, our control sequences (with natural nucleotides at the varied
position) show very little tendency for abortive synthesis, indicating that by
far the majority of the termination is caused by the non-natural nucleoside structures.
Another example of differential behavior internally and at the terminus is the
abasic nucleoside, which acts as a strong terminator internally but causes no
termination at all at the end of the template. It appears that in general it is
not safe to extrapolate results within a template to expectations at the
terminus. Many nucleoside analogues have been studied for their mutagenic
potential, and a large number of these cause pauses at internal sites (
18
-
21
); however, our results indicate that until they are studied at the terminus it
is unclear whether they can act as efficient end terminators.
Also interesting is the finding that in DNA synthesis, all three nucleoside
analogues in this study allow a natural nucleotide to be inserted opposite
themselves when they are internally located. However, when the analogues are at
the end of the template, they strongly inhibit this insertion. It is unclear at
present what is the origin of this difference in behavior with context. It
seems likely that end termination simply causes the enzyme to pause at the unusual template base after incorporating the last normal nucleotide, and that
this pause is long enough to allow enzyme dissociation from the template. In
the terminated RNA synthesis we observe substantial lowering of longer products
and a slight lowering of full-length product at early reaction times; this finding is consistent with
the terminator causing a pause which eventually leads to dissociation. In the
terminated DNA synthesis we observe a large decrease in
n
+1 products and a large increase in full-length products; we attribute this to the very strong tendency of the KF
enzyme to add an extra nucleotide. Causing dissociation prior to this event
allows the enzyme to make the desired product a greater fraction of the time.
In RNA synthesis, the 4-methylindole nucleoside
M
shows repeatable and significant (~2-fold) efficiency in two different nearest neighbor contexts. It gives
stronger end termination than do the naphthalene and pyrene analogues
N
and
P
. It is possible that the difference arises because
M
has the natural [beta]-anomeric configuration. It may be that yet larger non-hydrogen bonding base analogues which possess [beta]-configuration might further improve termination efficiency. One such possibility might
be a beta-anomeric analogue of pyrene nucleoside
P
; future studies will test the terminating ability of this non-natural nucleoside in comparison to the present results.
In DNA synthesis, all three terminators dramatically improved the amounts of
full-length products with the KF enzyme. It appears that both [beta]- and [alpha]-oriented bases can be effective. The fact that
the pyrene analogue is somewhat better at termination than the other two may be
because its size is larger, allowing it to occupy a greater volume in the
active site and excluding more effectively the incoming nucleoside
triphosphates.
The terminator nucleosides studied here are more successful in application to
DNA synthesis than to RNA synthesis, at least for the enzymes studied. It is
also true that the magnitude of the problem is greater for DNA synthesis, in
that the tendency for T7 RNA polymerase to add extra nucleotides is much less
than that for KF DNA polymerase. Nonetheless, a 2-fold reduction in
n
+1mer product during RNA synthesis, as seen for terminator
M
, would seem to make its use worthwhile. The application of such a terminator
adds very little effort to enzymatic oligonucleotide synthesis; indeed, it adds
only a single step to the synthesis (carried out automatically on the
synthesizer) and requires no other changes from standard methods. Since we
observed apparently poor termination in one of the four sequences, it would be
worthwhile to test this compound in additional sequences and in larger
preparative reactions to evaluate the general effects in RNA synthesis. At
present, since
M
is the best RNA terminator and is also a good DNA terminator, it would appear
to be the single analogue with the most general terminator utility. Future
studies may find new nucleosides with yet better termination abilities in RNA
synthesis.
We observe here that the KF DNA polymerase has a greater tendency to add an
extra nucleotide beyond the template than does T7 RNA polymerase. Enzymatic
synthesis of DNA oligonucleotides is often carried out with the KF (exo
-
) mutant, to limit degradation of the full-length products by the exonuclease activity under the reaction conditions.
