Nucleic acid process containing improved molecular switch

A probe for the detection of a nucleic acid target sequence containing a molecular switch comprising three essential elements: a probe sequence of 20-60 nucleotides surrounded by switch sequences of 10-40 nucleotides which are complementary to each other, wherein the state of the switch is useful for selectively generating a detectable signal if the probe is hybridized to a target; also, assays and kits utilizing such probes.

This invention relates to the field of bioassays that involve nucleic acid 
hybridization probes. These bioassays are useful for the detection of 
specific genes, gene segments or RNA molecules. The assays are useful 
clinically, for, e.g., tissue, blood and urine samples, as well as in food 
technology, agriculture, and biological research. 
BACKGROUND OF THE INVENTION 
The use of nucleic acid hybridization probes for bioassays is well known. 
One of the early papers in the field directed to assays for DNA is 
Gillespie, D. and Spiegelman, S., A Quantitative Assay for DNA-RNA Hybrids 
with DNA Immobilized on a Membrane, J. Mol. Biol. 12:829-842 (1965). In 
general terms such an assay involves separating the nucleic acid polymer 
chains in a sample, as by melting, fixing the separated DNA strands to a 
nitrocellulose membrane, and then introducing a probe sequence which is 
complementary to a unique sequence of the material being sought, the 
"target" material, and incubating to hybridize probe segments to 
complementary target segments, if targets are present. Non-hybridized 
probes are removed by known washing techniques, and then the amount of 
probe remaining is determined by one of a variety of techniques outlined 
below which provides a measurement of the amount of targets in the sample. 
A more recently developed form of bioassay that uses nucleic acid 
hybridization probes involves a second probe, often called a "capture 
probe." Ranki, M., Palva, A., Virtanen M., Laaksonen, M., and Soderlund, 
H., Sandwich Hybridization as a Convenient Method for the Detection of 
Nucleic Acids in Crude Samples, Gene 21:77-85 (1983); Syvanen, A.-C., 
Laaksonen, M., and Soderlund, H., Fast Quantification of Nucleic Acid 
Hybrids by Affinity-based Hybrid Collection, Nucleic Acids Res. 
14:5037-5048 (1986). A capture probe contains a nucleic acid sequence 
which is complementary to the target, preferably in a region near the 
sequence to which the radioactively labeled probe is complementary. The 
capture probe is provided with a means to bind it to a solid surface. 
Thus, hybridization can be carried out in solution, where it occurs 
rapidly, and the hybrids can then be bound to a solid surface. One example 
of such a means is biotin. Langer, P. R., Waldrop, A. A. and Ward, D. C., 
Enzymatic Synthesis of Biotin-Labeled Polynucleotides: Novel Nucleic Acid 
Affinity Probes, Proc. Natl. Acad. Sci. USA 78:6633-6637 (1981). Through 
biotin the capture probe can be bound to streptavidin covalently linked to 
solid beads. 
The present invention is directed to the methods and means, including 
assays and pharmaceutical kits containing requisite reagents and means, 
for detecting in an in vitro or ex vitro setting the presence of nucleic 
acid species. 
It is a goal in this art to detect various nucleic acid sequences in a 
biological sample, in which the said sequences, as so-called target 
sequences, are present in small amounts relative to its existence amongst 
a wide variety of other nucleic acid species including RNA, DNA or both. 
Thus, it is desirable to detect the nucleic acid encoding polypeptides 
that may be associated with pathological diseases or conditions, such as, 
for example, RNA of the human immunodeficiency virus. In addition to the 
detection of nucleic acids encoding the proteins of such viral particles, 
it is desirable to detect other nucleic acids characteristic of a 
pathological disease or condition such as a defective gene, as in the case 
of hemophilia. It is also desirable to detect other nucleic acids whose 
presence in the sample indicates that the organism is able to resist the 
action of a drug, such as an antibiotic. 
Several approaches have been used for detecting the probe. One is to link a 
readily detectable reporter group to the probe. Examples of such reporter 
groups are fluorescent organic molecules and .sup.32 P-labeled phosphate 
groups. These detection techniques have a practical limit of sensitivity 
of about a million targets per sample. 
A second approach is to link a signal generating system to the probe. 
Examples are enzymes such as peroxidase. Probes are then incubated with a 
color-forming substrate. Leary, J. J., Brigati, D. J. and Ward, D. C., 
Rapid and Sensitive Colorimetric Method for Visualizing Biotin-Labeled DNA 
Probes Hybridized to DNA or RNA Immobilized on Nitrocellulose: Bio-Blots, 
Proc. Natl. Acad. Sci. USA 80:4045-4049 (1983). Such amplification reduces 
the minimum number of target molecules which can be detected. As a 
practical matter, however, nonspecific binding of probes has limited the 
improvement in sensitivity as compared to radioactive tagging to roughly 
an order of magnitude, i.e., to a minimum of roughly 100,000 target 
molecules. 
Yet another approach is to make many copies of the target itself by in vivo 
methods. Hartley, J. L., Berninger, M., Jessee, J. A., Bloom, F. R. and 
Temple, G. S., Bioassay for Specific DNA Sequences Using a Non-Radioactive 
Probe, Gene 49:295-302 (1986). This can also be done in vitro using a 
technique called "polymerase chain reaction" (PCR). This technique was 
reported in Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. 
T., Erlich, H. A., and Arnheim, N., Enzymatic Amplification of Beta-globin 
Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle 
Cell Anemia, Science 230:1350-1354 (1985); Saiki, R. K., Gelfand, D. H. 
Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and 
Erlich, H. A., Primer-directed Enzymatic Amplification of DNA With a 
Thermostable DNA Polymerase, Science 239:487-491 (1988); Erlich, H. A., 
Gelfand, D. H., and Saiki, R. K., Specific DNA Amplification, Nature 
331:461-462 (1988), and Mullis et al., European Patent Application 
Publication Nos. 200362 and 201184 (see also U.S. Pat. Nos. 4,683,195 and 
4,683,202). In PCR, the probe is complementary only to the beginning of a 
target sequence but, through an enzymatic process, serves as a primer for 
replication of an entire target. Each repetition of the process results in 
another doubling of the number of target sequences until a large number, 
say, a million copies, of the target are generated. At that point 
detectable probes, e.g., radioactively labeled probes, can be used to 
detect the amplified number of targets. The sensitivity of this method of 
target amplification is generally limited by the number of "false positive 
signals" generated, that is, generated segments that are not true copies 
of the target. Nonetheless, this method is quite sensitive. The procedure 
requires at least two nucleic acid probes and has three steps for a single 
cycle. This procedure is cumbersome and not always reliable. 
Yet another method for amplification is to link to the probe an RNA that is 
known to be copied in an exponential fashion by an RNA-directed RNA 
polymerase. An example of such a polymerase is bacteriophage Q-beta 
replicase. Haruna, I., and Spiegelman, S., Autocatalytic Synthesis of a 
Viral RNA In Vitro. Science 150:884-886 (1965). Another example is brome 
mosaic virus replicase. March et al., POSITIVE STRAND RNA VIRUSES Alan R. 
Liss, New York (1987). In this technique, the RNA serves as a template for 
the exponential synthesis of RNA copies by a homologous RNA-directed RNA 
polymerase. The amount of RNA synthesized is much greater than the amount 
present initially. This amplification technique is disclosed in Chu, B. C. 
F., Kramer, F. R., and Orgel, L. E., Synthesis of an Amplifiable Reporter 
RNA for Bioassays, Nucleic Acids Res. 14:5591-5603 (1986); Lizardi, P. M., 
Guerra, C. E., Lomeli, H., Tussie-Luna, I. and Kramer, F. R., Exponential 
Amplification of Recombinant-RNA Hybridization Probes, Bio/Technology 
6:1197-1203 (October 1988), which is incorporated herein by reference and 
is attached hereto in manuscript form [hereinafter referred to as "Lizardi 
et al."]; published European Patent Application 266,399 (EP Application 
No. 87903131.8). After non-hybridized probes are removed by washing, the 
RNA polymerase is used to make copies of the replicatable RNA. According 
to the disclosure of published European Patent Application No. 266,399, 
replication of the RNA may take place while the RNA is linked to the 
probe. Alternatively, the replicatable RNA may be separated from the 
remainder of the probe prior to replication. That application also 
discloses a variety of chemical links by which a probe sequence can be 
joined to a replicatable RNA. In addition, it discloses that the probe 
sequence may be part of a replicatable RNA, as described in Miele, E. A., 
Mills, D. R., and Kramer, F. R., Autocatalytic Replication of a 
Recombinant RNA, J. Mol. Biol. 171:281-295 (1983). That European 
application also discloses that such recombinant RNAs must be able to 
hybridize specifically with the target sequence as well as to retain their 
ability to serve as a template for exponential replication by an 
appropriate RNA-directed RNA polymerase, as is demonstrated in the results 
obtained by Lizardi et al., supra. 
Replication of RNA, as opposed to target amplification using PCR, can be 
done in a single step. In that step one can synthesize as many as a 
billion copies of the replicatable RNA that was joined to the probe in as 
little as twenty minutes, which theoretically could lead to detection of a 
single target molecule. However, in practice the sensitivity of this type 
of probe replication is limited by the persistence of nonspecifically 
bound probes. Nonspecifically bound probes will lead to replication just 
as will probes hybridized to targets. 
A major problem in the implementation of bioassays that employ 
hybridization technology coupled to signal amplification systems is the 
background signal produced by nonspecifically bound probe molecules. These 
background signals introduce an artificial limit on the sensitivity of 
bioassays. In conventional bioassays this problem is sometimes alleviated 
by the utilization of elaborate washing schemes that are designed to 
remove nonspecifically bound probes. These washing schemes inevitably add 
to the complexity and cost of the assay. 
As a means to reduce the background noise level of assays employing probes 
linked to replicatable RNA by covalently joined linking moieties, European 
Patent Application No. 266,399 discloses what it refers to as "smart 
probes," that is, probes whose linked RNA is said not to serve as a 
template for replication unless and until the probe has hybridized with a 
target sequence. In that application two embodiments are disclosed for 
smart probes. 
In a first embodiment in that application, the smart probe comprises a 
probe portion consisting of about 75-150 deoxynucleotides, made by in 
vitro or in vivo methods known in the art. The smart probe also comprises 
a recombinant, replicatable RNA containing an inserted heterologous 
sequence of about 10-30 nucleotides, made by, e.g., the method of Miele, 
E. A., Mills, D. R., and Kramer, F. R., Autocatalytic Replication of a 
Recombinant RNA. J. Mol. Biol. 171:281-295 (1983). Joining those two 
portions at their 5' ends is a linking moiety of the formula 
--O(PO.sub.2)NH(CH.sub.2).sub.a SS(CH.sub.2).sub.b NH(PO.sub.2)O--, where 
a and b are each 2 to 20. Furthermore, the sequence at the 3' end of the 
DNA portion of the smart probe is capable of being (and very likely to be) 
hybridized to the heterologous sequence of the RNA portion of the smart 
probe. The enzyme ribonuclease H is said to be capable of cleaving the RNA 
portion of smart probes which have not hybridized to targets, but not be 
capable of cleaving the RNA portion of smart probes which have hybridized 
to targets, because when the probe sequence in the DNA portion of a smart 
probe is bound to its target, it is said to be incapable of also being 
hybridized to the heterologous sequence in the RNA portion of the smart 
probe, thereby providing a way to eliminate nonspecifically bound probes 
prior to amplification. Amplification via RNA replication is said to 
optionally include the preliminary step of cleaving the disulfide bond in 
the linking moiety. 
