Hi, can Trilink synthesize a RNA di-nucleotide containing at least one intrisically fluorescent ribo-nucleotide analogue? Thank you-Egor

Dear Egor,

We are able to synthesize an RNA dinucleotide containing an intrinsically fluorescent ribo-nucleotide analog.

I have included a link to the product page for a few options we can offer:

Isomorphic-Intrinsically Fluorescent

For a formal quotation, please contact us through sales@trilinkbiotech.com

Best Regards,

Tiffany Teng

Product Specialist I

How do I deprotect my 5’ maleimide oligonucleotide?

The protected maleimide modification is deprotected using a retro Diels-Alder reaction.  This can be done with microwave irradiation or by heating in toluene. The second approach is described below. Retro Diels-Alder reactions should be conducted in anhydrous conditions as exposure to significant levels of moisture can cause incomplete deprotection, hydrolysis and/or addition of water to the maleimide.
1.  Suspend lyophilized oligo in anhydrous toluene.
2. Let sit for 4 hrs at 90°C.
3. Cool to room temperature.
4. Evaporate toluene.
The oligonucleotide is now ready for conjugation to a thiol compound.

What is the machine validation charge for trimer modified oligonucleotides?

When trimer modified oligonucleotides require machine synthesis, a machine validation is performed prior to starting the job. This validation ensures that all aspects of the synthesizer are functioning properly and decreases the chance of synthesis failure. Since the trimer mix amidite is an expensive modification we take all necessary precautions to limit the chance of a synthesizer malfunction which could waste trimer mix amidite. A machine validation charge is required for each new synthesis run so it is beneficial to order all of your trimer modified oligonucleotides at once. This allows us to split the validation charge across all oligonucleotides in the order, reducing cost per oligonucleotide.

Why does the price of trimer modified oligonucleotides vary?

Trimer modification requires a highly custom synthesis protocol in which multiple additional factors are taken into account. Each trimer mix job is individually analyzed in order to provide the most cost effective pricing in terms of synthesis cost and trimer amidite usage. If a small number of trimer mix incorporations are needed then it is more cost effective to manually couple the trimer mix rather than machine couple. While manual coupling adds increased labor to the quote, the amidite usage is decreased since there is no need to prime the machine. Also, manual coupling eliminates the need for a machine validation requiring an additional charge.

Does TriLink provide LNA modified oligonucleotides?

Yes, we are able to synthesize LNA modified oligonucleotides, however there are a few stipulations. Since this material is under license by Exiqon we require that our customers obtain a license from Exiqon, purchase the LNA amidites and have the material shipped to our facility for synthesis.

The analysis of my purified thiol modified oligonucleotide shows three different species. Are these impurities?

No, this is generally not an indication of impurity. A thiol modified oligonucleotide which is not fully reduced will be in three states: free thiol (oligonucleotide-SH), protected thiol (oligonucleotide-S-S-C6-O-DMT) and dimerized oligonucleotide (oligonucleotide-S-S-oligonucleotide). We recommend that you reduce these oligonucleotides just prior to use to ensure that only the free thiol is present. DTT or TCEP are the preferred reductants.

Is it best to have my 5’ thiol linker modified oligonucleotide delivered with or without the thiol protected?

There is simple answer to this question, as it depends on your experience and what you plan to do with the molecule. In most applications, the thiol will be reduced just prior to use to ensure that unwanted dimers are cleaved. If the conjugation is to a maleimide, the reduction can actually be done in situ during conjugation. In that case, there is very little difference. Technically, it is easier to purify a protected thiol, but it is very difficult to keep it on completely intact.

How do I reduce my thiol modified oligo?

1. Make a solution of 60 mM TCEP. (18 mgs TCEP in 1 mL H2O)
2. Add 125 uL 0.1M NaHPO4, 0.15M NaCl, pH 7.26, vortex & make certain oligo is in solution.
3. Add 75 uL 60 mM TCEP to oligo solution.
4. Vortex.
5. Let sit for 2 hrs at room temperature.
6. Run size exclusion column, RP-HPLC.

What are the advantages of using 2’OMe RNA bases in an oligonucleotide?

2′ O-Methyl RNA is a more stable form of RNA with similar properties. It has the same low rate of depurination, but it is protected against base hydrolysis. 2′ OMe RNA is chemically more stable than RNA as well, and allows for greater flexibility in conjugation schemes.
2′ O-Methyl RNA:RNA duplexes are the most stable. RNA:RNA and DNA duplexes have lower melting temperatures. 2′ OMe RNA is most commonly used in therapeutic applications due to its nuclease resistance.

What’s the difference between a phosphodiester and a phosphorothioate backbone?

A phosphorothioate linkage has a sulfur in a non-bridging location on the phosphate backbone. This modification is known to greatly retard nuclease degradation of oligonucleotides. Phosphorothioates also have a lower melting temperature, which is a measure of the association and disassociation rates. A phosphodiester linkage contains all oxygens on the phosphate backbone.