However, as seen in our results, the enzyme lacking this activity has a strong
tendency to add an extra nucleotide at the end. In enzymatic DNA synthesis for
structural studies, this fact has caused other workers to choose the (exo
+
) variant of KF, which has a less strong tendency to add an extra nucleotide (
10
). In our experiments, however, even this latter enzyme gives a large amount of
the undesired longer product, and so terminators such as
M
,
N
and
P
should be of general value in DNA synthesis.
The present study has focused on short synthetic oligonucleotides as templates.
In the case of enzymatic synthesis of longer RNAs, it is common to generate
template duplexes from restriction cleavage of biologically derived DNAs. For
some applications it is necessary that the 3' terminus of the RNA be well-defined and homogeneous, such as when a subsequent ligation of the
RNA is to be performed. One approach to achieving this has been the
construction of cassettes in which a
cis
-cleaving ribozyme is included in order to generate a clean 3'-end. It seems possible that the present method might be a
useful alternative, in that a short sticky-ended segment of DNA carrying a terminator could be ligated onto the end
of any restriction fragment. This would also have the advantage of giving free
hydroxyl groups at the RNA terminus rather than phosphorylated hydroxyls which
result from ribozyme cleavage. This possibility awaits future experiments.
CONCLUSIONS
We conclude that simple placement of a non-hydrogen-bonding `terminator' nucleoside at the 5'-end of a given DNA template strand can be used to significantly improve the 3'-end homogeneity of products of RNA and DNA synthesis. Of
the structures studied to date, 4-methylindole nucleoside
M
is the most efficient terminator for T7 RNA polymerase. For DNA synthesis with
the Klenow fragment, the [alpha]-pyrene nucleoside
P
is the most effective terminator, with
M
also showing good efficiency. The application of such terminators may be useful
in simplifying purification and lowering the cost of enzymatic RNA and DNA
synthesis
in vitro.
MATERIALS AND METHODS
1
H and
13
C NMR spectra were recorded with a 300 MHz spectrometer unless otherwise noted,
and chemical shifts are reported in p.p.m. on the [delta] scale with the solvent as an internal reference, and the coupling
constants are in Hertz (Hz). The mass spectra were performed using fast atom
bombardment or electron impact.
All reactions were monitored by thin-layer chromatography (TLC) using EM Reagents plates with fluorescence indicator (SiO
2
-60, F-254). Flash column chromatography was conducted using EM Science
Silica Gel 60 (230-400 mesh). Mass spectral analyses were performed by the Riverside Mass
Spectrometry Facility (University of California, Riverside, CA).
All reactions were carried out under a nitrogen atmosphere in dry, freshly
distilled solvents under anhydrous conditions unless otherwise specified. THF
was distilled from sodium metal/benzophenone, methylene chloride was distilled
from NaH, and pyridine was distilled from BaO prior to use.
The abasic nucleoside ([Phi]) was synthesized according to the published procedure (
14
).
1
'
,2
'
-Dideoxy-1
'
-(4-methylindol-1-yl)-3
'
,5
'
-di-
O
-toluoyl-
[beta]
-D-ribofuranose (M)
4-Methylindole (0.92 g, 7 mmol) was added to a flame-dried, nitrogen flushed flask and dissolved in 35 ml acetonitrile.
The solution was cooled to 0o C when 60% sodium hydride dispersion (0.393 g, 9.8 mmol) was added and the
blue-green solution stirred for 30 min. Solid 1-[alpha]-chloro-3,5-di-
O
-toluoyl-2-deoxyribose (
22
) (2.781 g, 7.2 mmol) was added in several portions over 90 min. The reaction
was cooled for >= 2 h then stirred overnight while warming to room temperature. The
acetonitrile was evaporated and the brown residue redissolved in diethyl ether.
The ether solution was washed with saturated sodium bicarbonate solution and
brine before drying over anhydrous magnesium sulfate. Concentration and
chromatography on silica eluting with methylene chloride gave 54% yield as a
beige foam, R
f
= 0.47 (methylene chloride).
