Diagnostic Use of RNA Replication in Infectious Diseases 
zyme ligase is still relatively inefficient. (About 
25 percent of the ligation-competent binary 
probe molecules are joined in a 1-hour incuba- 
tion.) An actual assay would require a ligation 
efficiency closer to 85 percent. Fortunately, re- 
cent developments in RNA biochemistry suggest 
a possible solution to this problem. 
Directed Evolution of Ribozymes 
We propose to develop more-efficient ribo- 
zyme ligases for our assays by directed evolution 
in vitro. Rachel Green, in Jack Szostak's labora- 
tory, has selected novel ribozymes with altered 
catalytic efficiency by Darwinian selection in a 
test tube. A Darwinian selection experiment be- 
gins with the synthesis of a large number of DNA 
molecules, each containing the sequence of a ri- 
bozyme. Known as a mutant pool, the population 
of molecules contains point mutations generated 
during chemical synthesis. Each molecule con- 
tains just a few mutations, located at random po- 
sitions in the sequence, and thus the population 
contains over a trillion (10'^) variants. The en- 
zyme T7 RNA polymerase is then used to generate 
RNA copies of the DNA, creating over a trillion 
dififerent mutant ribozymes. These are briefly in- 
cubated under RNA ligation conditions, where 
they are given the opportunity to catalyze a chem- 
ical reaction that joins them to a special piece 
of RNA. 
Those ribozymes that succeed in carrying out 
the ligation reaction are chosen as survivors in a 
subsequent step, while those that fail to partici- 
pate in catalysis are lost by dilution. Survivors are 
allowed to increase in number by reverse tran- 
scription (which converts RNA into DNA), fol- 
lowed by PGR amplification of the DNA. This se- 
ries of steps effectively rewards all competent, 
efficient ribozyme mutants by reconverting them 
into DNA and copying them many times. 
At this point the DNA is transcribed again into 
RNA, and a second cycle of Darwinian selection 
begins, exactly as above. After four cycles of se- 
lection, a relatively small number of mutant ribo- 
zyme sequences will be present in the DNA popu- 
lation, instead of the original millions. These 
molecules are the fittest: they were able to cata- 
lyze ligation reactions in all four rounds of selec- 
tion. The molecules are then sequenced in order 
to compare them to the original parental 
ribozyme. 
Using the methods outlined above, Rachel 
Green isolated a number of interesting mutant 
ribozymes that display high efficiency in a spe- 
cific type of ligation reaction. The experiments 
used to generate these ribozymes mimic natural 
variation and selection. While it takes nature 
hundreds, thousands, or even millions of years to 
evolve better enzyme catalysts, RNA enzymes can 
be improved in the laboratory by a Darwinian se- 
lection process in a matter of months. The term in 
vitro genetics has been coined to describe these 
man-made selection schemes. We are working on 
improvements that should enable us to generate 
ribozyme ligases better suited to our assays, in 
order to improve the process of joining RNA bi- 
nary probes. 
Multiplexed Diagnostic Assays 
Epidemiological monitoring of infectious dis- 
eases is a complex undertaking in a developing 
country like Mexico. Diseases must be monitored 
both in large, modern urban settings, like Mexico 
City, and in sparsely populated, rural areas like 
the Ghiapas countryside. The list of diseases caus- 
ing significant mortality and morbidity includes 
AIDS, malaria, amebiasis, tuberculosis, hepatitis, 
typhoid fever, diverse intestinal infections, and 
more recently cholera. There is a need for devel- 
oping accurate, low-cost technologies to detect 
pathogens in the field, and also a need for ex- 
tremely efficient, high-tech tools for the epidemi- 
ological laboratory and the blood supply centers. 
A valuable epidemiological tool would be a diag- 
nostic assay capable of detecting any of a number 
of pathogens in a single clinical sample. Such a 
tool is known as a multiplex assay. 
Under the auspices of the Rockefeller Founda- 
tion and the World Health Organization, our insti- 
tute has established a collaborative research pro- 
gram with Stanford University School of Medicine 
to develop and implement state-of-the-art tech- 
niques for epidemiological assessment in Mex- 
ico. The Stanford group is headed by Gary 
Schoolnik from the Division of Geographic Medi- 
cine and HHMI. Using a multiplex PGR assay, 
they have conducted a series of experiments dem- 
onstrating the simultaneous detection of any of 
several bacterial pathogens in diarrheal stool. 
Assays based on RNA amplification could be 
multiplexed by using a combination of binary 
probes. Each binary probe pair would contain 
RNA sequences designed for binding to an indi- 
vidual pathogen, and as many as 10 probe pairs 
could be mixed together in a single assay. Eventu- 
ally multiplexed assays could be automated, and 
cost-effectiveness would be very favorable be- 
cause several candidate infectious agents would 
be assayed at once. 
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