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Locked Nucleic Acid
 
LNA14/05.2005
 
 
LNA Oligonucleotide Synthesis on Expedite™ Multiple Oligo Synthesis System (MOSS)
 
The use of LNA phosphoramidites on an Expedite™ instrument follows standard procedures for use of phosphoramidites.
 
The LNA-A, G and T amidites are used as 0.07M solutions in anhydrous acetonitrile, in the case of LNA-mC it is advised to dissolve the amidite in a 25% tetrahydrofurane/acetonitrile1 solution (0.07M) to avoid precipitation of the amidite.
 
A 250 seconds coupling time is normally found to be sufficient 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 optimal when synthesising standard oligonucleotides.

    
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
  

 
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.
 
 
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 */
 
 
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 */
 
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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