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Locked Nucleic Acid
 
LNA24/05.2005
 
By M. Meldgaard, C. Lomholt and H. M. Pfundheller
 
LNA Oligonucleotide Synthesis
 
 
Introduction

LNA oligonucleotides are synthesized by the phosphoramidite approach and can thus be synthesized with standard oligonucleotides synthesizers. However, it is recommended to use slightly altered coupling protocols in order to get the best coupling efficiency with the LNA phorphoramidites. In general, a longer coupling time and oxidation time compared to the coupling and oxidation times needed for standard DNA phosphoramidites is recommended.

Exiqon has developed some very efficient protocols for using LNA phosphoramidites on ABI DNA synthesizers – the Expedite™ and the ABI3900 synthesizers that are described in this flyer along with guidelines for synthesizing 3’-end modified LNA-oligonucleotides.
 
 

Protocols for Expedite™ synthesizer

The LNA-A, G and T amidites are used as 0.07M solutions in anhydrous acetonitrile.

For LNA-mC the amidite is first dissolved in anhydrous tetrahydrofurane. When a clear solution has been obtained anhydrous acetonitrile is added to a final THF/ACN1 ratio of 25:75 and an amidite concentration of 0.07M. It is highly recommended to check the THF for peroxides as these oxidize the phosphoramidite and hence irreversibly inactivate it.

It is recommended to increase the coupling time to 250 seconds for all LNA phosphoramidites.

It is recommended to use 0.5M dicyanoimidazole (DCI) as activator2.

LNA requires a longer oxidation time compared to DNA. 45 Seconds has been found to be optimal when synthesizing standard oligonucleotides.



Synthesis protocols are depicted on the following pages using an Expedite™ DNA synthesizer equipped with and without a MOSS unit. The protocol is given for a 0.2μmol synthesis scale with the LNA-A amidite either at position 5 or at position A.

The user should be aware that these protocols have been developed on one instrument using one type of support and holder. Changing instrument, support or holder might cause a change in the length of the reagents flow and therefore the addition or deletion of one or more pulses might be necessary in order to get the reagents to the support at the right time e.g. the step “slow pulse to couple” should be used when the amidite is at the column and neither when the amidite is before or after the support.

    
Molecular weight
g/mole
Product Nr.
CAS. Nr.
Dissolve in
To obtain a 0.07M solution
100mg           250mg
LNA-ABz
885.9
A-0063-
[206055-79-0]
Anhydrous Acetonitrile
1.6 mL
4.1 mL
LNA-mCBz
875.9
mC-0066-
[206055-82-5]
THF/Acetonitrile
25/75 (v/v)
1.6 mL
4.1 mL
LNA-GDMF
852.9
G-0082-
[709641-79-2]
Anhydrous Acetonitrile
1.7 mL
4.2 mL
LNA-T
772.8
T-0064-
[206055-75-6]
Anhydrous Acetonitrile
1.8 mL
4.6 mL
  
Table 1. Dissolution of LNA amidites. The 0.07M concentration of LNA amidites is recommended for the use on Expedite™ DNA synthesizers.



1 We recommend the use of anhydrous tetrahydrofurane (Aldrich 401757), as an alternative anhydrous dichloromethane (Aldrich 270997) can be used as a substitute for THF.
2 A 0.5M DCI solution is not commercial available. A 0.5M DCI solution can be made by dissolving 5.9g of DCI (Aldrich 67864) in 100mL of anhydrous acetonitrile.

Expedite™ with MOSS unit
Synthesizing LNA oligonucleotides on an Expedite™ DNA synthesizer with a MOSS unit requires altered protocols compared to the ones used when used without a MOSS unit. Given below are		examples of synthesis of LNA oligonucleotides on a 0.2μmol scale with LNA either on position 5 or position A.

Synthesis Cycle for LNA on an Expedite™ Multiple Oligo Synthesis System (MOSS)
The synthesis cycle has been written for position 5 on the Expedite™. If any other positions are used, third line in the coupling step has to be changed.

