Patent Application
20080084008
Scheme 1 shows a top and a bottom view of a 96-chamber synthesis plate.
Scheme 2 is a bottom view of a 96-chamber synthesis plate.
Scheme 3 is an enlarged, cross sectional view of a single chamber.
Scheme 4 is a cross sectional view of a synthesis column.
Scheme 5 is a schematic view of an 8-pin insertor.
Scheme 6 is a schematic view of an 8-pin extractor.
Scheme 7 is a schematic view of a combo 1-pin insertor/1-pin extractor.
Scheme 8 is a schematic view of loading and extracting frits from a 96-chamber synthesis plate with an 8-pin insertor and an 8-pin extractor.
Scheme 9 describes the preparation of an embedded catechol-based universal support.
To produce the frits of the invention, silane-modified CPG is advantageously used in order to control the said frit loading capacity prior to its manufacture. Bifunctional silanes, having a first functional group enabling covalent binding to the glass surface (a Si-halogen or Si-alkoxy group) and a second functional group that imparts the desired chemical modifications to the surface, are used to modify the CPG surface. Silane-modified CPG are controlled porous glass beads, which have been preferentially modified with aminoalkyltrialkoxysilane, [alkylamino]alkyl(trialkoxy)silane or mercaptoalkyl-(trialkoxy)silane and mixtures thereof. Preferentially, alkyl is selected from the group consisting of methyl, ethyl and propyl and wherein alkoxy is selected from the group consisting of methoxy, ethoxy and propoxy. In a preferred embodiment, low loading capacity (5 to 30 μmol/g) aminopropyl-CPG 1 is prepared by reacting CPG (500, 1000 or 2000 A pore diameter, preferably 1000 A, particle size 40/75 or 75/200 microns, preferably 75/200) with aminopropyltriethoxysilane in dichloromethane at room temperature.
A silane-modified CPG or a blend of two different silane-modified CPG is mixed with an aqueous-free polyalkylene in a solid weight ratio of 30 to 50%. Polyalkylenes are selected from the group consisting of ultrahigh molecular weight polyethylene, high density polyethylene, medium density polyethylene, low density polyethylene, polypropylene, and mixtures thereof. Preferentially, aminoalkyl-CPG 1 is mixed in a solid weight ratio of 35 to 45% with high-density polyethylene.
An aluminum plate drilled with 50 to 5000 wells, preferably 1000 to 2000, is filled with the said polyethylene/silane-modified CPG mixture. In one embodiment, the aluminum plate dimensions (X, Y, Z in inch) are respectively (14.0, 6.0, 0.50). Preferably, the said wells have a round cross sectional shape. In one embodiment, cylindrical wells with a diameter/length (in mm) of 3.90/6.0 or 3.90/9.0 or 3.90/12.0 have been drilled. Those wells yield cylindrical frits which sizes are optimal to contain 50 nmol, 200 nmol and 1 μmol of reactive moieties, respectively.
The said filled aluminum plate is heated at approximately 180 to 200° C. under a normal atmosphere for a predetermined time (5 to 20 min). Heating schedule is a function of the mixture composition, the size of the aluminum plate and the number of chambers. At these temperature, around 1 to 5% shrinkage uniformly occurs throughout the structure. For use with this invention, preferably the firing schedule, temperature and powder composition can be modified in such a way as to significantly control shrinkage. Upon cooling the aluminum plate, the frits are removed from the wells and are controlled for adequate mass and diameter.
It is a further object of this invention to provide accessories enabling convenient and reproducible uses of the synthesis frits.
Synthesis plates have been prepared and used as frit holders to carry out the high throughput synthesis of nucleic acids. The said plate is preferably made of Teflon. Preferably, the plate surface is modeled off the industry standard. This way, equipment such as multiple pipetters or robots designed for use with 96-well plates may be easily adjusted for use with the said synthesis plate. The synthesis plate may be of any height (Z), preferably between 1.5 and 2.0 inches. In one preferred embodiment, the plate dimensions (X, Y, Z) in inch are 4.98, 3.35, 1.60, respectively.
Any number of cylindrical open top and bottom ends chambers may be drilled into a synthesis plate. Preferably, the number of chambers is a multiple of 48 (i.e., 96, 384, 1536), especially 96 (see Scheme 1). Preferably, the spacing between chambers, both in the X and Y direction of the plate, is modeled off the industry standard 96-well plate (8×12 mutually perpendicular rows).