In that embodiment, cleavage of probes not hybridized to targets is said to 
be possible for ribonuclease H, because the 3' end of the DNA portion of 
the smart probe (which contains the probe sequence) is hybridized to the 
recombinant replicatable RNA portion, presumably thereby providing a site 
wherein ribonuclease H can cleave the RNA and render it inoperative as a 
template for amplification by an RNA-directed RNA polymerase. 
In the other embodiment of a smart probe disclosed in published European 
Patent Application 266,399, there is a probe portion, a linking moiety, 
and a replicatable RNA portion, linked as described above. Here, however, 
the probe portion comprises not only a probe segment of 50-150 
nucleotides, but also additional segments, called "clamp" segments, on 
either side of it, that is, a 5'-clamp segment and a 3'-clamp segment, 
each of about 30-60 nucleotides. Each clamp segment is said to hybridize 
with a segment of the replicatable RNA portion, rendering the RNA inactive 
as a template for replication, unless and until the probe is hybridized 
with a target. That hybridization causes the clamps to release, thereby 
rendering the RNA replicatable, either directly or after optional cleavage 
of the disulfide bond. 
The smart probes disclosed in published European Patent Application No. 
266,399 comprise a somewhat complicated linking moiety containing a weakly 
covalent and rather easily dissociable disulfide linkage. Disulfide bonds 
readily dissociate under reducing conditions. The two versions of smart 
probes disclosed in that application rely on distant intramolecular 
interactions to render the probe smart. This is a disadvantage which makes 
such probes difficult to design, particularly since distant interactions 
are not well understood. The second version, reported above, has a further 
complication that it utilizes two distant clamps which must displace a set 
of relatively strong neighboring complements. And, the design depends on 
both distant clamps hybridizing or none, which makes design very 
difficult. 
An object of the present invention is a simple molecular allosteric switch 
that renders a nucleic acid hybridization probe smart, that is, capable, 
in an appropriate assay, of generating a signal only if the probe is 
hybridized to a target sequence. 
It is a further object of this invention to couple the activity of a signal 
generating system to the state of such a switch. 
It is yet another object of this invention to develop probes containing 
such an allosteric switch that are linked to any of a number of different 
signal generating systems whose activity is dependent on the state of the 
switch. 
It is another object of this invention to develop assays of improved 
sensitivity that utilize the above constructs, as well as kits for 
performing such assays. 
SUMMARY OF THE INVENTION 
The present invention is predicated on a simple molecular allosteric switch 
that works on the principle that when a nucleic acid double helix is 
formed between a relatively short probe sequence and a target sequence, 
the ends of the double helix are necessarily located at a distance from 
each other due to the rigidity of the double helix. That rigidity is 
discussed in detail in Shore, D., Langowski, J. and Baldwin, R. L., DNA 
Flexibility Studied by Covalent Closure of Short Fragments into Circles, 
Proc. Natl. Sci. USA 78:4833-4837 (1981); and Ulanovsky, L., Bodner, M., 
Trifonov, E. N., and Choder, M., Curved DNA: Design, Synthesis, and 
Circularization, Proc. Natl. Acad. Sci. USA 83:862-866 (1986). 
This invention involves the use of a nucleic acid hybridization probe 
comprising at least the following essentials: a probe sequence of 
approximately 15-115 nucleotides in length surrounded on both sides by 
complementary nucleic acid sequences which are considerably shorter than 
the probe sequence, preferably not greatly in excess of one-half the 
length of the probe sequence. This combination of three sequences forms a 
simple molecular allosteric switch. When not hybridized to a target 
sequence, the switch sequences are hybridized to each other, which we 
refer to as a closed switch. When the probe sequence hybridizes to a 
predetermined complementary target sequence for which the probe is 
designed, the strong interaction between the probe and target sequences to 
form a rigid double helix necessarily results in the dissociation of the 
switch sequences, which we refer to as an open switch. In the open 
configuration, the switch sequences are unable to interact with each 
other. 
The invention comprises probe molecules containing the above switch wherein 
one of the switch sequences, or both switch sequences in combination, 
comprise a biologically functional nucleic acid moiety useful for 
selectively generating a detectable signal indicative of the hybridization 
of the probe with its predetermined target sequence. 
The invention further comprises bioassay methods which take advantage of 
the allosteric change in the switch sequences in the above probe molecules 
to generate a detectable signal indicative of the hybridization of the 
probe with its predetermined target sequence. The assay may be qualitative 
(a qualitative demonstration) or quantitative (a quantitative 
determination). It may include amplification, which may be linear or 
exponential in nature. 
The invention also includes kits of reagents and macromolecules for 
carrying out the above bioassays.

DETAILED DESCRIPTION OF THE INVENTION 
Shown in FIG. 1 is a probe, or probe portion, comprising the three 
essential ingredients of a probe according to this invention, namely, a 
probe sequence and complementary switch sequences on both sides of the 
probe. As depicted in FIG. 1, the switch is closed. FIG. 2 is the same 
probe or probe portion in its open state. 
Referring to FIG. 1, probe sequence 1 is a nucleic acid probe sequence 
extending from its 5' side 2 to its 3' side 3. Immediately adjacent to the 
5' side of the probe sequence is a acid first switch sequence 4. 