MOSS LNA-cycle (0.2μmole scale)

/* --------------------------------------------------------------------------- */
/* Function                   Mode     Amount     Time(sec)      Description */
/*                                             /Arg1         /Arg2                           */
/* ----------------------------------------------------------------------------*/
$Deblocking
12 /*Wsh A                */ PULSE      15              0          "Pre cycle wash"
144/*Index Fract. Coll.*/ NA             1              0          "Event out ON"
0 /*Default                 */ WAIT        0              1.5        "Wait"
16 /*Dblk                   */ PULSE      20             0           "Dblk to column"
141/*Trityl Mon. On/Off*/ NA            1              1          "START data collection"
16 /*Dblk                   */ PULSE      20             0           "Deblock"
16 /*Dblk                   */ PULSE       5            10           "Deblock"
16 /*Dblk                   */ PULSE      10             0           "Deblock"
16 /*Dblk                   */ PULSE       5            10           "Deblock"
16 /*Dblk                   */ PULSE      10             0           "Deblock"
16 /*Dblk                   */ PULSE       5            10           "Deblock"
16 /*Dblk                   */ PULSE      10             0           "Deblock"
38 /*Diverted Wsh A    */ PULSE      20           20           "Deblock"
38 /*Diverted Wsh A    */ PULSE      40             0           "Flush system with Wsh A"
141/*Trityl Mon. On/Off*/ NA            0             1           "STOP data collection"
144/*Index Fract. Coll. */ NA            2             0           "Event out OFF"
$Coupling
1 /*Wsh                     */ PULSE      10             0           "Flush system with Wsh"
2 /*Act                       */ PULSE        5             0           "Flush system with Act"
22 /*5 + Act                */ PULSE       5             0           "Monomer + Act to column"
2 /*Act                       */ PULSE        5             0           "Chase with Act"
1 /*Wsh                     */ PULSE      12             0           "Chase with Wsh"
1 /*Wsh                     */ PULSE      10          250           "Slow pulse to couple"
1 /*Wsh                     */ PULSE        4             0           "Flush with Wsh"
$Capping
13 /*Caps                  */ PULSE       12             0           "Caps to column"
12 /*Wsh A                */ PULSE       10             0           "Chase with Wsh A"
12 /*Wsh A                */ PULSE       10           45           "Slow pulse to cap"
$Oxidizing
15 /*Ox                     */ PULSE       15             0           "Ox to column"
12 /*Wsh A                */ PULSE       12             0           "Chase with Wsh A"
12 /*Wsh A                */ PULSE       10           45           "Slow pulse to Ox"
$Capping
13 /*Caps                  */ PULSE       10             0           "Caps to column"
12 /*Wsh A                */ PULSE       10             0           "Chase with Wsh A"
12 /*Wsh A                */ PULSE       10           10           "Slow pulse to Cap"
12 /*Wsh A                */ PULSE       20             0           "End of cycle wash"

Bottle/position codes:
18 /*A + Act */
19 /*C + Act */
20 /*G + Act */
21 /*T + Act */
22 /*5 + Act */
23 /*6 + Act */
24 /*7 + Act */
25 /*8 + Act */
26 /*9 + Act */


Synthesis Cycle for DNA on an Expedite™ Multiple Oligo Synthesis System (MOSS)
The synthesis cycle has been written for position A on the Expedite™. If any other positions are used, third line in the coupling step has to be changed.

MOSS DNA-cycle (0.2μmole scale)