1
H NMR
3
, p.p.m.) [delta] 8.03 (2H, d, J = 8), 8.00 (2H, d, J = 8), 7.42 (1H, d, J = 8.4), 7.33
(3H, m), 7.28 (2H, d, J = 8), 7.12 (1H, t, J = 8), 6.97 (1H, d, J = 7.2), 6.60
(1H, d, J = 3.2), 6.51 (1H, dd, J = 5.6, 8.4'), 5.75 (1H, m), 4.69 (2H, d, J = 6), 4.61 (1H, br s), 2.89 (1H, m), 2.69
(1H, m), 2.56 (3H), 2.48 (3H, s), 2.46 (3H, s);
13
C NMR
3
, p.p.m.) 18.6, 21.7, 37.8, 64.3, 75.1, 81.5, 85.6, 101.9, 102.1, 107.5, 120.5,
122.3, 123.2, 126.6, 126.9, 129.0, 129.2 (d),129.7 (d), 130.6, 135.6, 143.9,
144.4;
HRMS
30
H
29
NO
5
483.2045, found 483.2028.
General procedure for glycosidic coupling reaction and isolation of major
[alpha]
-epimers as bis-p-toluoyl esters of
N
and
P
Dry THF (5 ml) was placed in a round-bottomed flask equipped with a condenser, drying tube and addition funnel.
Magnesium turnings (0.3 g, 1.2 mmol) and a few crystals of iodine were added. 1-Bromopyrene (0.35 g, 1.2 mmol) was added to the mixture. Slight heating
was needed (40o C) to drive the reaction to completion. After formation of the Grignard
reagent was complete (~1 h), dry CdCl
2
(110 mg, 0.6 mmol) was added and the reaction mixture was continuously heated under reflux for 1 h. 1-[alpha]-chloro-3,5-di-
O
-toluoyl-2-deoxyribose (
22
) (0.51 g, 1.3 mmol) was then added to the above mixture in one portion. The
solution was stirred at room temperature for 4 h under an atmosphere of N
2
. The solution was poured into 10% ammonium chloride (2 * 50 ml) and extracted with methylene chloride. The organic layers were
washed with saturated sodium bicarbonate and brine and dried over anhydrous
magnesium sulfate. The solution was filtered, concentrated and purified by
flash silica gel chromatography eluting with hexanes-ethyl acetate (9:1). The major product 1',2'-dideoxy-1'-(1-pyrenyl)-3',5'-di-
O
-toluoyl-[alpha]-D-ribofuranose (
P
) was obtained as a pale yellow oil ([alpha]-epimer, 48% isolated yield) R
f
=
0.29 (ethyl acetate:hexanes 20:80):
1
H NMR
3,
p.p.m.) [delta] 8.80 (2H, d, J = 8.0), 8.72 (2H, d, J = 8.0), 8.05 (1H, s), 7.92-8.00 (2H, m), 7.72-7.60 (4H, m), 7.58 (2H, d, J = 8.0), 7.32 (2H, d, J = 8.0),
6.96 (2H, d, J = 8.0), 6.15 (1H, dd, J = 8.2, 6.0), 5.76 (1H, m), 4.98 (1H, m),
4.75-4.65 (2H, m), 3.30-3.22 (1H, m), 3.50-3.45 (1H, m), 3.44 (3H. s), 3.38 (3H, s);
13
C NMR
3
, p.p.m.) [delta] 21.3, 21.4, 39.2, 64.5, 76.2, 78.0, 82.5, 122.3, 122.9, 123.2, 123.6,
126.0, 126.3, 126.4, 126.6, 126.8, 127.0, 128.7, 128.8, 128.9, 129.0, 129.2,
129.4, 129.6, 129.8, 130.6, 131.4, 136.2, 143.5, 143.6, 165.8, 166.2;
HRMS
37
H
31
O
5
(M+1) 554.2093, found 554.2069.