Immediately adjacent to the 3' side of the probe sequence is a nucleic 
acid second switch sequence 5. Switch sequences 4 and 5 are complementary 
and hybridize to each other via hydrogen bonds 7, forming the stem 6 of a 
"hairpin" secondary structure. Referring to FIG. 2, probe sequence 1 is 
hybridized via hydrogen bonds 9 to its predetermined target sequence 8. 
Switch sequences 4 and 5 are apart and not interacting with one another. 
The probe may be RNA or DNA. The probe sequence 1 must be of sufficient 
length to ensure a very specific interaction with its predetermined target 
sequence 8. It should be at least about 15 nucleotides in length, although 
we prefer that it be at least about 20 nucleotides in length. 
The probe sequence 1 should be short enough to ensure that the sides 2, 3 
of probe sequence 1, when hybridized to the target sequence 8 (FIG. 2) are 
physically prevented by the rigidity of the hybridized region between 
sides 2 and 3 from approaching each other within a distance that would 
permit switch sequences 4, 5 from interacting with each other. In other 
words, when the probe sequence is hybridized, the switch sequences 
necessarily are not hybridized to each other. An additional force helps to 
drive the transition to an open state, namely, torsional forces tending to 
unwind stem 6 when the hybridized region shown in FIG. 2 forms a double 
helix. In practice, the probe sequence is no longer than about 100 
nucleotides. We prefer that the probe sequence be 20-60 nucleotides in 
length, and most preferably, about 30 nucleotides in length. 
The switch sequences are related to the length of the probe sequence. Most 
preferably, we prefer that the length of the switch sequences be no more 
than half the length of the probe sequence. The switch sequences should be 
at least about 10 nucleotides in length to permit formation of a stable 
stem 6. Turner, D. H., Sugimoto, N., Jaeger, J. A., Longfellow, C. E., 
Freier, S. M. and Kierzek, R., Improved Parameters for Prediction of RNA 
Structure, Cold Spring Harbor Symp. Quant. Biol. 52:123-133 (1987). The 
length of switch sequences for certain embodiments described below must 
also be sufficiently long to contain necessary functional sequences. We 
prefer switch sequences of about 10-30 nucleotides. 
Thus, this invention provides a probe for the detection of a predetermined 
nucleic acid target sequence comprising 
a. a probe sequence of from about 20 to about 60 nucleotides, having a 5' 
side and a 3' side, which probe sequence is complementary to said target 
sequence, 
b. a first switch sequence of from about 10 to about 40 nucleotides at the 
5' side of the probe sequence, 
c. a second switch sequence of from about 10 to about 40 nucleotides at the 
3' side of the probe sequence, said second switch sequence being 
complementary to said first switch sequence, 
wherein, when the probe sequence is not hybridized with said target 
sequence, the first switch sequence is hybridized to the second switch 
sequence but, when the probe sequence is hybridized with said target 
sequence, thereby forming a double helix, the rigidity of said double 
helix prevents the first switch sequence from hybridizing to the second 
switch sequence, and wherein said first switch sequence, said second 
switch sequence, or said first and second switch sequences in combination, 
comprise a biologically functional nucleic acid moiety useful for 
selectively generating a detectable signal if the probe sequence is 
hybridized with said target sequence. 
In designing a probe according to the invention, attention should be paid 
to the relative strengths of the open switch hybrid (FIG. 2) as compared 
to the closed switch hybrid (FIG. 1) under the assay conditions to be 
used: the former should be greater. There are assay conditions, however, 
in which the strengths of hybrids is only length-dependent. Wood, W. I., 
Gitschier, J., Lasky, L. A., and Lawn, R. M., Base Composition-independent 
Hybridization in Tetramethylammonium Chloride: A Method for 
Oligonucleotide Screening of Highly Complex Gene Libraries, Proc. Natl. 
Acad. Sci. USA 82:1585-1588 (1985). 
Switch design can be readily tested by digesting probes or probe portions 
(FIGS. 1, 2) with appropriate nucleases before and after hybridization to 
model nucleic acids containing target sequences and then analyzing the 
digestion products by polyacrylamide gel electrophoresis. This will be 
apparent to those skilled in the art and will not be described further. 
To help drive the transition from closed to open, one may take advantage of 
the principle of strand displacement to provide an additional force. 
Green, C., and Tibbetts, C., Reassociation Rate Limited Displacement of 
DNA Strands by Branch Migration, Nucleic Acids Res. 9:1905-1918 (1981). 
This may be accomplished by overlapping a switch sequence with a probe 
sequence, which means that at least one nucleotide of the switch sequence 
is also a nucleotide of the probe sequence. 
While the switch sequences must be adjacent to the probe sequence, they 
need not be immediately adjacent to it. A few nucleotides may separate the 
switch sequences from the probe sequences, but not so many that the 
functioning of the switch is materially affected, as those skilled in the 
art will readily appreciate. 
Probe molecules of this invention, containing the switch described above, 
can be of diverse design and still take advantage of the allosteric change 
that accompanies probe sequence hybridization (FIG. 2) in signal 
generation. 
For example, a switch sequence may, by virtue of the conformation it 
assumes in the open state, enable an interaction with another 
macromolecule, or even a different portion of the same molecule, which is 
required for the generation of a detectable signal. In Example I below, 
the second switch sequence, in the open state, is able to hybridize with a 
complementary nucleic acid strand. In Example III, the first switch 
sequence, in the open state, forms a hairpin structure that enables it to 
bind specifically to a viral protein. In Example IV, the second switch 
sequence, in the open state, is able to interact with an 
oligoribonucleotide or with an oligodeoxyribonucleotide. In Example V, the 
first switch sequence, in the open state, assumes a structured 
conformation that enables it to interact with a relatively distant region 
of the same probe molecule. 