/* ---------------------------------------------------------------------------- */
/* Function                   Mode     Amount     Time(sec)       Description */
/*                                              /Arg1         /Arg2                           */
/* ---------------------------------------------------------------------------- */
$Deblocking
12 /*Wsh A                */ PULSE     15               0             "Pre cycle wash"
144/*Index Fract. Coll.*/ NA            1               0             "Event out ON"
0 /*Default                 */ WAIT       0               1.5           "Wait"
16 /*Dblk                   */ PULSE     20              0              "Dblk to column"
141/*Trityl Mon. On/Off*/ NA           1              1              "START data collection"
16 /*Dblk                   */ PULSE     20              0              "Deblock"
16 /*Dblk                   */ PULSE       5            10              "Deblock"
16 /*Dblk                   */ PULSE     10              0              "Deblock"
16 /*Dblk                   */ PULSE       5            10              "Deblock"
16 /*Dblk                   */ PULSE     10              0              "Deblock"
16 /*Dblk                   */ PULSE       5            10              "Deblock"
16 /*Dblk                   */ PULSE     10              0              "Deblock"
38 /*Diverted Wsh A    */ PULSE     20            20              "Deblock"
38 /*Diverted Wsh A    */ PULSE     40              0              "Flush system with Wsh A"
141/*Trityl Mon. On/Off*/ NA           0              1              "STOP data collection"
144 /*Index Fract. Coll.*/ NA           2              0              "Event out OFF"
$Coupling
1 /*Wsh                     */ PULSE     10              0              "Flush system with Wsh"
2 /*Act                       */ PULSE       5              0              "Flush system with Act"
18 /*A + Act               */ PULSE       5              0              "Monomer + Act to column"
2 /*Act                       */ PULSE       5              0              "Chase with Act"
1 /*Wsh                     */ PULSE      12             0              "Chase with Wsh"
1 /*Wsh                     */ PULSE      10          120             "Slow pulse to couple"
1 /*Wsh                     */ PULSE       4              0             "Flush with Wsh"
$Capping
13 /*Caps                  */ PULSE      12             0              "Caps to column"
12 /*Wsh A                */ PULSE      10              0             "Chase with Wsh A"
12 /*Wsh A                */ PULSE      10            45             "Slow pulse to cap"
$Oxidizing
15 /*Ox                     */ PULSE      15             0              "Ox to column"
12 /*Wsh A                */ PULSE      12             0              "Chase with Wsh A"
12 /*Wsh A                */ PULSE      10            30             "Slow pulse to Ox"
$Capping
13 /*Caps                  */ PULSE      10             0              "Caps to column"
12 /*Wsh A                */ PULSE      10             0              "Chase with Wsh A"
12 /*Wsh A                */ PULSE      10            10             "Slow pulse to Cap"
12 /*Wsh A                */ PULSE      20             0              "End of cycle wash"

Bottle/position codes:
18 /*A + Act */
19 /*C + Act */
20 /*G + Act */
21 /*T + Act */
22 /*5 + Act */
23 /*6 + Act */
24 /*7 + Act */
25 /*8 + Act */
26 /*9 + Act */
Expedite™ without MOSS unit
Synthesizing LNA oligonucleotides on an Expedite™ DNA synthesizer without using a MOSS unit requires altered protocols compared to the ones given above, as there is a shorter distance from the
reagent bottle to the support. Given below are examples of synthesis of LNA oligonucleotides on a 0.2μmol scale with LNA either on position 5 or position A.

Synthesis Cycle for LNA on an Expedite™ Nucleic Acid Synthesis System without MOSS

The synthesis cycle has been written for position 5 on the Expedite™. If any other positions are used, third line in the coupling step has to be changed.
 
LNA-cycle (0.2μmole scale)

