TECHVAULT > FAQ > APPLICATION SUPPORT > ANTISENSE OLIGOS

I'm planning to transfect double strand oligo into prostate cancer cell line, can the oligo last in the cells for 4 days? Can you define the half life of oligo with partial modification?

You may need to do multiple treatments with the antisense oligo if you need to reduce expression for four days.  You may also want to consider using a chimeric oligo to extend the half life.  2'O Me RNA bases have been used successfully to extend the halflife of ASOs in cell lines.  The two articles listed below should be helpful in designing your oligos.   

Zellweger T, et al.  J Pharmacol Exp Ther 2001 Sep;298(3):934-40
Gleave M, et al.  Cancer Metastasis Rev 2002;21(1):79-92 

Would a phosphothioate modification to the end of a DNA primer allow a longer half-life of the PCR product when transfected into a cell? If so, how many? If not, what modification is recommended?

Phosphorothioate bonds increase the oligonucleotides resistance to [exo]nuclease degradation.  If you chose to modify PCR primers with PS bonds then the resulting double-stranded PCR product will have PS bonds only on the 5' ends of each strand of the dsDNA product, there will not be PS bonds on the 3' end of the dsDNA PCR product.  Endonucleases and Exonucleases with 3'-->5' activity will still be able to degrade your dsDNA product.

For single stranded oligos, nuclease-resistance is highest when every phosphodiester bond is changed to a PS bond.  In general we recommend at least 3 PS bonds on both 5' and 3' ends of the oligo, but more PS bonds will give greater exonuclease resistance.

Could you tell me about nuclease-resistent oligonucleotides, particularly DNase I-resistant?

There are a variety of ways to make oligonucleotides resistent to nuclease activity.  These include the use of phosphorothioated bonds, which can be used to form the entire backbone of an oligo or can be used to "cap" the ends of the sequence.  In addition, 2'O-Methyl RNA bases can also be used to make an oligo resistent to nucleases.  Finally, phosphorothioate bonds and 2'O-Methyl RNA bases can be combined to create further stability.

How long can phosphothioate modified oligo stay in cell? If I designed a chimeric oligo with modification only in 5' and 3' with intact core sequence, will the stability decrease? If yes, what will the half life be?

The halflife of an antisense oligo varies widely depending on the tissue type you are studying and whether you are injecting it into a live animal or using tissue cultures. If you plan to inject the oligo into a live animal, you would be better off using a full phosphorothioate backbone to increase the lifetime within a cell, however it will be more toxic to the cell then a "capped" sequence.  Because of some of the toxic byproducts of the synthesis of this type of oligo, you should strongly consider using a sodium salt exchange to purify the oligo before injecting it.  If you are using tissue cultures, you can successfully use a capped sequence with a few (2-4) thioate bonds at the 3' and 5' ends of the oligo. 

Where should I place thioate bonds to increase the stability of my oligo? Does a thioate modification interfere with annealing?

Adding phosphothioate linkages will inhibit RNase and Dnase degradation, for many antisense experiments investigators have found that adding as many as 3 phosphothioate linkages at the 5' and 3' end works well. The inclusion of these linkages does not interfere with the annealing of the oligos.

What are the benefits of using a 2'OMe/phosphorothioate chimera for antisense work?

Phosphorothioate bonds do tend to lead to non-specific effects because the sulfur can interact with proteins in the cell.  2'OMe avoids the non-specific binding and tends to bind tighter to its target.  Mixing the two can increase the lifespan of the oligo in the cell.  By limiting the number of thioate bonds to three, you can get the increased lifetime in the cell and limit some of the non-specific effects.  There is a very good review on antisense modifications, their advantages and drawbacks, in the following journal: Kurreck, J.  (2003)  Eur. J. Biochem 270:1628-1644 

In choosing the antisense oligo sequence is self-dimerization a concern? If so, what is considered an acceptable complementarity (bp or delta G terms)?

You will need to be concerned with self dimerization. If the oligo has a stronger affinity for itself than your target, your experiment may not work.  To test for strong self dimerization, you can analyze your sequence at with our OligoAnalyzer SciTool by pasting your sequence into the box provided and hitting the "Dimer" button. A delta G value of -9 or more negative is problematic.   