It is also possible to do the reverse. In Example II, the switch sequences 
can bind to a specific enzyme only when they are in the closed state. 
Signal generation using probe molecules and methods of this invention may 
vary widely. The state of the simple allosteric switch governs signal 
generation, which means that there is no signal generated unless the probe 
sequence hybridizes with its target sequence. We prefer signal generating 
systems that involve amplification, particularly exponential 
amplification, to increase sensitivity 
The Examples which follow illustrate a few of the myriad variations 
involving amplification. They all utilize the exponential replication of a 
replicatable RNA by an RNA-directed RNA polymerase to generate a readily 
detectable signal. The Examples utilize MDV-1 RNA, which is described in 
Kacian, D. L., Mills, D. R., Kramer, F. R., and Spiegelman, S., A 
Replicating RNA Molecule Suitable for a Detailed Analysis of Extracellular 
Evolution and Replication, Proc. Nat. Acad. Sci. USA 69:3038-3042 (1972). 
The Examples also use Q-beta replicase, which is the specific polymerase 
for replicating MDV-1 RNA. Q-beta replicase is described in Haruna, I. and 
Spiegelman, S., Specific Template Requirements of RNA Replicases, Proc. 
Nat. Acad. Sci. USA 54:579-587 (1965). Any replicatable RNA and its 
homologous replicase could, of course, be used. Other useful signal 
generating systems could employ enzymes, enzyme cofactors, ribozymes, DNA 
and RNA sequences required for biological activity (e.g., promoters, 
primers, or linkers required for the ligation of plasmids used to 
transform bacteria). Detectable signals are diverse and include, for 
example, radiation, light absorption, fluorescence, mass increase, and the 
presence of biologically active compounds. 
Assay techniques which can be used to detect hybridized probes of this 
invention are also diverse. In the following Examples, synthesis of a 
replicatable RNA is used to signal that hybridization of the probe 
sequence has occurred. The signal generating systems illustrated in the 
Examples fall into three broad classes: in Examples II-III, the switch is 
incorporated within a replicatable RNA; in Examples IV-V, a replicatable 
RNA sequence is joined with a probe portion but can only be replicated 
after cleavage, which is dependent upon the presence of an open switch; 
and in Example I, the transcription of a replicatable RNA from a template 
added after hybridization, can only occur when an open switch sequence 
forms a part of a functional promoter of transcription. 
Each of the specific embodiments set forth in the accompanying Examples 
satisfies the objective of generating a signal only if the probe is 
hybridized to a target sequence. Either the biological activity of the 
signal generating systems illustrated depends strictly on the state of the 
switch, or the state of the switch provides a means for rendering 
nonspecifically bound probes unable to generate signals, or the state of 
the switch provides a means for separating hybridized probes from 
nonspecifically bound probes. Thus, each of the specific embodiments 
markedly reduces the background caused by nonspecifically bound probes, 
thereby significantly improving the sensitivity of the assays, including 
assays which include amplification. 
EXAMPLE I 
In this example, the probe is a single DNA strand designed to contain three 
sequences: a probe sequence approximately 34 nucleotides in length; a 
first switch sequence of about 17 nucleotides immediately adjacent to the 
5' side of the probe sequence; and a second switch sequence of about 17 
nucleotides immediately adjacent to the 3' side of the probe sequence. The 
switch sequences are designed to be complementary to one another. When 
hybridized to each other, the hybridized switch sequences comprise a 
promoter for the DNA-directed RNA polymerase, bacteriophage T7 RNA 
polymerase. In this application, we refer to the first switch sequence as 
a "promoter sequence" and the second switch sequence as a 
"promoter-complement" sequence. In this example, the switch sequences 
comprise the ends of the probe molecule. The design of promoter and 
promoter-complement sequences is according to Osterman, H. L. and Coleman, 
J. E., "T7 Ribonucleic Acid Polymerase-Promoter Interactions," 
Biochemistry 20:4885-4892 (1981). The particular promoter-complement 
sequence we have chosen to work with is TAATACGACTCACTATA. 
The probe molecule, including a probe sequence complementary to a 
predetermined target sequence, can be made by chemical synthesis of 
oligodeoxyribonucleotides using methods well known in the art, e.g., Gait, 
M. J., OLIGONUCLEOTIDE SYNTHESIS, IRL Press, Oxford, United Kingdom 
(1984). 
The probe of this example can be used to detect a DNA or RNA target 
sequence which is complementary to the probe sequence. The target sequence 
may be in a sample containing other, unrelated nucleic acids and other 
materials, for example, proteins. The probe may be used to detect a gene 
segment of an infectious agent (virus, bacterium, protozoan, etc.) in a 
clinical sample of, for example, human blood or urine. 
The target sequence must be exposed to the probe. This is done by 
techniques well known to the art. Commonly, but not necessarily, nucleic 
acid is isolated from a sample before the probe is added. 
The probe and the sample, which may contain nucleic acid target sequences, 
are next incubated under conditions, including time and temperature, 
appropriate to cause hybridization of probe sequences with target 
sequences. Appropriate conditions are well known in the art. For 
quantitative determination of the number of target sequences present, an 
amount of probe in excess, preferably in substantial excess, of the 
highest anticipated target amount should be used. If only a qualitative 
demonstration of the presence of target sequences is desired, a lesser 
amount of probe can be used. 
Probes hybridized to targets are separated from unbound probes by methods 
well known to the art, for example, through the use of capture probes. 
