ABSTRACT
We report the design, synthesis and evaluation of a non-nucleosidic photocleavable biotin phosphoramidite (PCB
-
phosphoramidite) which provides a simple method for purification and
phosphorylation of oligonucleotides. This reagent introduces a photocleavable
biotin label (PCB) on the 5
'
-terminal phosphate of synthetic oligonucleotides and is fully compatible
with automated solid support synthesis. HPLC analysis shows that the PCB moiety
is introduced predominantly on full-length sequences and is retained during cleavage of the synthetic
oligonucleotide from the solid support and during subsequent deprotection with
ammonia. The full-length 5
'
-PCB-labeled
oligonucleotide can then be selectively isolated from the crude oligonucleotide
mixture by incubation with immobilized streptavidin. Upon irradiation with 300-350 nm light the 5
'
-PCB moiety is cleaved with high efficiency in <4 min, resulting in rapid release of affinity-purified 5
'
-phosphorylated oligonucleotides into solution. 5
'
-PCB-labeled oligonucleotides should be useful in a variety of
applications in molecular biology, including cassette mutagenesis and PCR. As
an example, PCB
-
phosphoramidite has been used for the synthesis, purification and
phosphorylation of 50- and 60mer oligonucleotides.
Biotin is widely used for non-radioactive DNA/RNA detection (
1
) due to the extremely high affinity of the biotin-streptavidin interaction (association constant 10
15
/M) (
2
). A biotin moiety can be introduced into the oligonucleotide: (i) during its
solid support synthesis using biotinyl phosphoramidite (
3
-
7
); (ii) enzymatically with biotinylated nucleoside triphosphate analogs, such as
biotin-11-dUTP (
8
); (iii) through post-synthetic modifications with suitable biotinylation reagents such as
biotin
N
-hydroxysuccinimide ester, biotin hydrazide (
1
,
9
) or photobiotin (
10
). If this biotinylation could be reversed, thereby releasing the
oligonucleotide from the streptavidin-biotin complex, biotinylation would provide a much more general approach
in molecular biology. For example, oligonucleotides could be isolated using
streptavidin affinity media and then released for subsequent use in recombinant
DNA methods. A particularly useful application is the isolation of full-length oligonucleotides from failure sequences that result during
automated solid support synthesis. A second application is the isolation of PCR
products resulting from incorporation of biotinylated primers.
The most common approach for the removal of biotinyl moieties is the
introduction of a chemically cleavable spacer arm. For example, a biotinylated
nucleoside triphosphate analog containing a disulfide bond has been synthesized
(
11
) and used in a variety of applications (
12
-
14
). Alternatively, a biotin reagent with a cleavable spacer arm can be used to
label 5'-amino-modified oligonucleotides (
15
). However, release of the oligonucleotide requires a reducing agent, such as
dithiotreitol (DTT), which can damage enzymes and DNA-protein complexes and also leaves a modified base which can interfere
with subsequent use of the oligonucleotide. Nucleosidic phosphoramidites
containing an acid-cleavable spacer arm on the 5'-end have also been described (
16
,
17
). However, these require long cleavage times (>3 h) under acidic conditions
(80% acetic acid).
We report here the synthesis of a photocleavable biotin phosphoramidite (PCB-phosphoramidite). This phosphoramidite incorporates a recently described
photocleavable biotin moiety (PCB) (
18
) on the 5'-end of a synthetic oligonucleotide. We show that: (i) the PCB-phosphoramidite reagent is fully compatible with automated
DNA/RNA synthesizers using phosphoramidite chemistry; (ii) the 5'-PCB moiety is retained during cleavage from the solid support and
deprotection of the oligonucleotide with ammonia; (iii) the 5'-PCB moiety allows streptavidin affinity purification of the
oligonucleotide from failure sequences; (iv) the PCB moiety is rapidly and
quantitatively photocleaved from the 5'-end upon irradiation with near-UV light (300-350 nm) to give a 5'-phosphorylated oligonucleotide.
Importantly, PCB-phosphoramidite provides a rapid method for the purification, isolation
and phosphorylation of synthetic oligonucleotides. As an example, we have used
PCB-phosphoramidite to synthesize, purify and phosphorylate 50- and 60mer oligonucleotides.
