Homogeneous detection of a target through nucleic acid ligand-ligand beacon interaction

The presence of a target in a test mixture or the concentration of such target can be determined by a method relying on fluorescence emission measurement. Such method utilizes a nucleic acid ligand to the target and a ligand beacon comprising: i) a nucleic acid sequence complimentary to at least a portion of said nucleic acid ligand, ii) a fluorescent group, and iii) a fluorescence-modifying group. The emission profile of the ligand beacon is altered based on the presence and/or concentration change of the target in the test mixture.

FIELD OF THE INVENTION 
This invention is directed to a novel method for the highly selective 
detection of specific target molecules. The binding of a nucleic acid 
ligand to a target molecule is accompanied by a change in the fluorescence 
spectrum of the assay solution. The subject invention will be useful in 
any application where it is desired to detect a target molecule. 
BACKGROUND OF THE INVENTION 
The ability to detect the presence of a specific target molecule, such as a 
nucleic acid or a protein, has proved to have increasing importance in a 
large number of applications. One of the most significant applications 
utilizing sensitive and selective detection of such target molecules is in 
diagnostic assays. In these assays, measurement of the concentration of a 
target molecule is used to yield diagnostic or prognostic medical 
information. 
A recently described reagent for nucleic acid detection is the "molecular 
beacon". A molecular beacon is a unimolecular nucleic acid molecule 
comprising a stem-loop structure (FIG. 1). The stem is formed by 
intramolecular base pairing of two complementary sequences such that the 
5' and 3' ends of the nucleic acid are at the base of the stem. The loop 
links the two strands of the stem, and is comprised of sequences 
complementary to those to be detected. A fluorescent group (star in FIG. 
1) is covalently attached to one end of the molecule, and a fluorescent 
quenching group (filled circle in FIG. 1) is attached to the other end. In 
the stem-loop configuration, these two moieties are physically adjacent to 
one another. When the molecular beacon is illuminated with light 
corresponding to the excitation wavelength of the fluorescent group, no 
fluorescence is observed. This is because energy transfer occurs between 
the fluorescent group and the quenching group such that light emitted from 
the fluorescent group upon excitation is absorbed by the quenching group. 
When the molecular beacon is contacted with sequences complementary to the 
loop, the loop hybridizes to this sequence. This process is energetically 
favored as the intermolecular duplex formed is longer, and therefore more 
stable, than the intramolecular duplex formed in the stem region. As this 
intermolecular double helix forms, torsional forces are generated that 
cause the stem region to unwind. As a result, the fluorescent group and 
the quenching group become spatially separated such that the quenching 
group is no longer able to efficiently absorb light emitted from the 
fluorescent group. Thus, binding of the molecular beacon to its target 
nucleic acid sequence is accompanied by an increase in fluorescence 
emission from the fluorescent group. 
It is possible to simultaneously use two or more molecular beacons with 
different sequence specificities in the same assay. In order to do this, 
each molecular beacon is labeled with at least a different fluorescent 
group. The assay is then monitored for the spectral changes characteristic 
for the binding of each particular molecular beacon to its complementary 
sequence. In this way, molecular beacons have been used to determine 
whether an individual is homozygous wild-type, homozygous mutant or 
heterozygous for a particular mutation. For example, using one 
quenched-fluorescein molecular beacon that recognizes the wild-type 
sequence and another rhodamine-quenched molecular beacon that recognizes a 
mutant allele, it is possible to genotype individuals for the 
.beta.-chemokine receptor (Kostrikis et al. (1998) Science 279:1228-1229). 
The presence of only a fluorescein signal indicates that the individual is 
wild-type, and the presence of rhodamine signal only indicates that the 
individual is a homozygous mutant. The presence of both rhodamine and 
fluorescein signal is diagnostic of a heterozygote. Tyagi et al. (1998) 
Nature Biotechnology 16: 49-53) have even described the simultaneous use 
of four differently labeled molecular beacons for allele discrimination. 
Although useful for the detection of nucleic acid targets, molecular 
beacons have not been used for detecting other types of molecules. Indeed, 
there has been no suggestion made in the extensive art that molecular 
beacons can be used for anything other than detecting specific nucleic 
acids in mixtures containing a plurality of nucleic acids. Detection of 
nucleic acids is undeniably important, but in many 
applications--especially medical diagnostic scenarios--detection of 
non-nucleic acid molecules, such as proteins, sugars, and small 
metabolites, is required. 
