Bioorganic Chemistry
11
which is unfavorable from the stand-
point of free energy. It is difFcult to
determine exactly the free-energetic cost
of losing rotational/translational entropy
upon complexation, but it has been esti-
mated through a variety of techniques to
be on the order of 7 to 11 kcal mol
1
.
3
Nucleotide Interactions
A wide variety of bioorganic investiga-
tions deal with molecules that interact with
DNA. The purposes behind these investi-
gations are manifold, ranging from the
development of therapeutics to the under-
standing of the forces required for molec-
ular recognition. The following section is
divided into two parts. The Frst deals with
synthetic oligonucleotides interacting with
DNA, the second with small molecules that
interact with DNA.
3.1
Oligonucleotides
The revolution in recombinant DNA tech-
nology relies critically upon the ability to
synthesize a variety of oligonucleotides
quickly and cheaply. The development
of solid-phase oligonucleotide synthesis
has made this process relatively simple.
In addition to the many uses of these
oligonucleotides in recombinant DNA ma-
nipulation (ranging from use as poly-
merase chain reaction (PCR) primers to
the generation of site-directed mutants),
they have potential as therapeutics in the
Fe
ldo
fan
t
i
sen
sethe
rap
y
,genethe
rap
y
,
and RNA interference. Paralleling that use,
in solid-phase peptide synthesis (SPPS), a
common synthetic strategy that is used
in oligonucleotide synthesis is depicted in
±ig. 8.
There are many variations on this chem-
istry, not only for the production of natural
DNA but also for the production of unnat-
ural polynucleotides that have alternative
backbone and nucleic acid structures. An
example of the latter is phosphorothioate-
containing DNA, in which one of the two
unesteriFed phosphate oxygens is replaced
with a sulfur. This may be achieved by re-
placing the hydrogen peroxide or iodine
in the phosphite oxidation step shown in
±ig. 8 with a solution of elemental sulfur.
Phosphorothioate DNA has been found to
have greater resistance to nucleases (en-
zymes that cleave the diester backbone
of DNA), which makes it more suitable
for antisense therapy than naturally occur-
ring DNA.
Other approaches to artiFcial DNA have
been tried that completely bypass the use
of phosphate derivatives. Once again, the
r
a
t
i
on
a
l
eb
e
h
in
dt
h
i
si
st
om
a
k
eas
e
-
lective agent that will bind to a speciFc
gene target but will be resistant to nu-
cleases. P.E. Nielsen and coworkers have
recently developed a group of DNA analogs
in which the bases are connected to a
poly
amide
backbone (±ig. 9). The result
is an uncharged species that is capable
of forming speciFc base-pairing hydro-
gen bonds with target genes. In addition
to having resistance to nucleases, these
peptide nucleic acids, or PNAs, form
complexes with their target nucleic acid
sequences that appear to be even tighter
than those found in natural DNA duplexes.
A probable cause of this phenomenon is
the lack of negative charge on the PNA
compared with the normal hybridization
partner. We previously described the en-
ergetic advantage of coulombic attraction
through, for example, the formation of
salt bridges between negatively and pos-
itively charged species. The flip side to
previous page 685 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online next page 687 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online Home Toggle text on/off