All chemicals used in the synthesis were purchased from Aldrich Chemical Co.
(Milwaukee, WI).
1
H NMR spectra were recorded in CDCl
3
on a Varian (Palo Alto, CA) Unity Plus spectrometer at 400 MHz with chemical
shifts ([delta], p.p.m.) reported relative to a tetramethylsilane internal standard.
31
P NMR spectra were recorded in CDCl
3
on a JEOL (Peabody, MA) JNM-GSX270 spectrometer at 109.36 MHz with chemical shifts ([delta], p.p.m.) reported relative to an 85% H
3
PO
4
external standard. Oligonucleotide synthesis was performed on an Applied
Biosystems (Foster City, CA) DNA/RNA synthesizer model 392. Samples were
irradiated with a Blak Ray XX-15 UV lamp (Ultraviolet Products Inc., San Gabriel, CA) at a distance of
15 cm (emission peak 365 nm, 300 nm cut-off, 1.1 mW intensity at 31 cm). UV-visible spectra were recorded on a Shimadzu 2101PC
spectrophotometer. HPLC analysis was performed on a Waters (Milford, MA) system
consisting of a U6K injector, 600 Controller, Novapak C18 (3.9 * 150 mm) column and a 996 photodiode array detector. Buffer A, 0.1 N
triethylamine acetate, pH 6.0; buffer B, acetonitrile. Elution was performed
using a linear gradient (8-45%) of buffer B in buffer A over 45 min at a flow rate of 1 ml/min.
Preparative purification of 5'-PCB-(dT)
7
was achieved on a Waters Novapak C18 RCM cartridge (8 * 100 mm) using conditions as specified above, except for flow rate, which
was increased to 2 ml/min. Fractions were then analyzed, pooled and freeze
dried. No special precautions were necessary to protect the reagent and the 5'-PCB-labeled oligonucleotides from light.
1-N-(4,4
'
-dimethoxytrityl)-5-(6-biotinamidocaproamidomethyl)- 2-nitroacetophenone (compound
2
).
5-(6-Biotinamidocaproamidomethyl)-2-nitroacetophenone (
1
) (
18
) (0.5 g, 0.94 mmol) was dried by co-evaporation with anhydrous pyridine (3 * 2 ml) and then dissolved in 5 ml of the latter. To this solution
was added 4,4'-dimethoxytrityl chloride (DMTr-Cl) (0.634 g, 1.87 mmol) followed by 4-dimethylaminopyridine (0.006 g, 0.046 mmol). The
reaction mixture was stirred at room temperature for 5 h and then an additional
0.317 g DMTr-Cl was added. After 24 h the reaction was quenched with methanol (1 ml),
poured into 100 ml 0.1 M sodium bicarbonate and extracted with methylene
chloride (3 * 50 ml). Evaporation of the combined extracts gave a yellow oil, which
was further purified on a silica gel column using a step gradient of methanol
in dichloromethane, 0.2% triethylamine. Appropriate fractions were pooled and
evaporated to give compound
2
as a white foam (0.73 g, 93% yield). TLC, CHCl
3
:MeOH 9:1 v/v;
R
f
= 0.45.
1
H NMR: 7.87-7.85 (d,2H), 7.23-7.20 (m,5H), 7.17-7.15 (d,1H), 7.11-7.05 (m,5H), 6.75-6.71 (m,4H), 5.92-5.86 (t,1H), 5.61 (s,1H), 4.43-4.36 (m,2H), 4.05-3.85 (m,2H), 3.73
(s,6H), 3.70 (s,3H), 3.40-3.30 (m,1H), 3.12-3.02 (m,2H), 2.98-2.89 (m,1H), 2.48 (s,1H), 2.32-2.20 (m,2H), 2.11-2.03 (m,4H), 1.63 (s,3H), 1.60-1.34 (m,7H), 1.26-1.23 (m,2H). Elemental analysis
(%): calculated (C
46
H
53
N
5
O
8
S), C 66.09, H 6.39, N 8.38; found, C 65.85, H 6.23, N 8.05.
