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Locked Nucleic Acid
 
LNA23/05.2005
 
By Christian Lomholt and Henrik M. Pfundheller
 
LNA phosphorothioate synthesis using an Expedite™ DNA synthesizer
 
The use of LNA phosphoramidites on an Expedite™ instrument to synthesize LNA phosphorothioates follows with minor changes the standard procedures for use of phosphoramidites. 
 
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/ACN ratio of 25:75 and an amidite concentration of 0.07M.
Increased coupling time and sulfur oxidation time is recommended for all LNA phosphoramidites.
 
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.

 

    
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
  

1We recommend the use of anhydrous tetrahydrofurane (Aldrich 401757). As an alternative anhydrous dichloromethane (Aldrich 270997) can be used as a substitute for THF.

On the following you will find protocols for the synthesis of LNA phosphorothioates using Expedite™ DNA synthesizers with and without MOSS.
 
 
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               "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               "LNA-A + Act to column"
18 /*A + Act                */ PULSE         2            75              "LNA-A + Act to column"
2 /*Act                       */ PULSE          3          150              "Couple monomer"
1 /*Wsh                     */ PULSE          7          200              "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™ with MOSS (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"
16 /*Dblk                   */ PULSE        20               0              "Deblock to column"
141/*Trityl Mon. On/Off*/ NA              1               1              "START data collection"
16 /*Dblk                   */ PULSE        20               0              "Deblock to column"
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"
38 /*Diverted Wsh A    */ PULSE        10               0              "Deblock"
38 /*Diverted Wsh A    */ PULSE         2              10              "Slow Deblock"
38 /*Diverted Wsh A    */ 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       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            250              "Slow pulse to couple"
1 /*Wsh                      */ PULSE         4               0              "Flush with Wsh"
$Oxidizing
17 /*Aux                     */ PULSE       30               0              "SOx to column"
12 /*Wsh A                 */ PULSE       20            180              "Slow pulse to thioate"
12 /*Wsh A                 */ PULSE       20               0               "Wsh A"
$Capping
13 /*Caps                   */ PULSE       20               0               "Caps to column"
12 /*Wsh A                 */ PULSE       20              45              "Wsh A"
12 /*Wsh A                 */ PULSE       60               0               "End of cycle wash"
 
 
Synthesis Cycle for LNA phosphorothioates on an Expedite™ with MOSS (1.0 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"
16 /*Dblk                   */ PULSE         20           0                  "Deblock to column"
141/*Trityl Mon. On/Off*/ NA               1            1                 "START data collection"
16 /*Dblk                   */ PULSE         20           0                  "Deblock to column"
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"
38 /*Diverted Wsh A    */ PULSE         10            0                 "Deblock"
38 /*Diverted Wsh A    */ PULSE          2           10                 "Slow Deblock"
38 /*Diverted Wsh A    */ 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          7            0                 "LNA-A + Act to column"
2 /*Act                        */ PULSE         5            0                  "Couple monomer"
1 /*Wsh                      */ PULSE        10            0                 "Couple monomer"
1 /*Wsh                      */ PULSE        15         425                 "Couple monomer"
1 /*Wsh                      */ PULSE        20             0                "Flush system with Wsh"
$Oxidizing
17 /*Aux                     */ PULSE        30             0                "SOx to column"
12 /*Wsh A                 */ PULSE         20         180                "Slow pulse to thioate"
12 /*Wsh A                 */ PULSE         20            0                "Wsh A"
$Capping
13 /*Caps                   */ PULSE         20            0                "Caps to column"
12 /*Wsh A                 */ PULSE         20           45                "Wsh A"
12 /*Wsh A                 */ PULSE         60            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 */
 
Trademarks and patents
 
Exiqon™ is a registered trademark of Exiqon A/S, Vedbaek,
Denmark. Locked nucleic acid (LNA™) is covered by patents/
patents applications, and corresponding worldwide
applications owned by Exiqon A/S and Prof. Imanishi.
Expedite™ is a trademark of Applied Biosystems.
 
 
Latest revision May 30, 2005
 
 
 


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