I'd like to get 2-O-methyl oligonucleotide(RNA) to use it as an antisense against miRNA. How do I order it on this website?

IDT recognizes 2-O-Methyl modified oligos by typing a lower case "m" to the left hand side of the base.  The following sequence, mCmGmACGACGAmGmCmA, has 2-O-methyl modification on the 3 terminal bases on both ends of the sequence.  The lowest scale of synthesis for this modification is the 100nmole scale.

I would like to use FAM to visualize the uptake of my oligo into a cell. Does it matter if the FAM is added to 5', 3', or an internal base (eg. does it affect binding)? Can FITC be added to a phosphorothioate-modified base?

Attaching a fluorescent label for this purpose is actually quite common. It does not matter if you attach it 5', 3' or internally to visualize the uptake. Other things to consider, however, are the synthesis and the price. The oligos are synthesized 3' to 5'. 3' modifications tend to lead to lower yields because the are a more unstable base on which to build the rest of the oligo. If you prefer an internal FAM, this dye would need to be attached to a T base. FAM can be added to phosphorothioate modified bases.

Is a chimer containing 5 phosphorthioate bonds on both the 5' and 3' ends and 17 phosphodiester lingages between them a suitable probe for a series of antisense experiments?

When performing antisense experiments capping the 5' and 3' ends with phosphorothioate bonds is very common. For a negative control we actually recommend that you use your sequence in the reverse order (not it's complement) without phosphorothioate bonds.

For example, if your antisense oligo (phosphorothioate bonds are indicated by *) is:

5' A*T*C*G*A*TCGGATC*G*A*T*C*G 3'   

Then the negative control would be:

5' GCTAGCTAGGCTAGCTA 3'

For phosphorothioated oligos we highly recommend HPLC purification and Sodium Salt Exchange to remove excess thioate residues that can be toxic to cell lines. 

I get an error message when ordering my thioate oligo online, can you help?

The asterisk included in the antisense sequences actually denotes a phosphorothioate linkage and should be placed between the two bases you would like to have linked with a phosphorothioate bond. These bonds can only be located on a Phos linkage and since our oligos have OH ends, thioate bonds can not be placed on the absolute 3' or 5' end of your oligo.

For example, you would need to enter a sequence like this: N*N*N*NNNNN*N*N*N

How do you calculate the molecular weight and extinction coefficients for phosphorothioated oligos?

The molecular weigh can be calculated by using the following formula:

Anhydrous MW = ‡" ( Individual Base MW ) - 63.980 + 2.016 + (16 x number of phosphorothioate bonds)

The last portion of the formula is needed to correct for the increase in weight when sulfur is substituted for one of the oxygens in the phosphorothioate bonds. The weights of each of the bases are as follows:

dA - 313.209
dC - 289.184
dG - 329.208
dT - 304.196
dU - 290.169
dI - 314.194

Here is an example of this calculation:

Example sequence: 5'- A*T*G* TAA TGT TTG GTC* C*G*C -3' nA=3, nG=5, nC=3, nT=7, nBonds=6;

Using the formula above, the molecular weight of this oligo is (313.209 x nA) + (329.208 x nG) + (289.184 x nC) + (304.196 x nT) - 63.980 + 2.016 + (16 x nBonds) = 5536.6 g/mole

The extinction coefficient for a phosphorothioate oligo would be the same as that of an unmodified oligo.

If I need to use micromolar concentrations in my reaction, do I need to order the 1 μM scale?

Concentrations confuse a LOT of researchers.  Micromolar is micromoles per liter. Depending on how much media you bathe your cells in the 1 μM scale may or may not be enough. For example, a 25mer phosphorothioate modified oligo should give you a minimum yield of 20 ODs after purification. This would translate into about 80 nmoles.  80 nmoles in 800 μL of media would be at a concentration of 100 μM. That same 80 nmoles in 8 mL would be at a concentration of 10 μM. If you can give us an idea of how much material you need, we can better advise you as to what scale to order.

Which is the best way to deliver oligos inside the cells in culture?