1
'
,2
'
-Dideoxy-1
'
-(1-naphthyl)-3
'
,5
'
-di-
O
-toluoyl-
[alpha]
-D- ribofuranose (N)
([alpha]-epimer, 52% isolated yield) R
f
= 0.36 (ethyl acetate:hexanes 20:80)
1
H NMR
3,
p.p.m.) [delta] 8.05 (2H, d, J = 8.0), 7.95 (2H, m), 7.83 (2H, overlapped d), 7.71 (2H,
d, J = 8.0), 7.55 (3H, m), 7.32 (2H, d, J = 8.0), 7. 19 (2H, d, J = 8.0), 6.10
(1H, dd, J = 8.0, 6.0), 5.69 (1H, m), 4.90 (1H,m), 4.76-4.65 (2H, m), 3.28-3.18 (1H, m), 2,52-2.45 (1H, m), 2.48 (3H, s), 2.42 (3H, s);
13
C NMR
3
, p.p.m.) [delta] 21.4, 21.5, 39.5, 64.5, 76.2, 77.8, 82.2, 122.1, 122.9, 125.1, 125.3,
125.8, 126.6, 127.0, 128.7, 128.8, 128.9, 129.2, 129.4, 129.5, 129.9, 133.6,
137.9, 143.6, 165.8, 166.2;
HRMS
31
H
29
O
5
(M+1) 481.2015, found 481.2025.
General procedure for the deprotection of 1
'
,2
'
-dideoxy-1
'
- (aryl)-3
'
,5
'
-di-
O
-toluoyl-D-ribofuranoses
1',2'-Dideoxy-1'-(4-methylindol-1-yl)-3',5'-di-
O
-toluoyl-[beta]-D- ribofuranose (
1
1
) (1.766g, 3.8 mmol) was dissolved in 25 ml MeOH and treated with 2 ml 0.75 M NaOMe solution. The reaction was stirred for 3
h at room temperature under a nitrogen atmosphere and quenched by adding solid
ammonium chloride. The mixture was filtered and concentrated before
chromatographing on silica eluting with ethanol:diethyl ether (5:95). The product 1',2'-Dideoxy-1'-(4-methylindol-1-yl)-[beta]-D-ribofuranose (
M
) was obtained in 88% yield as a light pink oil, R
f
= 0.30 [ethanol:diethyl ether (5:95)].
1
H NMR
3
, p.p.m.) [delta] 7.32 (2H, m), 7.18 (1H, t, J = 9), 6.97 (1H, d, J = 6), 6.60 (1H, br s),
6.33 (1H, t, J = 6), 4.40 (1H, m), 3.88 (1H, m), 3.64 (2H, m), 3.30 (2H, br s),
2.56 (3H, s), 2.51 (1H, m), 2.31 (1H, m);
13
C NMR
3
, p.p.m.) [delta] 18.6, 39.5, 62.3, 71.3, 84.5, 85.8, 101.9, 107.4, 120.6, 122.4, 123.2,
128.8, 130.6, 135.8;
HRMS
14
H
17
NO
3
247.1208, found 247.1196.
1',2'-Dideoxy-1'-(1-pyrenyl)-[alpha]-D-ribofuranose (
P
) was obtained by chromatography on silica eluting with ethyl acetate as an off-white solid in 69% yield, R
f
= 0.26 (ethyl acetate).
1
H NMR
3
, p.p.m.) [delta] 8.35 (1H, d, J = 8), 8.29-8.00 (8H, m), 6.19 (1H, dd, J = 7.2, 8), 4.63 (1H, m), 4.47 (1H,
m), 4.18 (1H, m), 3.85 (1H, m), 3.10 (1H, m), 2.23 (1H, m), 1.87 (2H, bs);
13
C NMR
6
DMSO, p.p.m.) [delta] 48.7, 67.0, 77.1, 81.3, 91.8, 128.2 (d), 129.1, 129.2, 130.1 (d), 130.3,
131.3, 131.8, 131.9, 132.4, 132.6, 134.9, 135.4, 136.0, 143.1;
HRMS
21
H
18
O
3
(M
+
) 318.1257, found 318.1256.
1
'
,2
'
-Dideoxy-1
'
-(1-naphthyl)-
[alpha]
-D-ribofuranose (N)
1',2'-Dideoxy-1'-(1-naphthyl)-[alpha]-D-ribofuranose (
N
) was obtained by chromatography on silica eluting with ethyl acetate as an off-white solid in 78% yield, R
f
= 0.22 (ethyl acetate).