In general, the detection of non-nucleic acid target molecules is a more 
complicated matter than the detection of nucleic acids, and no single 
method is universally applicable. Specific proteins may be detected 
through the use of antibody-based assays, such as an enzyme linked 
immunoassay (ELISA). In one form of ELISA, a primary antibody binds to the 
protein of interest, and signal amplification is achieved by using a 
labeled secondary antibody that can bind to multiple sites on the primary 
antibody. This technique can only be used to detect molecules for which 
specific antibodies exist. The generation of new antibodies is a time 
consuming and very expensive procedure, and many proteins arc not 
sufficiently immunogenic to generate antibodies in host animals. 
Furthermore, it is often necessary to measure and detect small molecules, 
such as hormones and sugars, that are generally not amenable to antibody 
recognition. In these cases, enzymatic assays for the specific molecule 
are often required. 
The dogma for many years was that nucleic acids had primarily an 
informational role. Through a method known as Systematic Evolution of 
Ligands by EXponential enrichment, termed the SELEX.TM. process, it has 
become clear that nucleic acids have three dimensional structural 
diversity similar to or even more than proteins. The SELEX.TM. process is 
a method for the in vitro evolution of nucleic acid molecules with highly 
specific binding to target molecules and is described in U.S. patent 
application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled "Systematic 
Evolution of Ligands by Exponential Enrichment," now abandoned; U.S. 
patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled 
"Nucleic Acid Ligands," now U.S. Pat. No. 5,475,096; U.S. patent 
application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled "Methods 
for Identifying Nucleic Acid Ligands," now U.S. Pat. No. 5,270,163 (see 
also, WO 91/19813), each of which is specifically incorporated by 
reference herein. Each of these applications, collectively referred to 
herein as the SELEX.TM. Patent Applications, describes a fundamentally 
novel method for making a nucleic acid ligand to any desired target 
molecule. The SELEX.TM. process provides a class of products which are 
referred to as nucleic acid ligands, each ligand having a unique sequence, 
and which has the property of binding specifically to a desired target 
compound or molecule. Each SELEX.TM. process-identified nucleic acid 
ligand is a specific ligand of a given target compound or molecule. The 
SELEX.TM. process is based on the unique insight that nucleic acids have 
sufficient capacity for forming a variety of two- and three-dimensional 
structures and sufficient chemical versatility available within their 
monomers to act as ligands (form specific binding pairs) with virtually 
any chemical compound, whether monomeric or polymeric. Molecules of any 
size or composition can serve as targets. 
The SELEX.TM. method involves selection from a mixture of candidate 
oligonucleotides and step-wise iterations of binding, partitioning and 
amplification, using the same general selection scheme, to achieve 
virtually any desired criterion of binding affinity and selectivity. 
Starting from a mixture of nucleic acids, preferably comprising a segment 
of randomized sequence, the SELEX.TM. method includes steps of contacting 
the mixture with the target under conditions favorable for binding, 
partitioning unbound nucleic acids from those nucleic acids which have 
bound specifically to target molecules, dissociating the nucleic 
acid-target complexes, amplifying the nucleic acids dissociated from the 
nucleic acid-target complexes to yield a ligand-enriched mixture of 
nucleic acids, then reiterating the steps of binding, partitioning, 
dissociating and amplifying through as many cycles as desired to yield 
highly specific high affinity nucleic acid ligands to the target molecule. 
It has been recognized that the SELEX.TM. method demonstrates that nucleic 
acids as chemical compounds can form a wide array of shapes, sizes and 
configurations, and are capable of a far broader repertoire of binding and 
other functions than those displayed by nucleic acids in biological 
systems. 
The basic SELEX.TM. method has been modified to achieve a number of 
specific objectives. For example, U.S. patent application Ser. No. 
07/960,093, filed Oct. 14, 1992, entitled "Method for Selecting Nucleic 
Acids on the Basis of Structure," now abandoned in favor of U.S. patent 
application Ser. No. 08/198,670, filed Feb. 22, 1994, now U.S. Pat. No. 
5,707,796, describes the use of the SELEX.TM. process in conjunction with 
gel electrophoresis to select nucleic acid molecules with specific 
structural characteristics, such as bent DNA. U.S. patent application Ser. 
No. 08/123,935, filed Sep. 17, 1993, entitled "Photoselection of Nucleic 
Acid Ligands," now abandoned in favor of U.S. patent application Ser. No. 
