Diagnostic Use of RNA Replication 
in Infectious Diseases 
Paul M. Lizardi, Ph.D. — International Research Scholar 
Dr. Lizardi is Professor of Biochemistry at the Biotechnology Institute, National Autonomous University 
of Mexico, Cuernavaca. He received his Ph.D. degree from the Rockefeller University and conducted 
postdoctoral research in embryology with Donald Brown at the Carnegie Institution of Washington, 
Baltimore. After serving as Associate Professor at Rockefeller as an Andrew Mellon Foundation fellow. 
Dr. Lizardi spent a sabbatical year at Massachusetts General Hospital, Boston, and held a visiting 
professorship in genetics at Harvard Medical School. 
INFECTIOUS diseases are frequently managed 
by health professionals without definitive 
identification of the pathogen. Classical ap- 
proaches to diagnosis, such as direct microscopic 
observation, cultivation, or infection of suscepti- 
ble hosts, are often too slow or cumbersome for 
routine medical care. Modern laboratory tests 
based on the use of antibodies provide effective 
tools for diagnosis of a number of infectious dis- 
eases, but most antibody tests are designed to de- 
tect the presence of a host immune response 
rather than the pathogen itself. Thus there is a 
need for techniques permitting the rapid and reli- 
able detection of infectious agents so that epide- 
miological monitoring and patient management 
can be more effective. 
A direct way to detect a pathogen is to identify 
its genetic material, which invariably contains 
unique sequence patterns. However, the genetic 
material is usually so minute in a biological sam- 
ple that its detection presents an extraordinary 
technical challenge. Molecular biologists have 
been up to the task, and techniques developed 
recently permit the generation of millions of cop- 
ies of DNA or RNA segments in the test tube by a 
process of exponential amplification. The best- 
known amplification method is the polymerase 
chain reaction (PCR) , in which DNA strands are 
sequentially separated by heating and then cop- 
ied with DNA polymerase. The PCR limit of de- 
tection is about 50 molecules of target, which is 
over 100,000 times as sensitive as a typical en- 
zyme-linked immunoassay. 
Amplified RNA Binary Probes 
My laboratory is developing alternative ampli- 
fication methods for the detection of RNA or DNA 
in biological samples. The work is being carried 
out in close collaboration with Fred Kramer at the 
Public Health Research Institute in New York 
City and Jack Szostak at Massachusetts General 
Hospital in Boston. Our methods exploit several 
interesting properties of RNA molecules: 
• Relatively short molecules of RNA (15-30 
nucleotides) have long been known to form 
stable helical structures when bound to perfectly 
complementary single strands of DNA or RNA. 
The thermodynamic stability of these helical 
complexes distinguishes them from similar com- 
plexes containing mismatched bases. Hence one 
can design an RNA probe that will bind very 
strongly, and uniquely, to a segment of the DNA 
or RNA of an infectious agent in a biological 
sample. 
• RNA probe molecules can be joined to other 
RNA molecules in a target-dependent manner. 
That is, joining will only take place if the mole- 
cules are aligned on a complementary target 
strand that serves as a guide for the joining event. 
We catalyze the joining by an enzyme called a 
ribozyme ligase, isolated in Jack Szostak's labora- 
tory. The ligase is itself an RNA, derived from a 
naturally occurring intron called group I, origi- 
nally discovered in Tetrahymena thermophyla 
by Thomas Cech (HHMI, University of Colorado 
at Boulder) . 
• A specific class of molecules known as repli- 
catable RNAs can be produced exponentially 
under natural conditions, so that millions of cop- 
ies are generated in minutes. An enzyme called 
RNA replicase catalyzes reactions in which RNA 
single strands, parent and daughter, are forced 
apart during synthesis, in contrast to DNA-depen- 
dent reactions, in which the two strands remain 
annealed. The best characterized of these en- 
zymes is Q-beta replicase. We have shown that 
replicatable RNAs, in the presence of this en- 
zyme, can harbor RNA probe inserts without loss 
of replicative efficiency. 
We have devised schemes in which probe bind- 
ing, RNA joining, and exponential replication of 
the joined RNA are used in an assay to detect the 
presence of a specific target sequence from an 
infectious agent. Probes that are not joined are 
not replicated. The signal in these assays is repli- 
cated RNA, generated by joined probes and 
readily quantitated by fluorescence staining. The 
intensity of the signal is proportional to the num- 
ber of targets present in the original sample. 
However, a number of technical problems re- 
main to be solved before optimal sensitivity and 
specificity can be achieved in these assays. For 
example, the ligation step involving the ribo- 
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