Capping and de-capping during oligonucleotide synthesis

Details Capping and de-capping during oligonucleotide synthesis Capping and de-capping during oligonucleotide synthesis

1999-10-18

2002-09-17

Richter, Johann (Department: 1623)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S025330, C536S025340

Reexamination Certificate

active

06451998

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to arrays, particularly polynucleotide arrays such as DNA arrays, which are useful in diagnostic, screening, gene expression analysis, and other applications.
BACKGROUND OF THE INVENTION
Polynucleotide arrays (such as DNA or RNA arrays), are known and are used, for example, as diagnostic or screening tools. Such arrays include regions of usually different sequence polynucleotides arranged in a predetermined configuration on a substrate. These regions (sometimes referenced as “features”) are positioned at respective locations (“addresses”) on the substrate. The arrays, when exposed to a sample, will exhibit an observed binding pattern. This binding pattern can be detected upon interrogating the array. For example all polynucleotide targets (for example, DNA) in the sample can be labeled with a suitable label (such as a fluorescent compound), and the fluorescence pattern on the array accurately observed following exposure to the sample. Assuming that the different sequence polynucleotides were correctly deposited in accordance with the predetermined configuration, then the observed binding pattern will be indicative of the presence and/or concentration of one or more polynucleotide components of the sample.
Biopolymer arrays can be fabricated by depositing previously obtained biopolymers onto a substrate, or by in situ synthesis methods. The in situ fabrication methods include those described in WO 98/41531 and the references cited therein. The in situ method for fabricating a polynucleotide array typically follows, at each of the multiple different addresses at which features are to be formed, the same conventional iterative sequence used in forming polynucleotides on a support by means of known chemistry. Typically these methods use a nucleoside reagent of the formula:
in which:
A represents H or an optionally protected hydroxyl group;
B is a purine or pyrimidine base whose exocyclic amine functional group is optionally protected;
Q is a conventional protective group for the 5′—OH functional group;
x=0 or 1 provided:
a) when x=1:
R
13
represents H and R
14
represents a negatively charged oxygen atom; or
R
13
is an oxygen atom and R
14
represents either an oxygen atom or an oxygen atom carrying a protecting group; and
b) when x=0, R
13
is an oxygen atom carrying a protecting group and R
14
is either a hydrogen or a di-substituted amine group.
When x is equal to 1, R
13
is an oxygen atom and R
14
is an oxygen atom, the method is in this case the so-called phosphodiester method; when R
14
is an oxygen atom carrying a protecting group, the method is in this case the so-called phosphotriester method.
When x is equal to 1, R
13
is a hydrogen atom and R
14
is a negatively charged oxygen atom, the method is known as the H-phosphonate method.
When x is equal to 0, R
13
is an oxygen atom carrying a protecting group and R
14
is either a halogen, the method is known as the phosphite method and, when R
14
is a leaving group of the disubstituted amine type, the method is known as the phosphoramidite method.
The conventional sequence used to prepare an oligonucleotide using reagents of the type of formula (I), basically follows the following steps: (a) coupling a selected nucleoside through a phosphite linkage to a functionalized support in the first iteration, or a nucleoside bound to the substrate (i.e. the nucleoside-modified substrate) in subsequent iterations; (b) optionally, but preferably, blocking unreacted hydroxyl groups on the substrate bound nucleoside; (c) oxidizing the phosphite linkage of step (a) to form a phosphate linkage; and (d) removing the protecting group (“deprotection”) from the now substrate bound nucleoside coupled in step (a), to generate a reactive site for the next cycle of these steps. The functionalized support (in the first cycle) or deprotected coupled nucleoside (in subsequent cycles) provides a substrate bound moiety with a linking group for forming the phosphite linkage with a next nucleoside to be coupled in step (a). Final deprotection of nucleoside bases can be accomplished using alkaline conditions such as ammonium hydroxide, in a known manner.
The foregoing methods of preparing polynucleotides are described in detail, for example, in Caruthers,
Science
230: 281-285, 1985; Itakura et al.,
Ann. Rev. Biochem
. 53: 323-356; Hunkapillar et al.,
Nature
310: 105-110, 1984; and in “Synthesis of Oligonucleotide Derivatives in Design and Targeted Reaction of Oligonucleotide Derivatives, CRC Press, Boca Raton, Fla., pages 100 et seq., U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 5,153,319, U.S. Pat. No. 5,869,643, EP 0294196, and elsewhere. The phosphoramidite and phosphite triester approaches are most broadly used, but other approaches include the phosphodiester approach, the phosphotriester approach and the H-phosphonate approach.
In the case of array fabrication, different monomers may be deposited at different addresses on the substrate during any one iteration so that the different features of the completed array will have different desired biopolymer sequences. One or more intermediate further steps may be required in each iteration, such as the conventional oxidation and washing steps.
While each iteration of the foregoing sequence can have a very high yield (over 90%), there is still a small portion of the substrate bound moiety with unreacted linking groups (referenced together herein as “failed sequences”). It is known to cap such failed sequences to avoid the growth of undesired polynucleotide sequences from them. Capping compounds are described in the above mentioned references. A conventional capping compound is acetic anhydride which forms an acetate in conjunction with the hydroxy group of the substrate bound moiety. However, the yield of the capping reaction using acetic anhydride is relatively low. U.S. Pat. No. 4,816,571 suggests using a phosphite monoester capping reagent to form, along with the free hydroxy of the failed sequence, a phosphite triester blocking group. However, the present invention recognizes that in the fabrication of addressable arrays, use of such a capping reagent can leave some portions of the surface not carrying the desired polynucleotide sequences, with a different terminal group (a phosphite triester) than other portions since removal of the phosphite (de-capping) is relatively inefficient. This is particularly the case where an array is formed by a method which leaves spaces between the individual features (“interfeature spaces”), such as deposition of droplets of reagents at the desired feature locations, and when capping is performed by exposing an entire functionalized substrate (such as by flooding) with the capping reagent. In such cases, some portions of the functionalized surface may be capped but not others. Due to such differences in interfeature surface composition (specifically, the functional groups left at the end the failed sequences or functionalizing group), background absorption of polynucleotides in a sample being tested onto interfeature areas may vary across the substrate, making identification of a features to which polynucleotides have bound, more difficult. This may be particularly the case where automated systems are used to detect such features, based on patterns observed on the array following exposure to a sample.
It is also known in the context of RNA hydrolysis generally, and in the context of preparing a “universal” solid support upon which oligonucleotides can be synthesized, that a &bgr;-phosphotriester group (in relation to a an ester group) of a molecule used to link the growing oligonucleotide to a support, can be hydrolyzed so as to cleave the linker from the support and the phosphate from the linker to provide a 3′ hydroxy on the growing oligonucleotide. Such a scheme is disclosed in U.S. Pat. No. 5,681,945 and is illustrated in FIG.
1
. Similarly, deBear et al. in
Nucleosides
&
Nucleotides
, 6(5), 821-830 (1987) also discloses preparation of a universal soli

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