08/612,895, filed Mar. 8, 1996, now U.S. Pat. No. 5,763,177, describes a 
SELEX.TM. process-based method for selecting nucleic acid ligands 
containing photoreactive groups capable of binding and/or 
photocrosslinking to and/or photoinactivating a target molecule. U.S. 
patent application Ser. No. 08/134,028, filed Oct. 7, 1993, entitled 
"High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline 
and Caffeine," abandoned in favor of U.S. patent application Ser. No. 
08/443,957, now U.S. Pat. No. 5,580,737, describes a method for 
identifying highly specific nucleic acid ligands able to discriminate 
between closely related molecules, which can be non-peptidic, termed 
Counter-SELEX.TM.. U.S. patent application Ser. No. 08/143,564, filed Oct. 
25, 1993, entitled "Systematic Evolution of Ligands by Exponential 
Enrichment: Solution SELEX.TM.," abandoned in favor of U.S. patent 
application Ser. No. 08/461,069, now U.S. Pat. No. 5,567,588, describes a 
SELEX.TM. process-based method which achieves highly efficient 
partitioning between oligonucleotides having high and low affinity for a 
target molecule. 
The SELEX.TM. method encompasses the identification of high-affinity 
nucleic acid ligands containing modified nucleotides conferring improved 
characteristics on the ligand, such as improved in vivo stability or 
improved delivery characteristics. Examples of such modifications include 
chemical substitutions at the ribose and/or phosphate and/or base 
positions. SELEX.TM. process-identified nucleic acid ligands containing 
modified nucleotides are described in U.S. patent application Ser. No. 
08/117,991, filed Sep. 8, 1993, entitled "High Affinity nucleic acid 
ligands Containing Modified Nucleotides," abandoned in favor of U.S. 
patent application Ser. No. 08/430,709, now U.S. Pat. No. 5,660,985, that 
describes oligonucleotides containing nucleotide derivatives chemically 
modified at the 5- and 2'-positions of pyrimidines. U.S. patent 
application Ser. No. 08/134,028, supra, describes highly specific nucleic 
acid ligands containing one or more nucleotides modified with 2'-amino 
(2'-NH.sub.2), 2'-fluoro (2'-F), and/or 2'-O-methyl (2'-OMe). U.S. patent 
application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled "Novel 
Method of Preparation of 2' Modified Pyrimidine Intramolecular 
Nucleophilic Displacement," now abandoned in favor of U.S. patent 
application Ser. No. 08/732,283 filed Oct. 30, 1996, describes 
oligonucleotides containing various 2'-modified pyrimidines. 
The SELEX.TM. method encompasses combining selected oligonucleotides with 
other selected oligonucleotides and non-oligonucleotide functional units 
as described in U.S. patent application Ser. No. 08/284,063, filed Aug. 2, 
1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment: 
Chimeric SELEX.TM.," now U.S. Pat. No. 5,637,459, and U.S. patent 
application Ser. No. 08/234,997, filed Apr. 28, 1994, entitled "Systematic 
Evolution of Ligands by Exponential Enrichment: Blended SELEX.TM.," now 
U.S. Pat. No. 5,683,867, respectively. These applications allow the 
combination of the broad array of shapes and other properties, and the 
efficient amplification and replication properties, of oligonucleotides 
with the desirable properties of other molecules. 
The SELEX.TM. method further encompasses combining selected nucleic acid 
ligands with lipophilic compounds or non-immunogenic, high molecular 
weight compounds in a diagnostic or therapeutic complex as described in 
U.S. patent application Ser. No. 08/434,465, filed May 4, 1995, entitled 
"Nucleic Acid Complexes". VEGF nucleic acid ligands that are associated 
with a lipophilic compound, such as diacyl glycerol or dialkyl glycerol, 
in a diagnostic or therapeutic complex are described in U.S. patent 
application Ser. No. 08/739,109, filed Oct. 25, 1996, entitled "Vascular 
Endothelial Growth Factor (VEGF) Nucleic Acid Ligand Complexes now U.S. 
Pat. No. 5,859,228." VEGF nucleic acid ligands that are associated with a 
lipophilic compound, such as a glycerol lipid, or a non-immunogenic, high 
molecular weight compound, such as polyethylene glycol, are further 
described in U.S. patent application Ser. No. 08/897,351, filed Jul. 21, 
1997, entitled "Vascular Endothelial Growth Factor (VEGF) Nucleic Acid 
Ligand Complexes." VEGF nucleic acid ligands that are associated with a 
non-immunogenic, high molecular weight compound or lipophilic compound are 
also further described in PCT/US97/18944, filed Oct. 17, 1997, entitled 
"Vascular Endothelial Growth Factor (VEGF) Nucleic Acid Ligand Complexes." 
