Nucleic Acids Facility (NAF) at North Carolina
State University (NCSU)
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
- 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.
- 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.
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
The Nucleic Acids
Facility (NAF) at North Carolina State University
Image Gallery Staff
following characterization services are available from NAF.
to the novel nature of synthetic RNA and the complex chemistries used to
produce it, characterization of the purified product may be desirable
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
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.
Circular Dichroism Spectroscopy
Some modified nucleotides have unique CD chromaphores. NAF has instrumentation
to confirm their incorporation by CD spectroscopy.
1D NMR Analysis
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.