Scheme 2 and 3 show a cross-sectional view of a 96-chamber plate and an enlarged cross-sectional view of a single chamber, respectively. A chamber is made of a top cylinder, a middle cylinder and a bottom cone. The sidewalls of the top and middle cylinders may be of any height, depending on the desired volume of reagents per chamber. Preferably, the height of each cylinder is between 0.40 and 0.80 inch. The top and middle cylinder cross diameters are wider than the cross diameter of a cylindrical frit. Preferably, the top cylinder is wider by 0.10 to 0.15 inch and the middle cylinder is wider by 0.01 to 0.05 inch. The bottom cone or frit holder has a cross diameter smaller than the cross diameter of a cylindrical frit. Preferably, the cone has a top cross diameter 0.001 to 0.003 inch smaller than the cross diameter of a cylindrical frit, preferably 0.002 to 0.003 and a bottom cross diameter 0.003 to 0.008 inch smaller than the cross diameter of a cylindrical frit, preferably 0.004 to 0.005 inch. A smaller diameter cone allows the frit to be held tightly which has a three-fold effect: (i) it prevents the frits from being extracted from the synthesis plate during the automated synthesis of nucleic acids when a gas (nitrogen, argon) surpressure is applied to drive the chemical reagents into the frits or to drain the reagents from the frits. (ii) It prevents dripping and draining of the reagents of the reagents along the chamber sidewalls. Thus, it ensures that the reagents are forced into the frits or are fully drained when a gas surpressure is applied. (iii) It maintains a homogenous backpressure from each frit-filled chamber regardless of its synthesis status (i.e. synthesis completed or not in the said chamber).
For low throughput nucleic acid synthesis single synthesis columns prepared by injection molding of polypropylene are used. The said columns are opened cone with open top and bottom ends (scheme 4). They are used to hold a single frit in a low throughput synthesis of nucleic acids. Notably, the said column has a holding cylinder 0.002 to 0.010 inch smaller than the cross diameter of a cylindrical frit, preferably 0.002 to 0.004 inch.
A one- to 96-steel pin insertor is used to insert from one to 96 frits into the synthesis chambers from their top ends and secured them reproducibly into the bottom cone of the chambers. Preferably, an 8-pin insertor is used to insert simultaneously eight frits into eight synthesis chambers. A detailed schematic view of an 8-pin insertor is shown scheme 5. The steel pin insertor length is slightly longer than the combined length of the top and middle cylinders of a synthesis chamber.
Upon completion of a synthesis, a one- to 96-steel pin extractor is used to extract one to 96 frits through the bottom ends of the synthesis chambers. Preferably, an 8-pin extractor is used to extract simultaneously eight frits from eight chambers into eight collection vials. A detailed schematic view of an 8-pin extractor is shown scheme 6. The steel pin extractor length is slightly longer than the synthesis chamber length.
A schematic view a combo 1-pin insertor/1-pin extractor is shown scheme 7. A 1-pin insertor/1-pin extractor is used to insert or extract a single frit in/from a synthesis column or a synthesis chamber.
Pushing the frits through the narrower bottom end of the synthesis chambers or the synthesis columns does not damage the frits or the synthesis chambers or the synthesis columns. Therefore, the synthesis plates and synthesis columns are advantageously reused, contrarily to currently available consumable DNA synthesis columns. Another advantage is that the frits once extracted into collection vials or a 96-well collection plate are easily manipulated for post synthesis treatments.
C. Methods Describing the Use of the Synthesis Frits
To illustrate the use of the frits in the synthesis of nucleic acids, frits functionalized with a catechol-based universal linker have been prepared from aminopropylCPG frits 2. Catechol-based universal linkers have been described in U.S. Pat. No. 6,590,092. They are used irrespective to the first nucleotide of the said nucleic acids to be synthesized onto the solid support and irrespective of the type of monomer reagent used during the synthesis.
In a preferred embodiment, aminopropylCPG-frits 2 are reacted with excess carbonate 3 (Scheme 9). Excess carbonate is used in order to ensure a complete reaction of the amino moieties. Disappearance of the amino groups is monitored by ninhydrin test. The resulting carbamate bound catechol (regioisomeric mixture, one isomer shown) and the remaining CPG silanol groups are capped simultaneously with excess trimethylsilylimidazole yielding frits 4.
Frits 4 are employed to synthesize nucleic acids on automated synthesizers using synthesis columns or preferably using a 96-chamber synthesis plate. A schematic loading of a 96-chamber synthesis plate with an 8-pin insertor is described in scheme 8. The synthetic cycle begins with a catechol deprotection step carried out with 3% trichloroacetic acid in dichloromethane, i.e. the reagent commonly used in the 5′-detritylation step. The first nucleotide is then attached to the catechol bound support using conventional phosphoramidite chemistry under the same conditions and with the same monomer reagent as the condensation of the second nucleotide with the desired first nucleotide bonded to the support. The said first nucleotide corresponds to the first nucleotide in the sequence of the said nucleic acid. Chain elongation occurs by sequential reaction of 5′-protected nucleoside phosphoramidites with the 5′-hydroxyl-end of the oligonucleotide bound polymer. Oxidation (I2/pyridine/acetonitrile/H2O), capping (Ac2O) and detritylation (3% trichloroacetic acid in dichloromethane) steps are carried out as usual.
