Oxidizing reagent for use in oligonucleotide synthesis

Oxidizing compositions particularly for use in automated oligonucleotide synthesis containing a mixture of KI and I.sub.2 in solution, in equilibrium with KI.sub.3. One preferred composition contains 1.75% KI.sub.3 (providing 0.69% KI and 1.06% I.sub.2) in tetrahydrofuran/pyridine/water (93/5/2, v/v). These formulations enable synthesis of oligonucleotides of significantly higher quality than the currently employed formulation comprising 3% I.sub.2 in tetrahydrofuran/pyridine/water (74/21/2, v/v).

BACKGROUND OF THE INVENTION 
This invention relates generally to the field of chemistry. More 
particularly, the present invention relates to compositions and methods 
for use in automated synthesis of oligonucleotides. 
DNA synthesis primarily involves sequential assembling of nucleotides on an 
insoluble solid support. Phosphoramidite chemistry is the most widely used 
coupling chemistry and the oxidation reaction is one of the important 
chemical steps in every synthesis cycle. 
##STR1## 
At present, 3% iodine in tetrahydrofuran/pyridine/water (74121/2, w/w) is 
employed as the oxidizing agent in some automated oligonucleotide 
synthesizers, such as the Beckman.RTM. Oligo 1000 and Oligo 1000M 
synthesizers. However, it has been determined that this formulation is 
detrimental to the stability of oligonucleotides, especially of the 
trivalent internucleotide phosphorous intermediate formed transiently 
during oligonucleotide synthesis. 
Optimization of the iodine formulation led to a 0.3% iodine in 
tetrahydrofuran/pyridine/water (93/5/2, v/v) as an efficient reagent; this 
reagent also provided an improved quality oligonucleotide product. 
However, this formulation was found not to be compatible with some 
automated oligonucleotide synthesizers, such as the Oligo 1000 synthesizer 
(Beckman Instruments, Fullerton, Calif.). In particular, the Oligo 1000 
synthesizer utilizes the absorbance of iodine to calibrate the instrument; 
0.3% iodine did not provide the absorbance which was required. 
Introducing acridine (0.03%) orange dye as an inert additive into the 
formulation maintained a higher quality of oligonucleotides without any 
negative effects. As acridine orange is known to be a DNA intercalator, 
however, this approach is not entirely satisfactory. 
There thus remains a need for an oxidizing solution which is safe and 
efficient for use in automated oligonucleotide synthesizers. 
It is an object of the present invention to provide compositions and 
methods which do not suffer from the drawbacks of the heretofore-known 
compositions. 
SUMMARY OF THE INVENTION 
In accordance with the present invention, there are provided oxidizing 
compositions for use in automated oligonucleotide synthesis comprising a 
mixture of KI and I.sub.2 in solution. One preferred composition comprises 
1.75% KI.sub.3 (providing 0.69%KI and 1.06 % I.sub.2) in 
tetrahydrofuran/pyridine/water (93/5/2, v/v). These formulations enable 
synthesis of oligonucleotides of significantly higher quality than the 
currently employed formulation comprising 3% I.sub.2 in 
tetrahydrofuran/pyridine/water (74/21/2, v/v).