/* ---------------------------------------------------------------------------- */
/* Function                   Mode    Amount     Time(sec)        Description */
/*                                            /Arg1         /Arg2                             */
/* ---------------------------------------------------------------------------- */
$Deblocking
12 /*Wsh A                 */ PULSE    10             0               "Pre cycle wash"
144/*Index Fract. Coll. */ NA           1             0               "Event out ON"
0 /*Default                 */ WAIT       0             1.5             "Wait"
141/*Trityl Mon. On/Off*/ NA           1             1               "START data collection"
16 /*Dblk                   */ PULSE     10             0               "Dblk to column"
16 /*Dblk                   */ PULSE     50           49               "Deblock"
38 /*Diverted Wsh A    */ PULSE     40             0               "Flush system with Wsh A"
141/*Trityl Mon. On/Off*/ NA           0             1               "STOP data collection"
144/*Index Fract. Coll. */ NA           2             0               "Event out OFF"
$Coupling
1 /*Wsh                     */ PULSE     10             0               "Flush system with Wsh"
2 /*Act                       */ PULSE       5             0               "Flush system with Act"
22 /*5 + Act               */ PULSE       5             0               "Monomer + Act to column"
2 /*Act                      */ PULSE       1              0               "Couple monomer"
2 /*Act                      */ PULSE       3            75               "Couple monomer"
1 /*Wsh                    */ PULSE       7           175              "Couple monomer"
1 /*Wsh                    */ PULSE       8              0              "Flush system with Wsh"
$Capping
12 /*Wsh A               */ PULSE      20              0              "Flush system with Wsh A"
13 /*Caps                 */ PULSE       8               0              "Caps to column"
12 /*Wsh A               */ PULSE       6              45             "Cap"
12 /*Wsh A               */ PULSE      14               0             "Flush system with Wsh A"
$Oxidizing
15 /*Ox                    */ PULSE      15               0             "Ox to column"
12 /*Wsh A               */ PULSE      10              45            "Slow pulse to Ox"
12 /*Wsh A               */ PULSE        4               0             "Flush system with Wsh A"
$Capping
13 /*Caps                 */ PULSE        7               0             "Caps to column"
12 /*Wsh A               */ PULSE      30                0            "End of cycle wash"
 
Bottle/position codes:
18 /*A + Act */
19 /*C + Act */
20 /*G + Act */
21 /*T + Act */
22 /*5 + Act */
23 /*6 + Act */
24 /*7 + Act */
25 /*8 + Act */
26 /*9 + Act */
 
 
The synthesis cycle has been written for position A on the Expedite™. If any other positions are used, third line in the coupling step has to be changed.  
 
DNA-cycle (0.2μmole scale)
 
/* ---------------------------------------------------------------------------- */
/* Function                     Mode   Amount    Time(sec)        Description */
/*                                             /Arg1         /Arg2                            */
/* ---------------------------------------------------------------------------- */
$Deblocking
12 /*Wsh A                 */ PULSE     10             0              "Pre cycle wash"
144/*Index Fract. Coll. */ NA            1             0              "Event out ON"
0 /*Default                 */ WAIT        0             1.5            "Wait"
141/*Trityl Mon. On/Off*/ NA            1             1              "START data collection"
16 /*Dblk                   */ PULSE      10             0              "Dblk to column"
16 /*Dblk                   */ PULSE      50           49              "Deblock"
38 /*Diverted Wsh A    */ PULSE      40             0              "Flush system with Wsh A"
141/*Trityl Mon. On/Off*/ NA            0             1              "STOP data collection"
144/*Index Fract. Coll. */ NA            2             0              "Event out OFF"
$Coupling
1 /*Wsh                     */ PULSE      10             0              "Flush system with Wsh"
2 /*Act                       */ PULSE       5              0              "Flush system with Act"
18 /*A + Act               */ PULSE       5              0              "Monomer + Act to column"
2 /*Act                       */ PULSE       1              0              "Couple monomer"
2 /*Act                       */ PULSE       3            36              "Couple monomer"
1 /*Wsh                     */ PULSE       7            84              "Couple monomer"
1 /*Wsh                     */ PULSE       8              0              "Flush system with Wsh"
$Capping
12 /*Wsh A                 */ PULSE      20             0              "Flush system with Wsh A"
13 /*Caps                   */ PULSE       8              0              "Caps to column"
12 /*Wsh A                 */ PULSE       6            45               "Cap"
12 /*Wsh A                 */ PULSE      14             0               "Flush system with Wsh A"
$Oxidizing
15 /*Ox                      */ PULSE      15             0               "Ox to column"
12 /*Wsh A                 */ PULSE      15             0               "Flush system with Wsh A"
$Capping
13 /*Caps                   */ PULSE       7              0               "Caps to column"
12 /*Wsh A                 */ PULSE      30             0                "End of cycle wash"
 
Bottle/position codes:
18 /*A + Act */
19 /*C + Act */
20 /*G + Act */
21 /*T + Act */
22 /*5 + Act */
23 /*6 + Act */
24 /*7 + Act */
25 /*8 + Act */
26 /*9 + Act */
 
 
Synthesis Cycle for LNA on an Expedite™ Nucleic Acid Synthesis System without MOSS
 
The synthesis cycle has been written for position 5 on the Expedite™. If any other positions are used, third line in the coupling step has to be changed.  
 