After separation, the treated sample will contain probes hybridized to 
targets (FIG. 2) and also nonspecifically bound probes. The two are not in 
the same form, however. In the hybridized probes the allosteric switches 
are open; that is, the switch sequences are not hybridized to each other. 
In the nonspecifically bound probes, however, the switch sequences remain 
hybridized to each other. 
Detecting those probes with open switches will now be described. This 
example includes amplification prior to detection. 
Referring to FIG. 3, the sample is incubated with a single-stranded DNA 
molecule 10 comprising a promoter sequence 11 and a template sequence 12 
for the transcription of a replicatable RNA. The promoter sequence 11 
allows hybridization via hydrogen bonds 13, under conditions known to the 
art, to the promoter-complement of the second switch sequence 5 of probes 
having open switches. Specifically, this DNA molecule consists of the 17 
deoxyribonucleotides of the promoter sequence (complementary to the 
promoter-complement set forth above) followed by the 244 
deoxyribonucleotides complementary to MDV-poly (+) RNA described in 
Lizardi et al., supra. This DNA molecule can be prepared by isolating one 
of the complementary strands of a suitable restriction fragment of a 
plasmid containing that sequence by methods known in the art. Maniatis, 
T., Fritsch, E. F., and Sanbrook, J., MOLECULAR CLONING: A LABORATORY 
MANUAL Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982). The 
suitable plasmid that we constructed contained (1) a unique restriction 
site (that is, one contained nowhere else in the plasmid) upstream from 
and close to the promoter, and (2) and Sma I restriction site at the end 
of the MDV-poly cDNA sequence distal to the promoter. 
Subsequently, the sample is incubated with commercially available cloned 
bacteriophage T7 RNA polymerase in order to synthesize about 50-200, or 
more, MDV-poly RNA transcripts for each open switch, using conditions 
known to the art. Milligan, J. F., Duncan, R. G., Witherell, G. W. and 
Uhlenbeck, O. C., Oligoribonucleotide Synthesis Using T7 RNA Polymerase 
and Synthetic DNA Templates, Nucleic Acids Research 15:8783-8798 (1987). 
Then, Q-beta replicase, an RNA-directed RNA polymerase, is added and 
incubated with the MDV-poly RNA transcripts, which are templates for that 
polymerase. We prepared Q-beta replicase by the method of Eoyang, L., and 
August, J. T., Q-beta RNA polymerase from phage Q-beta-infected E. coli. 
pp. 829-839. In: Procedures in Nucleic Acid Research, Volume 2. (Cantoni, 
G. L., and Davis, D. R., eds.). Harper and Row, New York (1971). 
Incubation is carried out under conditions suitable for exponential 
amplification of the transcripts. Kramer, F. R., Mills, D. R., Cole, P. 
E., Nishihara, T., and Spiegelman, S., Evolution in vitro: Sequence and 
Phenotype of a Mutant RNA Resistant to Ethidium Bromide. J. Mol. Biol. 
89:719-736 (1974). 
Detection of the exponentially amplified RNA can be done by any of a 
variety of physical and chemical means, as described earlier in this 
application. For a quantitative determination, the amount of RNA detected 
after a fixed time of incubation with the RNA-directed RNA polymerase is a 
measure of the number of target sequence present in the sample. 
EXAMPLE II 
Referring to FIG. 4, in this example, the probe is a replicatable 
recombinant RNA 14. Miele, E. A., Mills, D. R., and Kramer, F. R., 
Autocatalytic Replication of a Recombinant RNA, J. Mol. Biol. 171:281-295 
(1983). It may be prepared according to the method of Lizardi et al., 
supra. For purposes of preparing a probe according to this example, the 
heterologous sequence 15 contained within the replicatable recombinant RNA 
is designed to contain three sequences: a probe sequence 16 approximately 
46 nucleotides in length; a first switch sequence 17 of about 23 
nucleotides immediately adjacent to the 5' side of the probe sequence; and 
a second switch sequence 18 of about 23 nucleotides immediately adjacent 
to the 3' side of the probe sequence. The switch sequences are designed to 
form a double-stranded recognition site for Escherichia coli ribonuclease 
III when hybridized to each other. This recognition site will not be 
present when the switch sequences are not hybridized to each other. The 
particular recognition site we use is shown in FIG. 4 and is described by 
Rosenberg, M. and Kramer, R. A., Nucleotide Sequence Surrounding a 
Ribonuclease III Processing Site in Bacteriophage T7 RNA, Proc. Natl. 
Acad. Sci. USA 74:984-988 (1977). It can be made by transcription from a 
recombinant plasmid utilizing techniques described in Lizardi et al., 
supra. 
Exposure of the target sequence, hybridization of the probe with the target 
sequence, and separation from unbound probes, are as described in Example 
I. As shown in FIG. 5, probe sequence 16 of a hybridized probe 14 is 
hybridized to target sequence 8, thereby forcing apart switch sequences 
17, 18. 
The sample is then incubated with E. coli ribonuclease III under 
appropriate conditions known to the art to cleave all the nonspecifically 
bound probes (and any unbound probes which may remain), rendering them 
unable to serve as templates for exponential replication by Q-beta 
replicase. Nishihara, T., Mills, D. R., and Kramer, F. R., Localization of 
the Q-beta Replicase Recognition Site in MDV-1 RNA, J. Biochem. 93:669-674 
(1983). The ribonuclease III is then removed from the sample by methods, 
e.g., phenol extraction, well known in the art. 
We release the probe from the target sequence by a brief heating step, 
Lizardi et al. supra, although preliminary experiments have indicated that 
this step may be optional. 
Exponential replication of the probe by Q-beta replicase and detection 
proceed as described in Example I. 