There are several different ways to introduce oligos into a cell, including using liposomes, attaching the oligo to a peptide, and electroporation. The best method depends on the type of cell and types of modifications on the oligo.

Can a phosphorothioated oligo be used in medium containing serum when using for antisense studies? How long is the half life in vitro?

Antisense oligos can be used in medium containing serum.  However, you need to inactivate the exonucleases in the serum to decrease the chances of degredation (even though the oligos are protected by the phosphorothioate modification, there is always a chance, so better to err on the side of caution).  You can do this by heat inactivatation at 65°C (instead of 56°C) for 30 minutes.    As for a half life in vitro, that is totally dependent on the message, the cell type and the construct. There have been claims of 24 hours in vivo (mice), but again, there is no "set" or "average" half life.  

Can IDT make a DNA oligo with a mixture of phosphodiester and phosphorothioate bonds? If so, what would it cost?

IDT can make such an oligo. In order to get phosphorothioate bonds the oligo must first be ordered on at least the 100nmole scale. The phosphorothioate bonds are indicated by an * in the sequence. 

For Example: 5'A*C*T*GCATCAGCCTGCC*A*T*C 3'

Please see the Antisense Oligos Catalog Page for more information and current pricing.

How do you synthesize oligos with a phosphorothioate backbone? How do you control the placement of the phosphorothioate and phosphodiester linkages when a chimeric backbone is requested?

The addition of a new base to an oligo takes place in four steps: 

1.  The trityl group is removed from the 5' oxygen 

2.  The two bases are coupled together 

3.  The phos is oxidized, stablizing the bond between the two bases 

4.  Any oligos which did not participate in the coupling reaction are capped off. 

The difference between the synthesis of a normal oligo with a full phosphodiester backbone and a oligo with a partial or full phophorothioate backbone is the choice of oxidizing agent used in the third step. A phophodiester bond is produced by using iodine and water to add a fourth oxygen to the phos. A phosphorothioate bond is produced by using Beaucage reagent to add a sulfer to the phos. Once either the sulfer or the oxygen has been attached to the phos, the bond is stablized and will not be affected by the subsequent cycles of chemistry. By switching back and forth between the two oxidizing agents, a chimeric backbone can be constructed. Please see our technical bulletin on The Chemistry of Oligonucleotide Synthesis for more details on how oligos are synthesized. 

What modifications (if any) do you recommend for antisense experiments?

The first antisense experiments used unmodified oligos, but they found that the cellular nucleases degraded the oligos quickly, leaving only a short time for the oligo to find its target.  Oligos containing phosphorothioated bonds are more resistant to nuclease degradation.  There are two options for inserting phosphorothioate bonds into an antisense oligo:  either modifying the entire backbone or just capping the ends by putting three thioate bonds on each end of the oligo.  Thioate bonds lower the Tm of your oligo, and the more thioate bonds you have, the greater the affect on the final Tm.  Therefore, we usually recommend capping the oligo.  We also strongly recommend HPLC purification and a Sodium Salt Exchange because the chemical byproducts of the synthesis/purification can be toxic to your cells.    

Can you describe the different modifications routinely used in antisense experiments ?

Phosphothioate bonds are added to antisense oligos to protect them from nuclease degradation. You can have a full phosphothioate backbone, however, your Tm decreases with each phosphothioate bond you add. You can decrease this effect by 'capping' your oligo. This is accomplished by adding 2-5 bonds to each end of your oligo (most people add 3 to each end.) The 5-Me dC, 2' O-Me RNA bases and C5-Propyne Pyrimidine modifications are sometimes added to increase the nuclease resistance and binding affinity of your oligos, but they are not critical. You can start your experiments without them and add them to subsequent oligos if you need increased stability. Antisense oligos are generally 18-24 bases in length. The concentrations will need to be determined on a case-by-case basis as they vary between organisms. Usually a concentration of 2uMolar (or 10ug/ml) is a good starting point.

If you would like more information on Antisense oligo design and usage, please see our Technical Reports:

Designing Antisense Oligonucleotides

Introducing Antisense Oligonucleotides into Cells

Application Support Topics