LNA-cycle (1μmole scale)
 
/* ---------------------------------------------------------------------------- */
/* Function                      Mode     Amount   Time(sec)      Description */
/*                                                /Arg1        /Arg2                          */
/* ---------------------------------------------------------------------------- */
$Deblocking
12 /*Wsh A                  */ PULSE      15            0              "Pre Wsh A"
144 /*Index Fract. Coll. */ NA             1            0              "Event out ON"
0 /*Default                   */ WAIT        0            1.5            "Wait"
141 /*Trityl Mon. On/Off*/ NA             1            1              "START data collection"
16 /*Dblk                     */ PULSE      20            0              "Deblock to column"
16 /*Dblk                     */ PULSE        2          10              "Deblock"
16 /*Dblk                     */ PULSE      10            0              "Deblock"
16 /*Dblk                     */ PULSE        2          10              "Slow Deblock"
16 /*Dblk                     */ PULSE      10            0              "Deblock"
16 /*Dblk                     */ PULSE        2          10              "Slow Deblock"
16 /*Dblk                     */ PULSE      10            0              "Deblock"
16 /*Dblk                     */ PULSE        2          10              "Slow Deblock"
16 /*Dblk                     */ PULSE      10            0              "Deblock"
16 /*Dblk                     */ PULSE        2          10              "Slow Deblock"
16 /*Dblk                     */ PULSE      10            0              "Deblock"
16 /*Dblk                     */ PULSE        2          10              "Slow Deblock"
16 /*Dblk                     */ PULSE      10            0              "Deblock"
38 /*Diverted Wsh A      */ PULSE      40            0              "Flush system with Wsh A"
141 /*Trityl Mon. On/Off*/ NA             0            1              "STOP data collection"
144 /*Index Fract. Coll. */ NA             2            0              "Event out OFF"
$Coupling
1 /*Wsh                      */ PULSE       15            0              "Flush system with Wsh"
2 /*Act                        */ PULSE         5            0              "Flush system with Act"
22 /*5 + Act                */ PULSE          5            0              "LNA-A + Act to column"
22 /*5 + Act                */ PULSE          2          75              "LNA-A + Act to column"
2 /*Act                        */ PULSE          3          75              "Couple monomer"
1 /*Wsh                      */ PULSE          7        100               "Couple monomer"
1 /*Wsh                      */ PULSE        10            0              "Flush system with Wsh"
$Capping
13 /*Caps                   */ PULSE        10            0               "Caps to column"
12 /*Wsh A                 */ PULSE          6          45               "Cap"
12 /*Wsh A                 */ PULSE          4            0               "Flush system with Wsh A"
$Oxidizing
15 /*Ox                      */ PULSE        15            0               "Ox to column"
12 /*Wsh A                 */ PULSE          8          45               "Slow Ox Wsh A"
12 /*Wsh A                 */ PULSE        10            0               "Flush system with Wsh A"
$Capping
13 /*Caps                  */ PULSE         10            0               "Caps to column"
12 /*Wsh A                */ PULSE           8          10               "Wsh A"
12 /*Wsh A                */ PULSE         30            0               "End of cycle wash"
 
Bottle/position codes:
18 /*A + Act */
19 /*C + Act */
20 /*G + Act */
21 /*T + Act */
22 /*5 + Act */
23 /*6 + Act */
24 /*7 + Act */
25 /*8 + Act */
26 /*9 + Act */
 
 
The synthesis cycle has been written for position A on the Expedite™. If any other positions are used, third line in the coupling step has to be changed.
 