1-N-(4,4
'
-dimethoxytrityl)-5-(6-biotinamidocaproamidomethyl)- 1-(2-nitrophenyl)-ethanol (compound
3
).
1-
N
-(4,4'-Dimethoxytrityl)-5-(6-biotinamidocaproamidomethyl)-2-nitroacetophenone (
2
) (0.85 g, 1.016 mmol) was dissolved in 7 ml ethanol and sodium borohydride
(0.028 g, 0.74 mmol) was added with stirring. After 1 h the reaction was
quenched with 4 ml acetone and evaporated under reduced pressure to give a
yellow oil, which was redissolved in 10 ml methanol and the solution added to
120 ml water. The precipitate was isolated by centrifugation (7000 r.p.m., 45
min) and dried
in vacuo
over KOH to give compound
3
(0.7 g, 82%). TLC, CHCl
3
:MeOH 9:1 v/v;
R
f
= 0.39.
1
H NMR: 7.83-7.74 (m,2H), 7.32 (t,1H), 7.24-7.17 (m,5H), 7.13-7.00 (m,4H), 6.98 (d,1H), 6.73-6.69 (m,4H), 5.99-5.91 (d,1H), 5.86-5.78 (m,1H) (OH), 5.49-5.45 (m,1H), 4.50-4.24 (m,4H), 3.74
(s,6H), 3.58-3.25 (m,1H), 3.07-3.01 (m,1H), 3.00-2.85 (m,1H), 2.29-2.12 (m,2H), 2.01-1.96 (m,2H), 1.80-1.75 (m,1H), 1.64 (s,3H), 1.53-1.43 (m,12H), 1.38-1.11 (m,1H). Elemental
analysis (%): calculated (C
46
H
55
N
5
O
8
S), C 65.93, H 6.62, N 8.36; found, C 65.97, H 6.52, N 8.10.
[1-N-(4,4
'
-dimethoxytrityl)-5-(6-biotinamidocaproamidomethyl)- 1-(2-nitrophenyl)-ethyl]-2-cyanoethyl-N,N-diisopropylaminophosphoramidite (compound
4
).
1-
N
-(4,4'-Dime t h o x y trityl)- 5-(6-biotinamidocaproamidomethyl)-1-(2-nitrophenyl) ethanol (
3
) (0.186 g, 0.22 mmol) was placed in an oven-dried flask with a magnetic stirring bar, sealed with a septum and dried for at
least 6 h
in vacuo
. Anhydrous acetonitrile (0.003% water) (1 ml) was added through a septum under
argon. Subsequently
N
,
N
-diisopropylethylamine (0.15 ml, 0.88 mmol) was added, followed by 2-cyanoethoxy-
N
,
N
-diisopropylchlorophosphine (0.052 g, 0.22 mmol). After 1 h another 0.5 eq.
phosphine was added. After an additional 2 h at room temperature the reaction
mixture was treated with 0.3 ml ethyl acetate, followed by a saturated saline
solution (10 ml), and extracted with methylene chloride (3 * 10 ml). The organic layer was washed with water, dried over sodium
sulfate and evaporated under reduced pressure and purified on a silica gel
column using a step gradient (0-3%) of triethylamine in acetonitrile. Appropriate fractions were pooled
and evaporated to give compound
4
as a white foam (0.144 g, 62% yield). TLC, MeCN:Et
3
N, 95:5 v/v;
R
f
= 0.48.
1
H NMR (p.p.m.): 7.79-7.32 (m,1H), 7.65-7.61 (m,1H), 7.25-7.19 (m,5H), 7.13-7.05 (m,4H), 6.91-6.85 (m,1H), 6.75-6.73 (m,4H), 5.75-5.66 (br s,1H), 5.54-5.43 (m,1H), 5.22s,
5.12d (1H), 4.38-4.26 (m,3H), 4.23-4.11 (m,2H), 3.88-3.77 (m,1H), 3.73 (s,1H), 3.66-3.54 (m,2H), 3.46-3.37 (m,1H), 3.29-3.21 (m,1H), 3.10-3.02 (m,2H), 2.65-2.60 (m,1H), 2.54-2.44 (m,1H), 2.40-2.32
(m,1H), 2.26-2.20 (dd,1H), 2.10-2.11 (app. t,1H), 2.08-2.01 (m,2H), 1.62-1.58 (m,6H), 1.55-1.45 (m,4H), 1.39-1.35 (t,2H), 1.31-1.27 (t,2H), 1.16-1.07 (m,9H), 0.87-0.83 (dd,3H).