DETAILED DESCRIPTION OF THE INVENTION 
The formulations of the present invention provide significant advantages 
relative to prior art formulations. The oligonucleotides synthesized using 
the inventive oxidizing agents are substantially higher in quality than 
products obtained using prior art formulations; thus, there is a higher 
percentage of the desired full length sequences and a lower percentage of 
shorter failure sequences when using a reagent in accordance with the 
present invention. Further, these formulations are totally compatible with 
automated oligonucleotide synthesizers, such as the Beckman.RTM. Oligo 
1000. In addition, the formulations are stable during storage and in use 
on the instrument. 
KI.sub.3 is in equilibrium with KI+I.sub.3. At a given time only about 0.3% 
of I.sub.2 is present in the active form, whereas the rest of the I.sub.2 
is present in the KI.sub.3 form (which is non-detrimental to DNA). 
Moreover, the absorbance of KI.sub.3 makes it compatible with the 
diagnostic features of automated oligonucleotide synthesizers, such as the 
Oligo 1000. Suitably, KI.sub.3 may be generated in situ as hereinafter 
described. In any event, for purposes of the present invention KI.sub.3 is 
the equivalent of a corresponding mixture of KI and I.sub.2. 
In preferred formulations of the invention, the solution comprises the 
equivalent of about 1% to about 3% KI.sub.3, more preferably about 1.3% to 
about 2% KI.sub.3. Similarly, the ratio of the components in the 
tetrahydrofuran/pyridine/water mixture may be varied significantly: the 
percentage of tetrahydrofuran may be varied within the range of about 50% 
to about 98%, the pyridine from 1% to 40%, and the water from 0.5% to 30%. 
The current preferred formulation comprises 1.75% KI.sub.3 in 
tetrahydrofuran/pyridine/water (93/5/2, v/v); this corresponds to a 
solution comprising 0.69% KI and 1.06% I.sub.2. 
The preferred formulation showed consistently superior performance relative 
to the heretofore known formulation generally employed with automated 
oligonucleotide synthesizers such as the Beckman Oligo 1000 and Oligo 
1000M. FIGS. 1A (prior art) and 1B (invention) demonstrate this superior 
performance by providing a comparison of 101 mers as analyzed by gel 
filled capillary electrophoresis; FIGS. 2A (prior art) and 2B (invention) 
similarly provide a comparison of 35 mers as analyzed by reverse phase 
HPLC. An accelerated stability study showed the expected stability. 
The invention may be better understood with reference to the accompanying 
examples, which are intended for purposes of illustration only and should 
not be construed as in any sense limiting the scope of the invention as 
defined in the claims appended hereto. 
Example 1 
For preparation of an exemplary oxidizer formulation in accordance with the 
present invention, commercially-available agents as purchased and 
double-distilled water were employed. 6.9 g of potassium iodide was 
dissolved in 20 ml of water. 10.6 g of iodine was dissolved in 930 ml of 
tetrahydrofuran and 50 ml of pyridine. Both solutions were mixed and the 
mixture shaken for 1-2 minutes. This produced a solution of approximately 
1.75% KI.sub.3 (In equilibrium with KI +I.sub.2) in THF/pyridine/water 
(93/5/2, v/v). 
Example 2 
Capillary Electrophoresis (CE) was run on the Beckman.RTM. P/ACE 2000 on 
101 mers synthesized using an oxidizing solution according to the prior 
art and one in accordance with the present invention. The 101 mers had the 
following sequence: .sup.5' AAC-GTC-GGT- 
AAC-GTA-CAC-GGT-AGC-TAC-GGA-CAC-CGT-GGC-AAT-ACG-ACA-GGT-AAC- 
CTG-TGG-AAC-GTA-CAC-GGA-AGA-GAC-TAG-GGA-TGG-GAG-TAC-GGA-TGG- GT.sup.3 ' 
(Seq. ID No. 1). The capillary gel column (Beckman, V100P Urea Gel Column) 
was loaded and cut to 37 cm long. Tris-Borate, 7 M Urea buffer (Beckman, 
Gel Buffer Kit) was used according to directions. The absorbances of 
oligonucleotides were in the range of 0.05 to 2 OD 260 nm/mL, depending 
upon the quality and length of oligonucleotide. Injection was at 10 kv for 
3 sec, while separation was at 11 kv for 30-60 min, depending upon the 
length. The results for oligonucleotides prepared using the prior art and 
inventive oxidizing agents are shown in FIGS. 1A and lB, respectively. 
Example 3 
Reverse Phase HPLC separates the desired oligonucleotide which is expected 
to carry a lipophilic dimethoxytrityl (DMT) group from the failure 
sequences which are not expected to carry the DMT group. 35 mers were 
synthesized using the prior art and inventive formulations with the 
5'-terminal DMT group left on. The 35 mers had the following sequence: 
.sup.5 'GAT-GCC-AGT-TCG-GTC-ATA-CAC-GTA-CTA-CGA-CT.sup.3 '. The 
oligonucleotides were cleaved and deprotected with ammonia. Both of these 
oligonucleotides were analyzed by reverse phase HPLC following the 
conditions described below: 
______________________________________ 
HPLC column: 
C.sub.18 microsorb (Rainin Cat. #86-200-C5) 5.mu. particles, 
4.6 .times. 25 mm 
Bottle A: 0.1 M Ammonium acetate, pH 6.9 
Bottle B: Acetonitrile 
Program: Flow rate, 1 ml/min 
0-20 min gradient to 15% B 
20-25 min gradient to 25% B 
25-27 min gradient to 50% B 
27-30 min at 50% B 
30-35 min at 0% B 
______________________________________ 
Results of HPLC analysis are shown in FIGS. 2A (prior art) and 2B 
(invention). 
From the foregoing description, one skilled in the art can readily 
ascertain the essential characteristics of the invention and, without 
departing from the spirit and scope thereof, can adapt the invention to 
various usages and conditions. Changes in form and substitution of 
equivalents are contemplated as circumstances may suggest or render 
expedient, and although specific terms have been employed herein, they are 
intended in a descriptive sense and not for purposes of limitation. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 1 
(2) INFORMATION FOR SEQ ID NO: 1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 101 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(iii) HYPOTHETICAL: no 
(iv) ANTI-SENSE: no 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
AACGTCGGTAACGTACACGGTAGCTACGGACACCGT36 
GGCAATACGACAGGTAACCTGTGGAACGTACACGGA72 
AGAGACTAGGGATGGGAGTACGGATGGGT101 
__________________________________________________________________________