DNA-cycle (1μmole scale)
 
/* ---------------------------------------------------------------------------------- */
/* Function                          Mode     Amount     Time(sec)        Description */
/*                                                     /Arg1         /Arg2                            */
/* ---------------------------------------------------------------------------------- */
$Deblocking
12 /*Wsh A                      */ PULSE       15              0              "Pre Wsh A"
144 /*Index Fract. Coll.     */ NA              1              0              "Event out ON"
0 /*Default                       */ WAIT         0              1.5            "Wait"
141 /*Trityl Mon. On/Off    */ NA              1              1              "START data collection"
16 /*Dblk                         */ PULSE       20              0              "Deblock to column"
16 /*Dblk                         */ PULSE        2             10              "Deblock"
16 /*Dblk                         */ PULSE       10              0              "Deblock"
16 /*Dblk                         */ PULSE        2             10              "Slow Deblock"
16 /*Dblk                         */ PULSE       10              0              "Deblock"
16 /*Dblk                         */ PULSE        2             10              "Slow Deblock"
16 /*Dblk                         */ PULSE       10              0              "Deblock"
16 /*Dblk                         */ PULSE        2             10              "Slow Deblock"
16 /*Dblk                         */ PULSE       10              0              "Deblock"
16 /*Dblk                         */ PULSE        2             10              "Slow Deblock"
16 /*Dblk                         */ PULSE       10              0              "Deblock"
16 /*Dblk                         */ PULSE        2             10              "Slow Deblock"
16 /*Dblk                         */ PULSE       10              0              "Deblock"
38 /*Diverted Wsh A          */ PULSE       40              0              "Flush system with Wsh A"
141 /*Trityl Mon. On/Off    */ NA              0              1              "STOP data collection"
144 /*Index Fract. Coll.     */ NA              2              0              "Event out OFF"
$Coupling
1 /*Wsh                           */ PULSE         5              0             "Flush system with Wsh"
2 /*Act                             */ PULSE         5              0             "Flush system with Act"
18 /*A + Act                      */ PULSE         5              0             "DNA-a + Act to column"
23 /*6 + Act                      */ PULSE         2            35             "DNA-a + Act to column"
2 /*Act                             */ PULSE          3            35            "Couple monomer"
1 /*Wsh                           */ PULSE          7            50            "Couple monomer"
1 /*Wsh                           */ PULSE        10              0            "Flush system with Wsh"
$Capping
13 /*Caps                        */ PULSE        10              0            "Caps to column"
12 /*Wsh A                      */ PULSE          6            45            "Cap"
12 /*Wsh A                      */ PULSE          4              0            "Flush system with Wsh A"
$Oxidizing
15 /*Ox                           */ PULSE        15              0            "Ox to column"
12 /*Wsh A                      */ PULSE          8            15            "Slow Pulse to Ox"
12 /*Wsh A                      */ PULSE        10              0            "Flush system with Wsh A"
$Capping
13 /*Caps                        */ PULSE        10              0            "Caps to column"
12 /*Wsh A                      */ PULSE          8             10           "Flush system with Wsh A"
12 /*Wsh A                      */ PULSE         30              0           "End of cycle wash"
 
Bottle/position codes:
18 /*A + Act */
19 /*C + Act */
20 /*G + Act */
21 /*T + Act */
22 /*5 + Act */
23 /*6 + Act */
24 /*7 + Act */
25 /*8 + Act */
26 /*9 + Act */
 
LNA Phosphorthioates
As LNA amidites can be used as parent DNA amidites it is likewise possible to make LNA phosphorothioates in which one of the non-bridging oxygen atoms has been replaced with a sulphur. The amidites should be used as described above – only change is that the oxidizer should be replaced with Beaucage’s reagent on the synthesizer.
 
We recommend the use of Beaucage’s reagent as a 0.2 M (4%) solution:
Dissolve 4g in 100 mL of anhydrous acetonitrile.*
* Do not add sieves as this might promote decomposition of Beaucage’s reagent. Use silanized bottles as recommended by the manufacturer.

Synthesis Cycle for LNA phosphorothioates on an Expedite™ (0.2 μmol scale)
 
The synthesis cycle has been written for position A on the Expedite™. If any other positions are used, third line in the coupling step has to be changed, see below.