Each of the above described patent applications which describe 
modifications of the basic SELEX.TM. procedure are specifically 
incorporated by reference herein in their entirety. 
It is an object of the present invention to provide methods that can be 
used to detect virtually any non-nucleic acid target molecule in a test 
mixture, using nucleic acid reagents that are easily and cheaply 
manufactured. 
It is a further object of the instant invention to provide a method for 
adapting molecular beacons in order to detect non-nucleic acid target 
molecules in a test mixture. 
Another object of the instant invention is to provide a single, universal 
assay for virtually any non-nucleic acid target molecule in which 
measurements of fluorescence emission are used to determine the 
concentration of the target. 
SUMMARY OF THE INVENTION 
The present invention includes methods for detecting the binding of nucleic 
acid ligands to their cognate target molecules. The methods rely on the 
insight that nucleic acid ligands can be recognized by molecular beacons 
in a target-dependent context. The methods and reagents described herein 
allow, for the first time, virtually any target molecule to be detected 
through simple fluorescence emission measurements. 
The invention uses novel molecular beacons, termed ligand beacons, that 
hybridize to nucleic acid ligands only under preselected conditions. In 
some embodiments, the ligand beacon can only hybridize to nucleic acid 
ligands that are free of their cognate target; in other embodiments, the 
ligand beacon can only hybridize to nucleic acid ligands that are bound to 
their cognate targets. In either case, the binding of nucleic acid ligand 
to target is accompanied by a measurable change in the spectral properties 
of the ligand beacon. Conventional molecular beacons known in the art are 
used to recognize complementary nucleic acid sequences, e.g., genomic 
sequences and sequences specific to pathogens. By contrast, ligand beacons 
recognize nucleic acid ligands with both a particular sequence and a 
particular configuration. The configuration of the nucleic acid ligand 
changes when it is or is not bound to its cognate target. 
The methods described herein provide, for the first time, a single 
universal method for target molecule detection which simply involves 
analyzing fluorescence emission. The reagents and methods described herein 
are particularly suitable for diagnostic assays. Diagnostic assays that 
require quantitative measurements (e.g., measurements of a hormone or 
sugar level) are possible according to the present invention by simply 
comparing the fluorescence measurement with that obtained from a control. 
Similarly, diagnostic assays requiring qualitative detection of substances 
(e.g., presence of a mutated gene product, or presence of a pathogen) are 
also possible. The reagents can be used in assays for single substances, 
or they can be used to simultaneously monitor a variety of substances in a 
single assay. Using different fluorescent groups with spectroscopically 
resolvable emission spectra, this method allows for the simultaneous 
detection of multiple targets in a single vessel. In this homogeneous 
multiplexing approach, distinct fluorescent groups can be attached to 
different nucleic acid ligands specific to targets of interest. In 
particular, the invention provide methods for performing assays using 
reagents attached to solid supports. In these embodiments, a plurality of 
nucleic acid ligands are attached to spatially discrete regions on solid 
supports, and contacted with the solution to be assayed. Using the 
detection methods described herein, measurements of fluorescence at 
discrete sites on the solid support can reveal whether particular 
substances are present in the assay solution, and in what quantities. In 
this way, it is possible to assay for a plurality--potentially hundreds or 
even thousands--of different substances in a single test. Arrays of 
nucleic acid ligands that can be used with the methods and reagents 
described herein are detailed in copending and commonly-assigned U.S. 
patent application Ser. No. 08/990,436, filed Dec. 15, 1997, entitled 
"Nucleic Acid Ligand Diagnostic Biochip" which is incorporated herein by 
reference in its entirety.

DETAILED DESCRIPTION OF THE INVENTION 
The SELEX.TM. method encompasses the identification of high-affinity 
nucleic acid ligands containing modified nucleotides conferring improved 
characteristics on the ligand, such as improved in vivo stability or 
improved delivery characteristics. Examples of such modifications include 
chemical substitutions at the ribose and/or phosphate and/or base 
positions. SELEX.TM. process-identified nucleic acid ligands containing 
modified nucleotides are described in U.S. patent application Ser. No. 