31
P NMR (p.p.m.): 146.7, 147.9. Elemental analysis (%): calculated (C
55
H
72
N
7
O
9
PS), C 63.63, H 6.99, N 9.44; found, C 63.11, H 6.79, N 9.20.
A 0.1 M solution of the PCB-phosphoramidite (
4
) in anhydrous acetonitrile was attached to the extra port of the Applied
Biosystems 392 DNA/RNA synthesizer. The syntheses were carried out on a 0.2 [mu]mol scale using cyanoethyl phosphoramidites. For the last coupling
(introduction of
4
) the coupling time was increased by 120 s, as recommended for conventional
biotin phosphoramidite (
19
). Typical coupling efficiency (as determined by trityl cation conductance) was
between 95 and 97%. Standard detritylation (`trityl-off' option) as well as cleavage and deprotection procedures were used.
Control 5'-phosphorylated sequences were synthesized using chemical
phosphorylation reagent Phosphalinktm (Applied Biosystems) according to the manufacturer's instructions (
20
).
Crude 5'-PCB-oligonucleotide (16 nmol) was added to a suspension of
streptavidin-agarose beads (700 [mu]l, 24 nmol) (Sigma, Milwaukee, WI) and the suspension incubated at
room temperature for 1 h. It was then spin-filtered (5 min, 5000 r.p.m.) using a 0.22 [mu]m Ultrafree MC filter (Millipore, Bedford, MA). Beads on the filter
were washed with 100 [mu]l phosphate buffer, pH 7.2, and spin-filtered (three times). Finally, the beads were resuspended in 700 [mu]l phosphate buffer and irradiated for 5 min. After irradiation the
suspension was spin-filtered, the beads were washed with phosphate buffer (3 * 100 [mu]l) and the combined filtrate volume was adjusted to 1 ml and
analyzed by UV absorption spectroscopy or HPLC.
In order to calculate the time dependence of the photocleavage, HPLC-purified 5'-PCB
-
(dT)
7
(48 nmol) was incubated with 1.5 eq. streptavidin-agarose beads for 1 h. The beads were spin-filtered, washed, resuspended in phosphate buffer, pH 7.2, and
irradiated. Aliquots (200 [mu]l each) were withdrawn after 0, 0.25, 0.5, 1, 2, 4, 6 and 10 min
irradiation, spin-filtered and washed as described above. The filtrate volume was adjusted
to 1 ml and the absorbance at 260 nm measured. A sample of 700 [mu]l streptavidin which had not been incubated with oligonucleotide was spin-filtered and the UV absorption measured, serving as background. A
similar measurement was made on a sample of oligonucleotide not incubated with
streptavidin (16 nmol, 700 [mu]l phosphate buffer, serving as 100% control). The molar extinction
coefficient at 260 nm for the PCB
moiety was determined separately (4700/M/cm) and this value subtracted from the
estimated (assuming a molar extinction coefficient equal to 12 000 for each dT)
molar extinction coefficient of 5'-PCB
-
(dT)
7
(88 700) for photorelease efficiency calculations. In order to determine the
time course of photocleavage in solution (dT)
7
-5'-PCB (1 OD
260
) was dissolved in 1 ml phosphate buffer and irradiated at 300-350 nm. Aliquots (10 [mu]l) were withdrawn after 0, 0.25, 0.5, 1, 2, 4, 6 and 10 min
irradiation and injected onto an HPLC column. The percent conversion was
calculated from the ratio of the area of the particular peak (i.e. 5'-PCB
-
(dT)
7
or 5'-p-(dT)
7
) over the sum of the areas of the component peaks, with molar extinction
coefficients of the components adjusted as described above.