1
H NMR
3
, p.p.m.) [delta] 7.86 (1H, d, J = 8), 7.77 (3H, m), 7.45 (3H, m), 5.62 (1H, dd, J = 7.3,
8), 4.32 (1H, m), 4.13 (1H, m), 3.94-3.62 (4H, m), 2.65 (1H, m), 1.97 (1H, m)
13
C NMR
3
, p.p.m.) [delta] 42.5, 58.2, 72.9, 77.0 (obscured by solvent), 85.3, 121.5, 122.9, 125.4
(d), 126.0, 127.8, 128.8, 129.8, 133.6, 138.0;
HRMS
15
H
16
O
3
(M
+
) 244.1102, found 244.1099.
1
'
,2
'
-Deoxy-5
'
-(4,4
'
-dimethoxytrityl)-1
'
-(4-methylindol-1-yl)-
[beta]
-D-ribofuranose (M)
1',2'-Dideoxy-1'-(4-methylindolyl)-[beta]-D-ribofuranose (0.336 g, 1.4 mmol) was
dissolved in 20 ml methylene chloride in a flame dried, nitrogen flushed flask.
N
,
N
-Diisopropylethylamine (0.34 ml, 4.2 mmol) was added, followed by 4,4'-dimethoxytrityl chloride (0.745 g, 2.2 mmol). The reaction
was stirred for 3 h at reflux, then quenched with 1 ml methanol. After 30 min
methylene chloride (30 ml) was added and the mixture was washed with saturated
sodium bicarbonate solution and brine. Drying over anhydrous sodium sulfate and
silica gel chromatography eluting with ethyl acetate:hexanes (40:60 plus 5%
triethylamine), R
f
= 0.23, gave the product in 72% yield.
1
H NMR
3
, p.p.m.) [delta] 7.42 (2H, m), 7.31 (4H, m), 7.22 (5H, m), 7.06 (1H, t, J = 6), 6.91 (1H,
d, J = 6), 6.82 (4H, d, J = 9), 6.50 (1H, d, J = 3), 6.35 (1H, t, J = 6), 4.51
(1H, m), 4.03 (2H, m), 3.74 (6H, s), 3.31 (2H, m), 2.63 (1H, m), 2.54 (3H, s),
2.43 (1H, m), 1.63 (1H, br s);
13
C NMR
3
, p.p.m.) [delta] 11.1, 18.6, 40.1, 45.9, 55.1, 64.0, 72.4, 84.9, 86.4, 101.4, 107.6,
113.1, 120.3, 122.1, 123.5, 126.8, 127.8, 128.1, 129.0, 130.0, 130.3, 135.7,
144.6, 158.4;
HRMS
35
H
35
NO
5
549.2515, found 549.2514.
General procedure for preparation of tritylated
[alpha]
-C-nucleosides
1',2'-Dideoxy-1'-(1-naphthyl)-[beta]-D-ribofuranose (461 mg, 1.88 mmol) was dissolved in dry pyridine (20 ml) and a catalytic amount of DMAP (11 mg) was added. 4,4'-Dimethoxytrityl chloride was added to the above mixture and the reaction was stirred under a nitrogen atmosphere for 3 h and then quenched with 2 ml ethanol. The reaction was diluted with methylene chloride (75 ml) and washed once with 5% sodium bicarbonate solution. The organic mixture was dried over anhydrous sodium sulfate, filtered and concentrated. The product was purified by silica gel chromatography (pre-equilibrated with 5% triethylamine in hexanes) eluting with ethyl acetate:hexanes (25:75). The product 1',2'-Deoxy-5'-
(4,4'-dimethoxytrityl)-1'-(1-naphthyl)-[alpha]-D-ribofuranose (
N
) was obtained as a yellowish foam in 83% yield (859 mg, 1.57 mmol), R
f
= 0.25 (ethyl acetate:hexanes 1:2):
1
H NMR
3,
p.p.m.) [delta] 7.90 (2H, m), 7.83 (2H, m), 7.62-7.25 (12H, m), 6.92 (4H, d, J = 8.8), 5.95 (1H, unresolved m) 4.55
(1H, m), 4.02 (1H, m) 3.83 (6H, s), 3.45 (2H, m), 3.00 (1H, m), 2.17 (2H, m);
13
C NMR
3
, p.p.m.) [delta] 42.6, 55.2, 64.8, 74.7, 77.0 (obscured by solvent), 85.1, 86.4, 113.3,
122.0, 123.4, 125.6, 126.0, 126.9, 127.8, 128.0, 128.3, 128.9, 130.2, 130.3,
133.8, 136.1, 139.0, 145.1, 158.6;
HRMS
36
H
33
O
5
(M-1) 545.2298, found 545.2328.
