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Locked Nucleic Acid
 
LNA15/05.2005
 
By C. Lomholt
 
 
LNA Oligonucleotide Synthesis on an Expedite™ without 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.  
 
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 */
 
 
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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