The synthesis of PCB-phosphoramidite (
4
) is depicted in Scheme 1 (see Materials and Methods for more details). The
compound consists of a protected biotin moiety linked through a spacer arm (6-aminocaproic acid) to a photoreactive 1-(2-nitrophenyl)ethyl moiety (
21
), which is derivatized with
N
,
N
'-diisopropyl-2-cyanoethyl-phosphoramidite. The starting material, 5-(6-biotinamidocaproamidomethyl)-2-nitroacetophenone (
1
) was synthesized as described previously (
18
). The 4,4'-dimethoxytrityl (DMTr) group was introduced selectively onto the N1
nitrogen of biotin (
3
,
6
). The intermediate (
2
) was then selectively reduced using sodium borohydride to give compound
3
and, finally, the resulting hydroxyl group was phosphitylated using 1.5 eq. 2-cyanoethoxy-
N
,
N
-diisopropylchlorophosphine. No phosphitylation of biotin nitrogen N2 was
observed under the reaction conditions.
PCB-phosphoramidite (
4
) was designed for direct use in any automated DNA/RNA synthesizer employing
standard phosphoramidite chemistry. As shown in Scheme 2, the selective
reaction of compound
4
with the free 5'-OH group of a full-length oligonucleotide results in the introduction of a
phosphodiester group linked to a photocleavable biotin moiety. In contrast, all
capped failure sequences which lack a free 5'-OH group do not react with the PCB-phosphoramidite. The biotinyl moiety thus allows selective
isolation of only full-length sequences through streptavidin affinity media. Upon irradiation
with near-UV light the phosphodiester bond between the PCB moiety and the phosphate
is cleaved, resulting in the formation of a 5'-monophosphate on the released oligonucleotide. The 1-(2-nitrophenyl)ethyl moiety is converted into a 2-nitrosoacetophenone derivative.
Scheme 2.
The heptamer 5'-PCB
-
(dT)
7
was assembled using PCB-phosphoramidite (
4
) in an automated DNA/RNA synthesizer.
The unmodified sequence 5'-OH-(dT)
7
and a 5'-phosphorylated sequence, 5'-p-(dT)
7
, were prepared using standard procedures (see Materials and Methods). Figure
1
shows the HPLC trace of 5'-PCB
-
(dT)
7
(trace a). Two main peaks are observed in this trace, with retention times of
23.7 and 24.3 min. These two peaks can be attributed to the two
diastereoisomers generated by introduction of the PCB
moiety onto the 5'-end of the oligonucleotide (
22
). Compared with the unmodified oligonucleotide 5'-OH-(dT)
7
(trace d, retention time 14.5 min) the PCB-modified oligonucleotide (trace a) shows an increased retention time,
which is typical for biotinylated oligonucleotides (
5
,
16
). We conclude from these data that the 5'-PCB moiety is retained during cleavage and deprotection of the
oligonucleotide with ammonia [5'-phosphorylated oligonucleotide is not present in the 5'-PCB
-
(dT)
7
sample].
We measured the time dependence of the photoconversion of 5'-PCB
-
(dT)
7
into 5'-p-(dT)
7
in solution. For this purpose a 5'-PCB
-
(dT)
7
solution was subjected to irradiation with 300-350 nm light and the reaction mixture was analyzed by reversed phase HPLC
after different irradiation times (Fig.
2
). It can be seen from the decrease in the intensity of peaks at 23.7 and 24.3
min, assigned to 5'-PCB
-
(dT)
7
, and the increase in the intensity of the single peak at 12.6 min, assigned to
5'-p-(dT)
7
, that the photoreaction is complete in ~4 min. The appearance of additional small peaks with a retention time of ~33 min can be attributed to formation of the biotinyl-2-nitrosoacetophenone derivative and other minor
photoproducts identified previously (
23
,
24
).
In order to evaluate the usefulness of PCB-phosphoramidite for synthesis and affinity purification/phosphorylation of
longer oligonucleotides two 5'-PCB-labeled sequences, a 50mer and a 60mer, were prepared. After
deprotection the crude 5'-PCB-oligonucleotides were separately incubated with streptavidin-agarose beads. The beads were then washed, resuspended
and finally irradiated to obtain the full-length phosphorylated oligonucleotides. Figure
3
shows the results of polyacrylamide gel electrophoresis (PAGE) of the crude
50mer (lane 1) and 60mer (lane 4) oligonucleotides and the affinity-purified and photocleaved oligonucleotides (50mer, lane 2; 60mer, lane 5).