08/117,991, filed Sep. 8, 1993, entitled "High Affinity Nucleic Acid 
Ligands Containing Modified Nucleotides", abandoned in favor of U.S. 
patent application Ser. No. 08/430,709, filed Apr. 27, 1995, now U.S. Pat. 
No. 5,660,985, that describes oligonucleotides containing nucleotide 
derivatives chemically modified at the 5- and 2'-positions of pyrimidines. 
U.S. patent application Ser. No. 08/134,028, above, describes highly 
specific nucleic acid ligands containing one or more nucleotides modified 
with 2'-amino (2'-NH.sub.2), 2'-fluoro (2'-F), and/or 2'-O-methyl 
(2'-OMc). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 
1994, entitled "Novel Method of Preparation of Known and Novel 2' Modified 
Nucleosides by Intramolecular Nucleophilic Displacement", now abandoned in 
favor of U.S. patent application Ser. No. 08/732,283, filed Oct. 30, 1996, 
describes oligonucleotides containing various 2'-modified pyrimidines. 
The methods of the instant invention have the ability to detect virtually 
any target molecule of interest through the production of a highly 
specific spectral shift. Importantly, as the target molecule probes are 
nucleic acids, they are particularly useful in "biochip" applications well 
known in the art. This art provides many efficient methods for coupling 
nucleic acids to the surface of solid supports in spatially specific ways. 
The methods and embodiments disclosed herein will therefore be useful in 
biochip-based medical screening applications as described in the 
co-pending and commonly assigned U.S. patent application Ser. No. 
08/990,436, filed Dec. 15, 1997, entitled "Nucleic Acid Ligand Diagnostic 
Biochip", and specifically incorporated herein by reference. In addition, 
the methods of the invention will be useful in the diagnosis of blood 
clotting disorders as described in co-pending and commonly assigned U.S. 
patent application Ser. No. 09/157,228, filed Sep. 18, 1998, entitled 
"Factor V Leiden Detection". 
Definitions 
Various terms are used herein to refer to aspects of the present invention. 
To aid in the clarification of the description of the components of this 
invention, the following definitions are provided: 
As used herein, "nucleic acid ligand" refers to a non-naturally occurring 
nucleic acid having a desirable action on a target. A desirable action 
includes, but is not limited to, binding of the target, catalytically 
changing the target, reacting with the target in a way which 
modifies/alters the target or the functional activity of the target, 
covalently attaching to the target as in a suicide inhibitor, facilitating 
the reaction between the target and another molecule. In the preferred 
embodiment, the action is specific binding affinity for a target molecule, 
such target molecule being a three dimensional chemical structure other 
than a polynucleotide that binds to the nucleic acid ligand through a 
mechanism which predominantly depends on Watson/Crick base pairing or 
triple helix binding, wherein the nucleic acid ligand is not a nucleic 
acid having the known physiological function of being bound by the target 
molecule. Nucleic acid ligands include nucleic acids that are identified 
from a candidate mixture of nucleic acids, said nucleic acid ligand being 
a ligand of a given target, by the method comprising: a) contacting the 
candidate mixture with the target, wherein nucleic acids having an 
increased affinity to the target relative to the candidate mixture may be 
partitioned from the remainder of the candidate mixture; b) partitioning 
the increased affinity nucleic acids from the remainder of the candidate 
mixture; and c) amplifying the increased affinity nucleic acids to yield a 
ligand-enriched mixture of nucleic acids. 
As used herein, "candidate mixture" refers to a mixture of nucleic acids of 
differing sequence from which to select a desired ligand. The source of a 
candidate mixture can be from naturally-occurring nucleic acids or 
fragments thereof, chemically synthesized nucleic acids, enzymatically 
synthesized nucleic acids or nucleic acids made by a combination of the 
foregoing techniques. In a preferred embodiment, each nucleic acid has 
fixed sequences surrounding a randomized region to facilitate the 
amplification process. 
As used herein, "nucleic acid" means either DNA, RNA, single-stranded or 
double-stranded, and any chemical modifications thereof. Modifications 
include, but arc not limited to, those which provide other chemical groups 
that incorporate additional charge, polarizability, hydrogen bonding, 
electrostatic interaction, and fluxionality to the nucleic acid ligand 
bases or to the nucleic acid ligand as a whole. Such modifications 
include, but are not limited to, 2'-position sugar modifications, 
5-position pyrimidine modifications, 8-position purine modifications, 
modifications at exocyclic amines, substitution of 4-thiouridine, 
substitution of 5-bromo or 5-iodo-uracil; backbone modifications, 
methylations, unusual base-pairing combinations such as the isobases 
isocytidine and isoguanidine and the like. Modifications can also include 
3' and 5' modifications such as capping. 
