For most applications, synthetic DNA can be used after removal of the ammonia solution used in deprotection. If a higher level of purity is required, anion exchange HPLC purification is available.

For each oligo a purification process should be designed which will be compatible with subsequent handling steps. The generalized processes which all RNAs must undergo in purification are deprotection, purification and desalting.
Deprotection - All RNA must undergo a multi-step deprotection process. Cleavage from the solid support and removal of exo-cyclic amine protecting groups is accomplished by using ammonia saturated ethanol. The ethanolic ammonia is removed by vacuum. After this step, the sample is stable and can shipped. Removal of the 2'OH protecting group is made by the fluoro anion by the addition of 1N TBAF solution. For some chemistries a deprotection step using DBU is required to fully deprotect the oligonucleotide.

Purification - Various methods of purification are available, however the method chosen should be compatible with the final use of the RNA. While purification may be omitted, the lower coupling efficiency of RNA produces significantly lower purity than a DNA of comparable length. NAF offers anion exchange HPLC purification because of the high recovery, flexibility and reproducibility this method provides.

Desalting - After ion exchange HPLC or even without purification, a desalting step is required. High recovery is obtained by reverse phase chromatography. The sample is eluted in organic solvent that is easily removed. Samples are usually shipped in dried form.

Yield - The amount of purified material recovered is variable. The incorporation of modified units frequently lowers yield. The empirical range of purified product we have obtained for 17 mer containing modified bases is 100 to 700 µg.

Due to the novel nature of synthetic RNA and the complex chemistries used to produce it,  characterization of the purified product may be desirable or necessary.

The following characterization services are available from NAF.

 

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To confirm the incorporation of modified nucleotides, hydrolysis of a small portion of the purified product is made. By reversed phase HPLC separation of the hydrolysate and comparison with analytical standards, proper composition can be confirmed. Using UV detection with a photodiode array detector allows for identification by both retention time and spectral character.

In collaboration with the North Carolina State University Mass Spec Lab., MALDI analysis of purified oligos is available. MALDI protocol has been demonstrated over the mass range of 1000 to 9000 + 0.5% MW.

Much thermodynamic information about a sequence can be determined by monitoring the UV melt of an RNA. NAF is able to determine the basic parameters of deltaH, deltaS, deltaG and Tm for a purified oligonucleotide.

Some modified nucleotides have unique CD chromaphores. NAF has instrumentation to confirm their incorporation by CD spectroscopy.

Magnetic resonance is a powerful means to confirm secondary and tertiary interactions in RNA. In collaboration with the North Carolina State University NMR Facility, proton NMR analysis of purified oligos is available.

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