1
'
,2
'
-Dideoxy-5
'
-(4,4
'
-dimethoxytrityl)-1
'
-(1-pyrenyl)-
[alpha]
-D-ribofuranose (P)
1',2'-Dideoxy-5'-(4,4'-dimethoxytrityl)-1'-(1-pyrenyl)-[alpha]-D-ribo-
furanose (
P
) was obtained as a white foam in 50% yield, R
f
= 0.23 (ethyl acetate:hexanes 1:2):
1
H NMR
3,
p.p.m.) [delta] 8.42 (1H, m), 8.32-8.01 (8H, m), 7.72-7.28 (9H, m), 6.94 (4H, d, J = 8), 6.26 (1H, unresolved m),
4.65 (1H, m), 4.55 (1H, m), 3.84 (6H, s) 3.54 (2H, m), 3.09 (1H, m), 2.25 (2H,
m);
13
C NMR
HRMS
42
H
36
O
5
(M+) 620.2577, found 620.2563.
General procedure for the preparation of 1
'
,2
'
-dideoxy- 5
'
-(4,4
'
-dimethoxytrityl)-1
'
-(aryl)-D-ribofuranose-3
'
-(cyano-ethyl)-
N
,
N
-diisopropyl-phosphoramidites
1',2'-Dideoxy-5'-(4,4'-dimethoxytrityl)-1'-(4-methylindolyl)-[beta]-
D-ribofuranose (0.311 g, 0.6 mmol) was dissolved in 3 ml methylene chloride under nitrogen. With stirring,
N
,
N
-diisopropylethylamine (0.040 ml, 2.3 mmol) was added via syringe followed
by 2-cyanoethyl-
N
,
N
-diisopropylchlorophosphoramidite (0.19 ml, 0.85 mmol). The pale yellow solution was stirred at room temperature for
20 min and then was diluted with 10 ml ethyl acetate. Washing the solution with
saturated sodium bicarbonate solution and brine before drying over anhydrous
sodium sulfate gave a pale yellow oil. The oil was chromatographed on silica
gel eluting with ethyl acetate:hexanes:triethylamine (40:55:5), R
f
= 0.66, to give 90% yield of 1',2'-dideoxy-5'-(4,4'-dimethoxytrityl)-1'-(4-methyl-
indolyl)-[beta]-D-ribofuranose-3'-(cyanoethyl)-
N
,
N
-diisopropylphosphoramidite (
M
) as an off-white foam.
1
H NMR
3
, p.p.m.) [delta] 7.47 (2H, m), 7.37 (4H, m), 7.27 (5H, m), 7.11 (1H, t, J = 6), 6.96 (1H,
d, J = 6), 6.80 (4H, d, J = 9), 6.56 (1H, d, J = 3), 6.42 (1H, t, J = 6), 4.74
(1H, m), 4.26 (2H, m), 3.80 (6H, s), 3.67 (3H, m), 3.39 (2H, m), 2.71-2.55 (1H, m), 2.56 (3H, s), 2.49 (2H, t), 2.41 (3H, s), 1.23 (12H, t);
13
C
3
, p.p.m.) [delta] 11.2, 18.6, 20.2, 24.5, 39.5, 43.3, 46.0, 55.1, 58.4, 63.6, 73.5, 73.9,
85.2, 86.4, 101.4, 107.7, 113.0, 120.2, 121.8, 123.55, 126.5, 127.7, 128.5,
129.1, 130.1, 130.6, 135.7, 144.7, 158.4;
31
P
3
PO
4
, p.p.m.) 146.3, 146.7;
HRMS
44
H
52
N
3
O
6
P 749.3594, found 749.3582.