As used herein, "SELEX.TM." methodology involves the combination of 
selection of nucleic acid ligands which interact with a target in a 
desirable manner, for example binding to a protein, with amplification of 
those selected nucleic acids. Optional iterative cycling of the 
selection/amplification steps allows selection of one or a small number of 
nucleic acids which interact most strongly with the target from a pool 
which contains a very large number of nucleic acids. Cycling of the 
selection/amplification procedure is continued until a selected goal is 
achieved. The SELEX.TM. methodology is described in the SELEX.TM. Patent 
Applications. 
As used herein "target" means any compound or molecule of interest for 
which a diagnostic test is desired and where a nucleic acid ligand is 
known or can be identified. A target can be a protein, peptide, 
carbohydrate, polysaccharide, glycoprotein, hormone, receptor, antigen, 
antibody, virus, substrate, metabolite, transition state analog, cofactor, 
inhibitor, drug, dye, nutrient, growth factor, etc. without limitation. 
As used herein, "solid support" means any microfabricated solid surface to 
which molecules may be attached through either covalent or non-covalent 
bonds. This includes, but is not limited to, Langmuir-Bodgett films, 
functionalized glass, membranes, charged paper, nylon, germanium, silicon, 
PTFE, polystyrene, gallium arsenide, gold, and silver. Any other material 
known in the art that is capable of having functional groups such as 
amino, carboxyl, thiol or hydroxyl incorporated on its surface, is 
contemplated. This includes surfaces with any topology, such spherical 
surfaces and grooved surfaces. 
As used herein, "bodily fluid" refers to a mixture of molecules obtained 
from an organism. This includes, but is not limited to, whole blood, blood 
plasma, urine, semen, saliva, lymph fluid, meningal fluid, amniotic fluid, 
glandular fluid, sputum, and cerebrospinal fluid. This also includes 
experimentally separated fractions of all of the preceding. Bodily fluid 
also includes solutions or mixtures containing homogenized solid material, 
such as feces, tissues, and biopsy samples. 
As used herein, "test mixture" refers to any sample that contains a 
plurality of molecules. This includes, but is not limited to, bodily 
fluids as defined above, and any sample for environmental and toxicology 
testing such as contaminated water and industrial effluent. 
As used herein, "fluorescent group" refers to a molecule that, when excited 
with light having a selected wavelength, emits light of a different 
wavelength. Fluorescent groups include, but are not limited to, 
fluorescein, tetramethylrhodamine, Texas Red, BODIPY, 
5-[(2-aminoethyl)amino]napthalene-1-sulfonic acid (EDANS), and Lucifer 
yellow. Fluorescent groups may also be referred to as "fluorophores". 
As used herein, "fluorescence-modifying group" refers to a molecule that 
can alter in any way the fluorescence emission from a fluorescent group. A 
fluorescence-modifying group generally accomplishes this through an energy 
transfer mechanism. Depending on the identity of the 
fluorescence-modifying group, the fluorescence emission can undergo a 
number of alterations, including, but not limited to, attenuation, 
complete quenching, enhancement, a shift in wavelength, a shift in 
polarity, a change in fluorescence lifetime. One example of a 
fluorescence-modifying group is a quenching group. 
As used herein, "energy transfer" refers to the process by which the 
fluorescence emission of a fluorescent group is altered by a 
fluorescence-modifying group. If the fluorescence-modifying group is a 
quenching group, then the fluorescence emission from the fluorescent group 
is attenuated (quenched). Energy transfer can occur through fluorescence 
resonance energy transfer, or through direct energy transfer. The exact 
energy transfer mechanisms in these two cases are different. It is to be 
understood that any reference to energy transfer in the instant 
application encompasses all of these mechanistically-distinct phenomena. 
As used herein, "energy transfer pair" refers to any two molecules that 
participate in energy transfer. Typically, one of the molecules acts as a 
fluorescent group, and the other acts as a fluorescence-modifying group. 