EXAMPLE III 
In this example the probe 19 (FIG. 6) is a replicatable recombinant RNA as 
in Example II, except that the probe sequence 20 is about 38 nucleotides 
in length and that the complementary switch sequences 21, 22, of about 19 
nucleotides, are designed such that when they are hybridized to one 
another they do not form a binding site for the coat protein of 
bacteriophage R17, but when not so hybridized, as shown in FIG. 7, the 
first switch sequence 21 organizes so as to comprise a secondary structure 
which is a strong binding site for that coat protein. Carey, J., Cameron, 
V., de Haseth, P. L. and Uhlenbeck, O. C., Sequence-Specific Interaction 
of R17 Coat Protein With Its Ribonucleic Acid Binding Site, Biochemistry 
22:2601-2610 (1983). 
Exposure of the target sequence, hybridization of the probe with the target 
sequence, and separation from unbound probes are as described in Example 
I. 
The bacteriophage R17 coat protein is covalently linked to a solid support, 
such as, for example, Sephadex or Sepharoic beads, magnetic beads, or 
microtiter plates, by methods well known in the art. An example of such a 
method of linkage is described in Alagon, A. J., and King, T. P., 
Activation of Polysaccharides with 2-Iminothiolane and Its Uses, 
Biochemistry 19:4331-4345 (1980). The washed sample, containing probes 
bound to target sequences and nonspecifically bound probes, is added to 
the insolubilized R17 coat protein. Nonspecifically bound probes are 
removed by washing. 
We release the probe from both the R17 coat protein and the target sequence 
by a brief heating step, and remove the solid support. 
Exponential replication of the probe by Q-beta replicase and detection 
proceed as described in Example I. 
EXAMPLE IV 
In this example, the probe 23 (FIG. 8) is a single strand of RNA designed 
to contain four functionally distinct sequences: a probe sequence 24 
approximately 34 nucleotides in length; a first switch sequence 25 of 
about 17 nucleotides immediately adjacent to the 5' side of the probe 
sequence; a second switch sequence 26 complementary to, and of the same 
length as, the first and located immediately adjacent to the 3' side of 
the probe sequence; and a replicatable RNA sequence 27 extending from the 
3' side of the second switch sequence, wherein at least five nucleotides 
of said replicatable RNA sequence are also nucleotides of the 3' side of 
the second switch sequence; that is, the replicatable RNA sequence can be 
considered to overlap the second switch sequence. 
Exposure of the target sequence, hybridization of probes to target 
sequences and separation of unbound probes are performed under appropriate 
conditions known to the art, as in Example I. As shown in FIG. 9, probe 
sequence 24 is hybridized to target sequence 8, and switch sequences 25, 
26 are forced apart, thereby freeing replicatable RNA sequence 27. The 
replicatable RNA sequences of bound probes are, at this point, not subject 
to exponential replication by RNA polymerase even though the switches are 
open. The replicatable RNA sequences 27 must be cleaved at their 5' sides 
to render them subject to exponential replication. Nishihara, T., Mills, 
D. R., and Kramer, F. R., Localization of the Q-beta Replicase Recognition 
Site in MDV-1 RNA, J. Biochem. 93:669-674 (1983). 
There are two means, at least, to cleave the replicatable RNA sequences. 
One is ribozyme cleavage. Another is cleavage by ribonuclease H. We prefer 
the former, which will be described first. 
A. Ribozyme Cleavage 
Ribozymes are structured RNA molecules that are capable of catalyzing a 
chemical reaction, such as particularly the cleavage of a phosphodiester 
bond. It is well known in the art that a ribozyme can be constructed by 
the interaction of two separate oligribonucleotides, one of which is 
cleaved at a particular phosphodiester bond when incubated under known, 
appropriate conditions. Uhlenbeck, O. C., A Small Catalytic 
Oligoribonucleotide, Nature 328:590-600 (1987); Haseloff, J. and Gerlach, 
W. L., Simple RNA Enzymes with New and Highly Specific Endoribonuclease 
Activities, Nature 334:585-591 (1988). 
The requirements for the two segments of an active ribozyme are outlined in 
the two references cited above. For purposes of this invention, the second 
switch sequence of our probe is designed to satisfy the requirements of 
the sequence that is cleaved. The replicatable RNA sequence with which we 
have chosen to proceed is MDV-poly (+) RNA according to Lizardi et al. 
supra. Our preferred design is shown in FIG. 8. As shown there, the second 
switch sequence is 17 nucleotides in length, and 11 nucleotides of the 5' 
side of the MDV-poly (+) RNA are also nucleotides of the 3' side of the 
second switch sequence. The second switch sequence includes the required 
GUC sequence needed for cleavage of the phosphodister bond on the 3' side 
of the GUC sequence, that is, on the 5' side of the replicatable RNA 
sequence. In designing the second switch sequence, care is taken to ensure 
that the subsequent hybridization to form the ribozyme will be more likely 
to occur than the interaction that can occur between the sides of the 
replicatable RNA sequence. 
The probe can be made by transcription from a suitable recombinant plasmid. 
Such a plasmid is designed utilizing methods known to the art with the 
criteria of Lizardi et al., supra. It is constructed by methods well known 
to the art. Maniatis, T., Fritsch, E. F., and Sambrook, J., MOLECULAR 
CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, Cold Spring 
Harbor N.Y. (1982). 
The non-cleaved strand 28, which is capable of forming the required 
ribozyme, is also shown in FIG. 9. It is made by methods well known to the 
art. Milligan, J. F., Duncan, R. G., Witherell, G. W. and Uhlenbeck, O. 
