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Octopus Molecule
Steric inhibition using sequence complementarity for targetting. | |
This nonenzymatic macromolecule is a generic, protein with two covalently attached flanking arms (or "tentacles," if you will) comprised of custom, 30 nucleotide oligos. This reaction component is designed to bind to the DNA, each arm binding the region flanking the restriction site. The generic protein
sits atop to the restriction site, providing steric inhibition.
Before the digest, this macromolecule is added to the reaction, and temperature is raised to 70 or so to partially denature the DNA. The restriction enzyme is then added. Since the restriction enzyme cannot access the site underneath the octopus molecule, it will not cut at that site, or it will cut at a reduced rate compared to other, unobstructed sites.
The idea here is to stop a restriction enzyme from cutting in an inappropriate location, such as in the middle of your gene.
Octopus proteins...visualised, not in this talk but in the project.
http://www.ted.com/...p/talks/view/id/147
please peruse the project, this is great effort. [4whom, Jun 10 2008]
this guy has his finger on the button...
http://www.ted.com/talks/view/id/80
@ 16: 30 he talks of the data and who should be using it. [4whom, Jun 10 2008]
Restriction protection using PNAs
http://pubs.acs.org.../abs/bi000675e.html
Similarish method using a DNA analogue in place of the protein/oligo construct. [MaxwellBuchanan, Jun 10 2008]
[link]
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{wishing I were remotely qualified to comment on this} |
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"MaxwellBuchanan to the Science aisle please! We have a biochemistry spillage!" |
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Some clarification for those who would
like it. Restriction enzymes cut DNA a
particular (usually very short) sequence,
such as "GAATTC" - a bit like a pair of
scissors that cuts printed text wherever
it finds the word "the". |
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Often, you want to cut DNA with a
restriction enzyme, but want to prevent
its cutting at some specific points (for
example, you don't want it to cut at the
"GAATTC" that's in the middle of a
particular gene, but you do want it to
cut all the other GAATTC positions). |
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Cuit's idea is (a) partly denature the
DNA (partly peel apart the two strands
so that things can get in) (b) add a
protein (which is just being used as a
"lump of stuff") to which are glued two
DNA "arms"; their sequence matches
the sequence either side of the GAATTC
that you *don't* want to cut. (c) Cool
things down again so the DNA strands
re-unite. The hope is that the "arms"
will unite with the corresponding
sequences in the target DNA, thereby
gluing the protein in place and
protecting that part of the DNA from
the restriction enzyme. |
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The analogy would be a sort of metal
strip that sticks to the phrase "then the
boat sank", thereby protecting that
particular "the" (but not others
elsewhere) from the scissors. |
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So, basically quite a good idea. Some
possible flaws: |
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(a) it's not trivial to couple the DNA
'arms' to a protein (though not difficult
either). But it needn't be a protein; it
could just be a stretch of 'uncuttable'
DNA between the two arms |
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(b) The DNA is only partly denatured at
the beginning. This is tricky, but
essential if it is to easily zip back
together afterwards. However, this "re-
zipping" is very fast - probably faster
than the binding of the "arms" to the
DNA. So, the DNA will zip back
together before the "arms" can get in
and stick. |
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A better solution would be to use DNA-
like molecules which recognize their
matching sequence, but bind more
strongly to the DNA strand than a
"normal" DNA strand would do. There
are some molecules like this, but I can't
for the life of me remember what they
are; I think they're PNAs (peptide
nucleic acids; synthetic molecules with
some properties of DNA and some of
proteins; yet they are neither). PNAs (or
PNA/DNA paired strands) aren't cut by
restriction enzymes. |
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You rely on oligos to hold your molecule in place. I am not sure why you want or need a protein in the middle. Just make it one oligo. In fact, if this worked, you could do exactly what you propose with your own gene sequence: make a bunch of oligos complementary to your gene and throw them in. |
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If (you protest) your oligos will not be sticky enough to protect your gene, then your custom molecule would not stick either. |
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It occurs to me that one could denature the DNA, throw in the oligos complemetary to your gene, partly renature, then add some DNA crosslinkers to stick the oligo there. If you have a great excess of oligos probably some will be where you want them even if the whole thing has not renatured. You will want to use a reversible DNA crosslinker. |
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Or maybe not. Presumably you are trying to figure out what is next to your gene. For that it would not really matter of these gene itself was crosslinked to an oligo. |
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You could label your oligos, making it easier to recover the fragment of interest after cutting with enzyme. |
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//Just make it one oligo. // No, not if
you mean a single fully complementary
oligo. It will just anneal to one strand,
and the resulting double-stranded DNA
will be cut by
the enzyme. The net result will be that
you'll cut one of the two strands, within
your gene; may not be a problem, but
then again it may. You could always
use an oligo which was mis-matched at
the restriction site (so, no double-
stranded restriction target is created).
But better to use a non-
cleavable analogue, which could have
the advantage of binding more strongly
(see PNA in anno
above). |
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I believe there a number of strategies
for protecting chosen restriction sites,
though I've never needed to use them. |
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[EDIT] Just google "restriction cleavage
protection" for some examples. I've
posted a link to the first one I hit, which
does indeed use PNAs. |
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//Just google "restriction cleavage protection" for some examples.//
I bet there were couple of dodgy results in that search! |
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