The preferred energy transfer pair of the instant invention comprises a 
fluorescent group and a quenching group. In some cases, the distinction 
between the fluorescent group and the fluorescence-modifying group may be 
blurred. For example, under certain circumstances, two adjacent 
fluorescein groups can quench one another's fluorescence emission via 
direct energy transfer. For this reason, there is no limitation on the 
identity of the individual members of the energy transfer pair in this 
application. All that is required is that the spectroscopic properties of 
the energy transfer pair as a whole change in some measurable way if the 
distance between the individual members is altered by some critical 
amount. 
"Energy transfer pair" is used to refer to a group of molecules that form a 
single complex within which energy transfer occurs. Such complexes may 
comprise, for example, two fluorescent groups which may be different from 
one another and one quenching group, two quenching groups and one 
fluorescent group, or multiple fluorescent groups and multiple quenching 
groups. In cases where there are multiple fluorescent groups and/or 
multiple quenching groups, the individual groups may be different from one 
another e.g., one complex contemplated herein comprises fluorescein and 
EDANS as fluorescent groups, and DABCYL as a quenching agent. 
As used herein, "quenching group" refers to any fluorescence-modifying 
group that can attenuate at least partly the light emitted by a 
fluorescent group. We refer herein to this attenuation as "quenching". 
Hence, illumination of the fluorescent group in the presence of the 
quenching group leads to an emission signal that is less intense than 
expected, or even completely absent. Quenching occurs through energy 
transfer between the fluorescent group and the quenching group. The 
preferred quenching group of the invention is 
(4-dimethylamino-phenylazo)benzoic acid (DABCYL). 
As used herein, "fluorescence resonance energy transfer" or "FRET" refers 
to an energy transfer phenomenon in which the light emitted by the excited 
fluorescent group is absorbed at least partially by a 
fluorescence-modifying group. If the fluorescence-modifying group is a 
quenching group, then that group can either radiate the absorbed light as 
light of a different wavelength, or it can dissipate it as heat. FRET 
depends on an overlap between the emission spectrum of the fluorescent 
group and the absorption spectrum of the quenching group. FRET also 
depends on the distance between the quenching group and the fluorescent 
group. Above a certain critical distance, the quenching group is unable to 
absorb the light emitted by the fluorescent group, or can do so only 
poorly. 
As used herein "direct energy transfer" refers to an energy transfer 
mechanism in which passage of a photon between the fluorescent group and 
the fluorescence-modifying group does not occur. Without being bound by a 
single mechanism, it is believed that in direct energy transfer, the 
fluorescent group and the fluorescence-modifying group interfere with each 
others electronic structure. If the fluorescence-modifying group is a 
quenching group, this will result in the quenching group preventing the 
fluorescent group from even emitting light. Quenching groups and 
fluorescent groups are frequently close enough together in the stem of 
ligand beacons that direct energy transfer can take place. For example, 
when DABCYL is located on one terminus of a ligand beacon, this quenching 
group can efficiently quench almost all fluorescent groups on the other 
terminus through direct energy transfer. 
In general, quenching by direct energy transfer is more efficient than 
quenching by FRET. Indeed, some quenching groups that do not quench 
particular fluorescent groups by FRET (because they do not have the 
necessary spectral overlap with the fluorescent group) can do so 
efficiently by direct energy transfer. Furthermore, some fluorescent 
groups can act as quenching groups themselves if they are close enough to 
other fluorescent groups to cause direct energy transfer. For example, 
under these conditions, two adjacent fluorescein groups can quench one 
another's fluorescence effectively. For these reasons, there is no 
limitation on the nature of the fluorescent groups and quenching groups 
useful for the practice of this invention. 
As used herein, "ligand beacon" refers to a nucleic acid molecule, labeled 
with an energy transfer pair, that can specifically hybridize to a nucleic 
acid ligand under preselected conditions. Upon doing so, the ligand beacon 
undergoes a conformational change that causes the members of the energy 
transfer pair to move relative to one another such that the emission from 
the fluorescent group is modified. Preferred energy transfer pairs 
comprise a fluorescent group and a quenching group. In preferred 
embodiments, the ligand beacon comprises a unimolecular stem-loop nucleic 
acid, wherein the fluorescent group and the quenching group are at the 
termini of the nucleic acid, and the loop comprises sequences that are at 
least partially complementary to sequences within the nucleic acid ligand. 
In some embodiments, the ligand beacon can only hybridize to the nucleic 
acid ligand when the nucleic acid ligand is not bound to its target. In 
other embodiments, the ligand beacon can only hybridize when the nucleic 
acid ligand is bound to its cognate target. In either case, hybridization 
of the ligand beacon to the nucleic acid ligand is accompanied by a change 
in the fluorescence emission intensity of the ligand beacon. 