/*------------------------------------------------------------------------------- */
/* Function                     Mode      Amount   Time(sec)         Description */
/*                                                 /Arg1       /Arg2                             */
/*--------------------------------------------------------------------------------*/
$Deblocking
12 /*Wsh A                   */ PULSE      15             0              "Pre Wsh A"
144 /*Index Fract. Coll.  */ NA             1             0              "Event out ON"
0 /*Default                   */ WAIT         0             1.5            "Wait"
141 /*Trityl Mon. On/Off */ NA             1             1              "START data collection"
16 /*Dblk                     */ PULSE       20             0              "Deblock to column"
16 /*Dblk                     */ PULSE         2           10              "Deblock"
16 /*Dblk                     */ PULSE       10             0              "Deblock"
16 /*Dblk                     */ PULSE         2           10              "Slow Deblock"
16 /*Dblk                     */ PULSE       10             0              "Deblock"
16 /*Dblk                     */ PULSE         2           10              "Slow Deblock"
16 /*Dblk                     */ PULSE       10             0              "Deblock"
16 /*Dblk                     */ PULSE         2           10              "Slow Deblock"
16 /*Dblk                     */ PULSE       10             0              "Deblock"
16 /*Dblk                     */ PULSE         2           10              "Slow Deblock"
16 /*Dblk                     */ PULSE       10             0              "Deblock"
16 /*Dblk                     */ PULSE         2           10              "Slow Deblock"
16 /*Dblk                     */ PULSE       10             0              "Deblock"
38 /*Diverted Wsh A      */ PULSE       50             0              "Flush system with Wsh A"
141 /*Trityl Mon. On/Off */ NA             0              1             "STOP data collection"
144 /*Index Fract. Coll. */ NA              2              0             "Event out OFF"
$Coupling
1 /*Wsh                       */ PULSE       15              0             "Flush system with Wsh"
2 /*Act                         */ PULSE         5              0             "Flush system with Act"
18 /*A + Act                 */ PULSE         5               0             "Monomer + Act to column"
2 /*Act                         */ PULSE         3             75             "Couple monomer"
1 /*Wsh                       */ PULSE         7            175            "Couple monomer"
1 /*Wsh                       */ PULSE       10               0             "Flush system with Wsh"
$Oxidizing
17 /*Aux                      */ PULSE       15               0             "SOx to column"
17 /*Aux                      */ PULSE       15            120            "SOx to column"
12 /*Wsh A                  */ PULSE        10              60            "Slow pulse to thioate"
12 /*Wsh A                  */ PULSE        20               0             "Wsh A"
$Capping
13 /*Caps                    */ PULSE        10               0             "Caps to column"
13 /*Caps                    */ PULSE        10              30            "Caps to column"
12 /*Wsh A                  */ PULSE          8              15            "Wsh A"
12 /*Wsh A                  */ PULSE        30                0            "End of cycle wash"
 
Synthesis Cycle for LNA phosphorothioates on an Expedite™ (1.0 μmol scale)
 
The synthesis cycle has been written for position A on the Expedite™. If any other positions are used, third and fourth line in the coupling step has to be changed, see below.
 
/*------------------------------------------------------------------------------- */
/* Function                        Mode     Amount    Time(sec)      Description */
/*                                                   /Arg1        /Arg2                          */
/*--------------------------------------------------------------------------------*/
$Deblocking
12 /*Wsh A                   */ PULSE        15             0             "Pre Wsh A"
144 /*Index Fract. Coll.  */ NA               1             0             "Event out ON"
0 /*Default                   */ WAIT           0             1.5           "Wait"
141 /*Trityl Mon. On/Off */ NA               1             1             "START data collection"
16 /*Dblk                     */ PULSE         20             0             "Deblock to column"
16 /*Dblk                     */ PULSE           2           10             "Deblock"
16 /*Dblk                     */ PULSE         10             0             "Deblock"
16 /*Dblk                     */ PULSE           2           10             "Slow Deblock"
16 /*Dblk                     */ PULSE         10             0             "Deblock"
16 /*Dblk                     */ PULSE           2           10             "Slow Deblock"
16 /*Dblk                     */ PULSE         10             0          &