1
'
,2
'
-Dideoxy-5
'
-(4,4
'
-dimethoxytrityl)-1
'
-(1-pyrenyl)-
[alpha]
- D-ribofuranose-3
'
-cyanoethyl-
N
,
N
-diisopropylphosphoramidite (P)
1',2'-Dideoxy-5'-(4,4'-dimethoxytrityl)-1'-(1-pyrenyl)-[alpha]-D-ribofuranose-3'-cyanoethyl-
N
,
N
-diisopropylphosphoramidite (
P
) was obtained as a white foam in 96% yield, R
f
= 0.23 (ethyl acetate:hexanes 1:2):
1
H NMR
3,
p.p.m.) [delta] 8.50 (1H, m), 8.36-8.05 (8H, m), 7.76 (2H, m), 7.55 (4H, m), 7.44 (2H, m), 7.30 (1H,
m), 6.96 (4H, m), 6.39 (1H, unresolved m), 4.94-4.80 (1H, m), 4.77 (1H, m), 3.85 (6H, s), 3.76-3.40 (4H, m), 3.34 (1H, m), 3.22 (1H, m), 2.44 (1H, m), 2.20 (1H,
m), 1.15-1.05 (12H, m), 1.02 (2H, d, J = 6.7);
13
C NMR
HRMS
51
H
54
N
2
O
6
P (M+1) 821.3762, found 821.3720.
1
'
,2
'
-Dideoxy-5
'
-(4,4
'
-dimethoxytrityl)-1
'
-(1-naphthyl)-
[alpha]
-D-ribofuranose-3
'
-cyanoethyl-
N
,
N
-diisopropylphosphor-amidite (N)
1',2'-Dideoxy-5'-(4,4'-dimethoxytrityl)-1'-(1-naphthyl)-[alpha]-D-ribo-furanose-3'-cyanoethyl-N,N-diisopropylphosphoramidite (
N
) was obtained as a white foam in 92% yield, R
f
= 0.35 (ethyl acetate:hexanes 1:2):
1
H NMR
3,
p.p.m.) [delta] 8.06 (1H, m), 7.95-7.80 (2H, m), 7.66-7.42 (9H, m), 7.38 (2H, m), 7.35 (1H, m), 6.92 (4H, m),
6.05 (unresolved m), 4.83-4.63 (1H, m), 4.58 (1H, m), 3.84 (6H, s), 3.63 (1H, m), 3.60-3.40 (3H, m), 3.37 (1H, m), 3.05 (1H, m), 2.42 (1H, m), 2.37 (1H,
m), 1.18 (6H, d, J = 6.6), 1.11 (3H, d, J = 7), 1.05 (3H, d, J = 7);
13
C NMR
HRMS
45
H
52
N
2
O
6
P (M+1) 747.3590, found 747.3563.
Oligonucleotide synthesis
Oligonucleotides were synthesized on an ABI 392 DNA/RNA synthesizer using [beta]-cyanoethyl phosphoramidites. The coupling time used for the non-polar phosphoramidites was the same as for conventional Watson-Crick bases. Intact incorporation of residues
M
,
N
,
P
was confirmed by synthesis of short oligomers of sequence T-X-T (where X =
M
,
N
,
P
); proton NMR (400 MHz) indicated the presence of the intact structures with the
expected integration. The 5' dimethoxytrityl group was removed at the end of the synthesis, and the
oligomers were deprotected with concentrated NH
4
OH at 55o C overnight. Following lyophilization, oligonucleotides were purified by
electrophoresis on 2.5 mm denaturing (8 M urea) 15 or 20% polyacrylamide gels.
