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Ribozyme
containing several different monomers (2’-O-Me
ribonucleotides to stabilize the ribozyme against RNase
degradation, various 3’ and 5’ modifications
for protection against exonuclease degradation, RNA
monomers and all sorts of commercially available linkers
and modifiers) are produced on a routine basis by RNA-TEC.
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Purity: For the ribozyme all purities ranging
from 90 to 98 % are available. This corresponds to 1
or 2 HPLC purifications. For each request we use a customized
purification protocol to meet the needs of the customer
in a cost efficient way.
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Yield: For our ribozymes we guarantee high
yields, see example below. This is because the incorporation
of 2’-O-Me RNA and DNA monomers gives higher coupling
efficiencies than RNA monomers.
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Typical products: For in vivo experiments
with ribozymes we are providing 25 to 30 mg or about
55 mg as standards. Other scales are available on request.
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Transcribed RNA versus chemically synthesized RNA:
If you are planning to do in vivo experiments where
you will need more of the ribozyme please bear in mind
that chemical synthesis will be much more cost effective.Also
ribozymes are better. This is largely due to the cost
of the T7 RNA polymerase. Moreover, some sequences are
difficult to transcribe due to their secondary structure
and sequence whereas chemical synthesis does not suffer
from such limitations.
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Yield examples: A 18 µmol scale synthesis of
a 28 mer ribozyme containing 13 different monomers and
internal linkers resulted in 27 mg of purified ribozyme.
The ribozyme was
purified by reversed phase HPLC (see below). The second
peak is the product and the first peak contains all
the shorter sequences. After this purification the product
was analyzed by analytical anion-exchange HPLC which
gave a purity of around 98 %. The 2 peaks on the chromatogram
below correspond to different secondary structures of
the same molecule (both peaks were analyzed by mass
spectrometry). This again shows RNA-TEC state-of-the-art
purification methods.
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