Genomics- The Science and Technology Behind the Human Genome Project. Charles R. Cantor, Cassandra L / genomics11-15 / 14

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520 SEQUENCE-SPECIFIC MANIPULATION OF DNA

Figure 14.48 General construct needed for encoded combinatorial chemistry. Adapted from Brenner and Lerner (1992).

monomeric	units in	the oligomer will determine the complexity of the coding scheme
needed in the DNA. A particularly nice trick, if small numbers of monomer units are in-
volved, is	to use a	comma-less code. For example to specify the 20 amino acids, only a

subset of 20 of the 64 possible triplet codons is needed. One can choose these, for example, so that if AAT and CAG represent two different amino acids, which can occur in ei-

ther order: AATCAG and CAGAAT, then ATC, TCA, AGA, and GAA are not assigned to

any amino acids. This makes the code resistant to frame shift errors and other ambiguities.

The chimeric DNA-oligomer compounds (Fig. 14.48) are screened for whatever activ-

ity is desired in the oligomer; then PCR is used just as in the select strategy in order to identify those components that have the desired afﬁnity for a target. It is relatively easy to synthesize the full set of sequence identiﬁers and oligomers in a systematic way. This is

shown in Figure 14.49. The actual efﬁciency of such schemes needs to be tested experimentally. Undoubtedly new schemes and variations on existing schemes will proliferate.

However,	the important		feature of all of	these approaches is that they	illustrate	the im-
mense power that DNA analysis can bring to conventional chemistry.
OLIGONUCLEOTIDES		AS	DRUGS
A large	number of	young biotechnology		companies are betting their	futures on	the

prospect that nucleic acids or nucleic acid analogs will function effectively as drugs. Most

of	this effort is not based on conventional ideas about gene therapy, where a underactive
or	inactive defective gene might be supplemented by an active one, or an overactive or in-

appropriately active gene might be substituted with a normal one. Such unconventional therapies are attractive, especially for many tissue-speciﬁc disorders, and such somatic

OLIGONUCLEOTIDES AS DRUGS

521

Figure		14.49	Split pool scheme for synthesizing a set of oligomeric compounds linked to their
speciﬁc oligonucleotide identiﬁers. Adapted from Brenner and Lerner (1992).
gene therapy is already being tested in				a few clinical trials. Here we are concerned with
the much more limited and conventional approach of attempting to use short pieces of nu-
cleic acids or their analogues as drugs.
	The intrinsic attractiveness of nucleic acids as drugs is their sequence speciﬁcity. One
can	imagine that		placed in the appropriate cell, an oligonucleotide could interfere with
RNA or DNA function either by binding				directly to these species or by competing with
them for targets such as proteins. One approach is to simply use the sequence-speciﬁc
binding		afﬁnity of	the oligonucleotide to	physically occlude a site or target. Here DNA is
a potentially attractive target because				it exists in very low copy numbers. An ideal anti-
gene scenario would be to design an oligonucleotide that would form a very stable triplex
under physiological conditions with an unwanted promoter and so turn off the transcrip-
tion	of	the gene	controlled by this promoter. Alternatively, the oligonucleotide could be
used	as	an afﬁnity	reagent to carry a photochemical or other covalent modiﬁer to a target
of interest. Again this seems potentially most effective with a DNA target. An extreme
version of oligonucleotide therapy would be to use catalytic RNAs to ﬁnd and destroy
multiple target molecules. If this can be realized, it will be an extremely effective way to
deal with infections by viruses with RNA genomes, or to attack other RNA targets.
	A number of		obstacles must be overcome before successful oligonucleotide therapy
will	be	achieved.	First, the materials	must be delivered effectively to the correct target
cells and in sufﬁcient quantities to be therapeutically					active. Side reactions with other
cells must be kept to a minimum. If cells lacked receptors for uptake of oligonucleotides,
the	problem would		be to develop a targeting mechanism for		the speciﬁc cells of interest.

522 SEQUENCE-SPECIFIC MANIPULATION OF DNA

Unfortunately, at least some cells in the body, T lymphocytes, have a natural pathway for oligonucleotide uptake. How general this phenomenon is remains to be seen. The implication is that unless cells with intrinsic uptake pathways are the desired target, this uptake may have to be suppressed, possibly by competition with a harmless oligonucleotide and possibly by shielding the therapeutic compound in some way.

Once cellular uptake is achieved, the oligonucleotide must then be targeted successfully to the desired intracellular location. This will be the nucleus for a reagent directed against DNA, and it might be the nucleus or endoplasmic reticulum for reagents directed against RNA. Such targeting is not a simple matter. Most extracellular macromolecules taken up by cells are automatically targeted to the lysosome where they are destroyed. This natural pathway must be interfered with to successfully deliver a nucleic acid elsewhere. There is no doubt that one should be able to do this by exploiting the same sorts of processes that various viruses use to enter cells and infect the nucleus or the cytoplasm. However, many of these processes are not yet well understood, and we may have to learn

much more about them before successful oligonucleotide delivery mechanisms can be created.

Within the cell, or in intercellular ﬂuids, a plethora of agents exist that can destroy or inactivate foreign nucleic acids. This is not surprising. Such agents must have evolved as antiviral defense mechanisms. To circumvent the action of these agents, oligonucleotide

drugs would either have to be introduced in large quantities or rendered resistant to a variety of nucleases and other enzymes of nucleic acid metabolism. For example, antisense messenger RNAs have been proposed as therapeutic agents to interfere with the translation of an unwanted message, arising perhaps from a virus or a tumor cell. These would be expected to act by binding to the normal mRNA and inactivating it for translation by occlusion or by a more active destructive process. The difﬁculty in vivo is the presence of

substantial amounts of RNA helicase activity. This is an enzyme that speciﬁcally recognizes double-stranded RNAs and unwinds the double helix. To be an effective drug under most circumstances, the backbone or bases of an antisense RNA will have to be altered so

that this molecule is no longer recognized by RNA helicases.
Given the	constrants mentioned above, the ideal oligonucleotide	drug is	probably
likely to have an	altered backbone to render it immune to normal nucleases	and other	en-

zymes and to increase its binding afﬁnity to natural nucleic acids. Compounds with uncharged backbones like PNAs seem particularly attractive in this regard. It may also be desirable to equip potential oligonucleotide-analog drugs with additional chemical func-

Figure 14.50	A potential	oligonucleotide drug, designed by Claude Helene, that can bind to a
DNA duplex and	permanently inactivate	it by photocrosslinking. For examples of results with this

kind of approach, see Giovannangeli et al. (1992).

			SOURCES AND ADDITIONAL		READINGS		523
tionalities in order to further enhance their binding and effectiveness at inactivating the
cellular target. An interesting example of such a potential drug is shown in Figure 14.50.
Designed by Claude Helene, this compound consists of a triplex-forming oligonucleotide
attached to a psoralen by a ﬂexible chain. The psoralen enhances				binding to DNA du-
plexes because it is an intercalator. More importantly, psoralen is a DNA photocrosslink-
ing, so near-UV irradiation, after formation of the triplex, results in irreversible crosslink-
ing of the target DNA duplex. This procedure has			been demonstrated to	work effectively
in cells. It may be a prototype of the sorts of materials that we will eventually see in ac-
tual therapeutic use.
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