After elution with Milli-Q H
2
O and desalting with a Waters C-18 Sep-Pak column, the purity of the oligonucleotide product was checked by
5'-end-labeling with [[gamma]-
32
P]ATP and T4 polynucleotide kinase and then using analytical (0.4 mm) denaturing gel electrophoresis. Oligomers with terminators at their 5'-end ran one nucleotide slower than their unterminated control
oligomer with the same sequence. Most sequences were gel purified twice to
reduce the contaminating
n
-1 oligonucleotide to <10 % of full-length synthesis product; most had no
n
-1 product visible after purification.
Enzymatic RNA synthesis and quantitation
T7 RNA polymerase reactions were run in 10 [mu]l volumes (final concentrations are listed). One [mu]M each of the template and the 18-nt T7 promoter (Fig.
1
B) were mixed in transcription buffer [40 mM Tris-HCl (pH 8.3 at room temperature), 6 mM MgCl
2
, 10 mM dithiothreitol (DTT), 2 mM spermidine] and Milli-Q H
2
O for 5 min (heating the promoter-template duplex to 80o C for 5 min to anneal had no effect on the yield or product distribution of
the transcription reaction). Following addition of 0.5 mM rNTPs and 10 [mu]Ci [[gamma]-
32
P]GTP, [[alpha]-
32
P]ATP or [[alpha]-
32
P]UTP (New England Nuclear), the polymerization reaction was initiated by adding
T7 RNA polymerase (New England BioLabs) to 5 U/[mu]l [decreasing the cold (unlabeled) concentration of the rNTP carrying the [alpha]-radiolabel increased the specific activity of products but did
not affect product distribution]. Increasing Mg
2+
and DTT concentrations to 20 mM and using 1 mM each of the rNTPs increased the
yield of the transcription reaction but did not affect the distribution of
product oligonucleotides. Reaction time was 30 min at 37o C.
Both RNA transcription and DNA elongation reactions were stopped by adding 8 [mu]l gel loading buffer [80% formamide, 1* TBE buffer (89 mM Tris-borate, 2 mM EDTA), 0.1% xylene cylanol, 0.1%
bromophenol blue]. Reactions were assayed by analytical (0.4 mm thickness)
denaturing gel electrophoresis (15 or 20% polyacrylamide). Following autoradiography to visualize reaction products, the intensities of various product bands were quantified with a
PhosphorImager (Molecular Dynamics Model 400 Series).
Enzymatic DNA synthesis
Reactions with the large fragment of
E.coli
DNA polymerase I [Klenow fragment, both normal (wild type) and
exo
-
mutant lacking the 3'-5' exonuclease activity, US Biochemical] were also run in 10 [mu]l volumes. The 18-nt T7 promoter was 5'-end labeled with [[gamma]-
32
P]ATP and T4 polynucleotide kinase and purified via denaturing gel
electrophoresis (0.4 mm thickness). Following elution from gel slices with
Milli-Q H
2
O and dialysis to desalt, the radiolabeled oligomer (~5 nM) was mixed with cold template (0.2 [mu]M), Klenow buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl
2
, 1 mM DTT, 50 mg/ml bovine serum albumin), and Milli-Q H
2
O for 5 min at room temperature. After addition of cold primer (0.18 [mu]M) and another 5 min room-temperature incubation, Klenow was added to 0.3 U/[mu]l (0.2 [mu]M) and the reaction mixture was incubated 10 min at 37o C. Elongation of the DNA primer was initiated by the
addition of 20 [mu]M dNTPs. Reaction times are 2 and 15 min at 37o C.
ACKNOWLEDGEMENTS
We thank the National Institutes of Health (GM52956) and the Army Research Office for support. E.T.K gratefully acknowledges an Alfred P. Sloan Foundation Fellowship and a Dreyfus Foundation Teacher-Scholar Award.
REFERENCES
1 Milligan ,J.F. , Groebe,D.R., Witherell,G.W. and Uhlenbeck,O.C. (1987 ) Nucleic Acids Res. 15
2 Milligan ,J.F. and Uhlenbeck,O.C. (1989 ) Methods Enzymol. 180
3 Stump ,W.T. and Hall,K.B. (1993 ) Nucleic Acids Res. 21
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