PAGE of the supernatant obtained after isolation of the oligonucleotides with
streptavidin-agarose beads is also shown (50mer, lane 3; 60mer, lane 6). In agreement
with earlier studies, the biotinylated oligonucleotides migrate more slowly
than non-biotinylated oligonucleotides, while 5'-phosphorylated sequences migrate faster than sequences with 5'-OH (
3
). It can be further seen that affinity purification and photocleavage results
in a compact band, indicative of high purity and homogeneity, in contrast to
the crude material, which exhibits a much broader band with additional material
appearing at lower molecular weight. Since this latter material did not bind to
streptavidin, it is likely to correspond to the failure sequences.
In a previous paper (
18
) we described a photocleavable biotin
N
-hydroxysuccinimide (PCB-NHS) derivative which reacts with primary aliphatic amino groups and
can be used for isolation of biomolecules. In the present work we further
expand this approach and describe the synthesis of a phosphoramidite reagent
which incorporates a photocleavable biotin moiety on the 5'-end of an oligonucleotide during solid support DNA synthesis.
The addition of a 5'-PCB moiety onto oligonucleotides provides a
photoremovable
affinity tag for fast and efficient purification and phosphorylation of
synthetic DNA/RNA. The wavelength and the low intensity of the light used for
photocleavage minimizes possible irradiation-induced damage to DNA, which typically occurs at wavelengths below 300 nm
(
25
). The approach described is especially important for the removal of failure
sequences in synthetic DNA, which are formed as a result of imperfections
during the synthesis cycle. Note, however, that this method would not eliminate
deletion sequences, which are also known to occur (
26
,
27
).
Current methods for purification and analysis of synthetic oligonucleotides
include reversed phase high performance liquid chromatography (HPLC) (
28
), ion exchange HPLC (
29
), high performance capillary electrophoresis (HPCE) (
30
) and PAGE (
31
). Alternatively, a DMTr group on the 5'-end of an oligonucleotide can facilitate purification by non-specific adsorption on a reversed-phase silica gel (
32
). However, none of these methods is able to remove failure sequences
quantitatively. In contrast, 5'
-
PCB
provides an efficient and simple method for isolation of full-length product and removal of failure sequences.
The ability to 5'-phosphorylate an oligonucleotide through photocleavage is a second
important advantage of this approach. Many applications of synthetic
oligonucleotides require phosphorylation on the 5'-end, including gene construction, cloning, oligonucleotide ligation
assay (
33
), the ligation chain reaction (
34
) and total cDNA sequencing. Typically, 5'-phosphorylation is achieved by either enzymatic or chemical
methods. The use of enzymes involves several time consuming steps and often
results in non-quantitative phosphorylation. Chemical phosphorylation is possible during
oligonucleotide synthesis using commercially available phosphoramidites such as
Phosphalinktm (
20
), however, the resulting product still requires additional purification.
In addition to PCB
-
mediated affinity purification and phosphorylation, PCB
-
oligonucleotides should also be useful in a number of other applications. For
example, they could be used as primers for PCR, thereby simplifying the
streptavidin-mediated affinity purification of PCR products from a reaction mixture
containing template DNA, polymerases and other components. Importantly, this
procedure would yield unmodified amplified fragments suitable for sequencing or
cloning. Other possible applications include isolation of DNA/RNA
macromolecular complexes (
11
-
15
) and controlled photorelease of oligonucleotides for the triggering of DNA-protein interactions and for therapeutic purposes.
We thank Lincoln Scott for help with
31
P NMR experiment. We also thank Sanjay Sonar and Matthew Coleman for helpful
discussions and Cheryl and Gary Ludlam for critical reading of the manuscript.
This work was supported by a grant from the Army Research Office (ARO) (DAAL03-92-G-0172) to KJR. JO was supported by a J.William Fulbright post-doctoral fellowship.
Department of Physics, Boston University, 590 Commonwealth Avenue, Boston, MA
02215, USA
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