C., Oligoribonucleotide Synthesis Using T7 RNA Polymerase and Synthetic 
DNA Templates, Nucleic Acids Research 15:8783-8798 (1987). FIG. 10 shows 
the nucleotide sequences of the ribozyme formed by switch sequence 26 and 
strand 28 of FIG. 9. 
Following separation of unbound probes, which we prefer, the non-cleaved 
strand 28 described above is incubated with the sample under conditions, 
known to the art, that will promote hybridization of that strand with the 
second switch sequence in probes hybridized to target sequences to form 
the desired ribozyme. Incubation under known conditions referred to above 
cleaves the replicatable RNA from those probes and permits replicatable 
RNA to serve as a template for exponential replication by Q-beta 
replicase. Referring to FIG. 10, cleavage occurs in strand 26 between the 
sixth and seventh nucleotides from the left as shown in the figure. 
Exponential replication and detection proceed as described in Example I. 
B. Ribonuclease H Cleavage 
The probe for this embodiment may be identical to the probe shown in FIG. 8 
and described above. In this embodiment we use commercially available E. 
coli ribonuclease H, which cleaves an RNA strand when it is hybridized to 
a short DNA oligonucleotide within the hybridized region. Donis-Keller, 
H., Site Specific Enzymatic Cleavage of RNA, Nucleic Acids Res. 7:179-192 
(1979). 
To take advantage of this, we synthesize a short DNA oligonucleotide 29 
(FIG. 11) of about 12 nucleotides that will hybridize to the second switch 
sequence on both sides of the GUC sequence. 
Following separation of unbound probes, which we prefer, the short DNA 
oligonucleotide 29 is incubated with the sample under well known 
conditions that will promote its hybridization (FIG. 11) to the second 
switch sequence. Then the ribonuclease H is added to catalyze cleavage 
during an incubation under known conditions. (Donis-Keller, supra). 
Exponential replication by Q-beta replicase and detection proceed as 
described in Example I. 
EXAMPLE V 
This example resembles Example IV-A except that the ribozyme sequences are 
both part of the probe. The probe 30 (FIG. 12) is a single-stranded RNA, 
prepared as described in Example IV but designed to contain five 
sequences: a probe sequence 31 approximately 34 nucleotides in length; a 
first switch sequence 32 of about 17 nucleotides having the sequence of 
the non-cleaved strand 28 shown in FIG. 9; a second switch sequence 33 of 
about 17 nucleotides complementary to the first sequence, as in Example I; 
a spacer sequence 34 of approximately 45 nucleotides extending from the 3' 
side of the second switch sequence, and a replicatable RNA moiety 35. The 
six nucleotides at the 3' side of the spacer sequence are identical to the 
six nucleotides at the 5' side of the second switch sequence shown in FIG. 
9. Thus, the region in which the spacer sequence is joined to the 
replicatable RNA sequence comprises the cleavable strand of a ribozyme, 
just as does the second switch sequence 26 in Example IV-A. In the unbound 
probe, the first switch sequence 32 is hybridized to the second switch 
sequence 33. In probes hybridized to target sequences, where the switch is 
open, however, the first switch sequence 32 is available to hybridize with 
the region in which the 3' side of the spacer sequence 34 is joined to the 
5' side of the replicatable RNA sequence 35, thereby forming a ribozyme. 
The spacer 34 is designed to be long enough to permit that hybridization. 
Exposure of the target sequence, hybridizing of probes to target sequences, 
and separation of unbound probes, which we prefer, is as described in 
Example I. Upon hybridization of a probe to a target sequence (FIG. 13), 
the switch sequences 32, 33 are not hybridized to each other and the 
ribozyme 36 is formed. 
Release of the replicatable RNA, exponential replication and detection 
proceed as in Example IV-A. 
As stated above the assays of this invention may be qualitative or 
quantitative. As one skilled in the art will readily appreciate, for a 
qualitative demonstration of a predetermined target sequence by the 
methods described above, biological and chemical reagents used in the 
assays must be used in readily determinable quantities sufficient to 
generate a reproducible, detectable signal in a sensitive assay. 
For a quantitative determination, the amount of probe added should be 
substantially in excess of the highest amount of target sequence expected 
and incubation should be carried out under conditions such that virtually 
all target sequences hybridize with probes. By "virtually all" we mean a 
very high percentage sufficient to impart reproducibility to the assay. In 
subsequent steps through signal detection, each step should be similarly 
quantitative. For example, destruction of unbound probes should destroy 
virtually all of the unbound probes for reproducibility and also to 
eliminate background noise. Transcription and replication steps should 
utilize sufficient reagents to be quantitative and should be carried out 
for set times for the sake of reproducibility. 
Often, both qualitative and quantitative assays will include parallel 
assays of at least a negative control, that is, one not containing target 
sequence, and at times will also include a series of samples containing 
known amounts of target sequence, such as a geometrically increasing 
series. 
The present invention is also directed to assay kits useful for the 
qualitative detection or quantitative determination of at least one 
specific, predetermined nucleic acid target sequence using probe molecules 
of this invention. Assay kits will include quantities of one or more 
probes which comprise at least the three essential sequences described 
above and at least one additional biologically active molecule, for 
example, a DNA strand, a ribozyme former, an RNA strand or an enzyme, 
useful for generating a signal indicative of switch opening. Kits may also 
include additional reagents such wash solutions, insolubilizing reagents 
and materials, amplification reagents and detection reagents. 
Amplification reagents may include enzymes and nucleotides. Detection 
reagents may include labeled nucleotides and color-forming substrates. 
Kits designed for research may include plasmids which will enable a 
researcher to prepare probes according to this invention containing any 
desired probe sequence.