Apparatus for the synthesis of oligonucleotides and process for the preparation thereof
The present invention provides apparatuses and methods for the synthesis of oligonucleotides and related compounds. In particular, the present invention allows to effectively prepare reagents to be fed into an apparatus for the synthesis of such oligomers.
1 . An apparatus for the automated synthesis of oligonucleotides, comprising:
a) a reaction vessel ( 6 ) connected via a liquid conduit ( 9 ) to a waste container ( 7 );
b) a liquid supply unit ( 19 ) for delivering liquid reagents to the reaction vessel ( 6 );
c) a bypass conduit ( 11 ), which allows to direct liquid flow from the liquid supply unit ( 19 ) into the waste container ( 7 ) without passage through the reaction vessel ( 6 ); and
d) a control unit ( 8 ),
wherein the liquid supply unit ( 19 ) comprises:
b-1) at least one mixing device ( 5 ), connected to
b-2) at least two liquid supply lines ( 1 , 2 ), each comprising at least one liquid conduit (l 1 , l 2 ) with n liquid inlets (i 1 - n , i 2 - n ), where n is an integer between 1 and 25, and at least one pump (p 1 , p 2 ),
wherein the control unit ( 8 ) is configured to individually define the composition of a deprotection reagent for each coupling cycle, and
wherein the composition of the deprotection reagent is different between at least two coupling cycles.
2 . The apparatus of claim 1 , wherein at least the inner surfaces of the at least one mixing device ( 5 ) and of one of the at least two liquid supply lines ( 1 , 2 ) are made from an acid-resistant material and/or comprise an acid resistant coating.
3 . The apparatus of claim 2 , wherein at least the inner surfaces of the at least one mixing device ( 5 ) and of one of the at least two liquid supply lines ( 1 , 2 ) are made from an acid-resistant alloy and/or comprise an acid resistant polymeric coating.
4 . The apparatus of claim 1 , wherein the liquid supply unit ( 19 ) further comprises a third liquid supply line ( 3 ) connected to the at least one mixing device ( 5 ), the third liquid supply line ( 3 ) comprising at least one liquid conduit ( 13 ) with n liquid inlets (i 3 - n ), where n is an integer between 1 and 25, and at least one liquid pump (p 3 ).
5 . The apparatus of claim 1 , wherein the at least one mixing device ( 5 ) comprises a static mixer.
6 . The apparatus of claim 1 , further comprising n sensors (s 1 to sn), where n is an integer equal to or larger than 1, which sensor(s) (s 1 to sn) is/are positioned downstream of the at least one mixing device ( 5 ) and determine(s) at least one property of the liquid emerging from the mixing device ( 5 ).
7 . The apparatus of claim 6 , wherein at least one readout provided by at least one of the sensor(s) (s 1 to sn) is used as a feedback signal to regulate the activity of one or more of the pumps comprised in the liquid supply unit ( 19 ).
8 . The apparatus of claim 6 , wherein at least one readout provided by at least one of the sensor(s) (s 1 to sn) is used to control whether the liquid flow is directed into the reaction vessel ( 6 ) or into the waste container ( 7 ) without passage through the reaction vessel ( 6 ).
9 . The apparatus of claim 1 , further comprising at least one flow sensor ( 24 ) integrated into each of the at least two liquid supply lines ( 1 , 2 ) upstream of the at least one mixing device ( 5 ), wherein at least one readout provided by the flow sensors is used as a feedback signal to regulate the activity of one or more of the at least one pump (p 1 , p 2 ).
10 . The apparatus of claim 1 , wherein the liquid supply unit ( 19 ) further comprises at least one heat exchanger ( 13 ).
11 . The apparatus of claim 6 , comprising a conductivity sensor, a temperature sensor, and a UV absorption sensor.
12 . The apparatus of claim 1 , wherein the reaction vessel ( 6 ) is a packed column reactor or a batch reactor.
13 . The apparatus of claim 1 , further comprising a recycling circuit directing flow from the liquid conduit ( 9 ), which connects the reaction vessel to the waste container, back into the reaction vessel ( 6 ) by a pump ( 17 ).
14 . The apparatus of claim 13 , wherein the recycling circuit is comprised in one of the at least two liquid supply lines ( 1 , 2 ), which is connected via one of its inlets (i 1 - n , i 2 - n ) and a multi way valve ( 12 ) to the liquid conduit ( 9 ), and whose pump (p 1 , p 2 ) may drive the recirculation of the fluid from the liquid conduit ( 9 ) via the at least one mixing device ( 5 ) back into the reaction vessel.
15 . The apparatus of claim 1 , wherein the control unit ( 8 ) is configured to control the execution of a synthesis protocol comprising at least two iterations of a coupling cycle, wherein the protocol individually defines the composition of an acidic deprotection reagent for each coupling cycle.
16 . The apparatus of claim 1 , wherein the control unit ( 8 ) directs the action of the at least one pump (p 1 , p 2 ), so as to achieve a specific composition of the deprotection reagent in each of the coupling cycles.
17 . A method of assembling an oligonucleotide chain in automated fashion by repeated cycles of building block coupling, wherein the following steps 1 through 6 are carried out anew in each of the cycles of building block coupling:
1. providing a n-mer oligonucleotide, where n is an integer equal to or larger than 1, bound to a solid support and comprising a first reactive group free to extend the oligonucleotide backbone by reaction with a second reactive group comprised in a building block to be incorporated;
2. providing the building block to be incorporated, which comprises the second reactive group free to react with the first reactive group of the n-mer oligonucleotide, and which further comprises a first reactive group blocked by an acid sensitive temporary protecting group;
3. contacting the n-mer oligonucleotide with the building block to be incorporated under conditions that allow for binding of the first reactive group of n-mer oligonucleotide to the second reactive group of the building block to be incorporated, and generating a protected, extended n-mer oligonucleotide, which is blocked from further extension by the acid sensitive temporary protecting group;
4. preparing an acidic deprotection reagent by mixing at least two liquid compositions, wherein the composition of the acidic deprotection reagent is defined individually for each coupling cycle, and wherein the composition of the acidic deprotection reagent is different between at least two coupling cycles;
5. contacting the protected, extended n-mer oligonucleotide of step 3 with the acidic deprotection reagent of step 4, thereby cleaving the acid sensitive temporary deprotecting group from the extended n-mer oligonucleotide; and
6. removing the deprotection reagent and soluble cleavage product from the extended n-mer oligonucleotide, which may then be used as the n-mer oligonucleotide in step 1 of the following coupling cycle.
18 . The method of claim 17 , wherein the content of acid in the deprotection reagent is selected from the range of 0.1% (w/w) to 50% (w/w) acid individually for each coupling cycle.
19 . The method of claim 17 , wherein the acid sensitive temporary protecting group is a trityl type protecting group.
20 . The method of claim 17 , wherein the first reactive group is a hydroxyl group.
21 . The method of claim 20 , wherein the second reactive group is selected from the group consisting of a phosphoamidite group and a H-phosphonate monoester group.
22 . The method of claim 21 , wherein step 3 comprises contacting the building block and the n-mer oligonucleotide under conditions, which allow for formation of a phosphite triester group, and mixing the resulting compound with an oxidizing reagent or a sulfurizing reagent to convert the phosphite triester group into a phosphate triester group or a thiophosphate triester group, respectively.