After the reagents are delivered into the synthesis chambers or the synthesis columns, a brief application of pressure is required to drive the reagents into the frits. Indeed, at ambient pressure, a wetting of a frit is sufficient to prevent entry of chemical reagents. This allows an efficient pre-mixing of the chemical reagents such as activator and 3′-phosphoramidite (or the synthesis columns) prior to their entry into the frit. The reagents stay inside the frit as long as needed and are flushed when a full draining surpressure is applied. To deliver the reagents into the frits or drain the reagents, an optimal pressure of 2.5 to 4.0 PSI at the chamber pressure is recommended. Delivery of the reagents into the frits requires a short pulse of pressure (one second for acetonitrile and dichloromethane solutions or two seconds for tetrahydrofuran solutions) while draining requires applying a surpressure for a longer time, at least 8 s and preferably 15 s.
Upon completion of a nucleic acid synthesis, a frit extractor is used to push down the oligonucleotide-bound frits without damaging them into vials or into a collecting 96-well plate (see scheme 8). The post-synthesis cleavage of the oligonucleotide-bound CPG and deprotection steps are carried out simultaneously by heating the frits with 33% ammonium hydroxide (6 h at 80° C.), 40% aq. methylamine or aq. ammonia-methylamine (1:1, v/v) (4 h at 80° C.) to yield 3′-hydroxyoligonucleotides free of any residual terminal phosphate group. After discarding the frits, the basic solutions containing the oligonucleotides are evaporated.
The following examples illustrate the invention without limiting it:
A mixture of high-density polyethylene (66 g) and aminopropylCPG 1 (44 g, 10 μmol/g, 1000-angstrom pore size, and particle size 75/200 microns) is prepared. The mixture is poured onto an aluminum plate drilled with 1100 cylindrical wells. The well dimensions are diameter/length 3.90 mm/9.0 mm, respectively. The plate is heated at 190° C. for 15 min and cooled before releasing the frits 2. Excess carbonate 3 is added to a thousand frits suspended in dichloromethane under inert atmosphere at room temperature. After gently stirring for 48 hours, the frits are filtrated and washed successively with acetone and dichloromethane. The frits are resuspended in dichloromethane and trimethylsilylimidazole (0.80 mL) is added. After stirring for 2 hours, frits 4 are filtrated, washed with methanol and dichloromethane, and dried under vacuum.
Seventy-two frits 4 (200 nmol loading capacity) are inserted into 72 chambers of a 96-chamber synthesis plate of the invention using an 8-pin insertor. All 24 unused chambers of the synthesis plate are sealed with duct tape. Oligonucleotides having three different lengths (25-mers, 50-mers, and 75-mers) are synthesized on a high throughput synthesizer (BLP-192 from Biolytic Lab Performance, Ca) using conventional phosphoramidite chemistry that is in current use and will thus be known to those skilled in the art.
The following protocol is developed for a synthesizer using positive pressure for reagent delivery and draining. The gas pressure to drive the reagents into the frits and to drain the reagents from the frits is manually set at 2.5 PSI.
The 200 nmol frit has a dead volume around 60 μl. To get the best reaction yields with this frit, the total volume of reagents delivered for each step of the synthesis must be around 70-80 μl. To ensure a complete DMT removal, the delivery of 2×150 μl of 3% TCA in dichloromethane is recommended. Instead of using Tetrazol as activator, dicyanoimidazole (DCI) or ethyl thiotetrazol (ETT) is recommended for an optimal coupling efficiency. Upon completing the syntheses, the resulting oligonucleotide bound frits are pushed into vials using a frit extractor. Ammonium hydroxide is added and the vials are sealed and heated at 65° C. overnight. All the oligonucleotides obtained were of good to high purity as shown by HPLC of their crude and of correct sequences as inferred by mass spectrometry. The quality and consistency of all three-length nucleic acids were excellent.
Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.
4,153,661May 1979Ree et al264/120.4,373,519February 1983Errede et al602/43.4,415,732,November 1983Caruthers et al536/264,458,066;July 1984Caruthers et al536/25.4,565,663January 1986Errede et al264/120.4,725,677February 1988Kosterb et al536/25.4,971,736November 1990Hagen et al264/4615,904,848May 1999Wong et al.210/500.36.6,416,716July 2002Shukla et al422/101.6,590,092July, 2003Ngo536/25.