Although the ligand beacon comprises a unimolecular stem-loop nucleic acid 
in preferred embodiments, there is no limitation on the structure of the 
ligand beacon. Any nucleic acid that can hybridize to a nucleic acid 
ligand, and in doing so undergo a conformational change that alters the 
distance between nucleotides, is contemplated in the instant invention. 
For example, nucleic acid configured as G-quartets may be useful in this 
invention. These nucleic acid structures are formed by hydrogen bonding 
between the Hoogsteen and Watson-Crick faces of four spatially adjacent 
guanosines. Adjacent quartets can stack on top of one another to form a 
highly symmetric and regular complex. Similarly, ligand beacons that 
undergo conformational changes in which initially separated nucleotide 
positions become adjacent upon hybridizing to nucleic acid ligands are 
also included in the invention. These latter ligand beacons, when labeled 
with fluorescent groups and quenching groups at the appropriate nucleotide 
positions, undergo a decrease in fluorescence intensity upon binding to 
the nucleic acid ligand. 
In the preferred embodiment, the nucleic acid ligands of the present 
invention are derived from the SELEX.TM. methodology. The SELEX.TM. 
process is described in U.S. patent application Ser. No. 07/536,428, 
entitled "Systematic Evolution of Ligands by EXponential Enrichment," now 
abandoned, U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 
1991, entitled "Nucleic Acid Ligands," now U.S. Pat. No. 5,475,096, U.S. 
patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled 
"Methods for Identifying Nucleic Acid Ligands", now U.S. Pat. No. 
5,270,163 (see also, WO 91/19813). These applications, each specifically 
incorporated herein by reference, are collectively called the SELEX.TM. 
Patent Applications. 
The SELEX.TM. process provides a class of products which are nucleic acid 
molecules, each having a unique sequence, and each of which has the 
property of binding specifically to a desired target compound or molecule. 
Target molecules are preferably proteins, but can also include among 
others carbohydrates, peptidoglycans and a variety of small molecules. 
SELEX.TM. methodology can also be used to target biological structures, 
such as cell surfaces or viruses, through specific interaction with a 
molecule that is an integral part of that biological structure. 
In its most basic form, the SELEX.TM. process may be defined by the 
following series of steps: 
1) A candidate mixture of nucleic acids of differing sequence is prepared. 
The candidate mixture generally includes regions of fixed sequences (i.e., 
each of the members of the candidate mixture contains the same sequences 
in the same location) and regions of randomized sequences. The fixed 
sequence regions are selected either: (a) to assist in the amplification 
steps described below, (b) to mimic a sequence known to bind to the 
target, or (c) to enhance the concentration of a given structural 
arrangement of the nucleic acids in the candidate mixture. The randomized 
sequences can be totally randomized (i.e., the probability of finding a 
base at any position being one in four) or only partially randomized 
(e.g., the probability of finding a base at any location can be selected 
at any level between 0 and 100 percent). 
2) The candidate mixture is contacted with the selected target under 
conditions favorable for binding between the target and members of the 
candidate mixture. Under these circumstances, the interaction between the 
target and the nucleic acids of the candidate mixture can be considered as 
forming nucleic acid-target pairs between the target and those nucleic 
acids having the strongest affinity for the target. 
3) The nucleic acids with the highest affinity for the target are 
partitioned from those nucleic acids with lesser affinity to the target. 
Because only an extremely small number of sequences (and possibly only one 
molecule of nucleic acid) corresponding to the highest affinity nucleic 
acids exist in the candidate mixture, it is generally desirable to set the 
partitioning criteria so that a significant amount of the nucleic acids in 
the candidate mixture (approximately 5-50%) arc retained during 
partitioning. 
4) Those nucleic acids selected during partitioning as having the 
relatively higher affinity for the target are then amplified to create a 
new candidate mixture that is enriched in nucleic acids having a 
relatively higher affinity for the target. 
5) By repeating the partitioning and amplifying steps above, the newly 
formed candidate mixture contains fewer and fewer unique sequences, and 
the average degree of affinity of the nucleic acids to the target will 
generally increase. Taken to its extreme, the SELEX.TM. process will yield 
a candidate mixture containing one or a small number of unique nucleic 
acids representing those nucleic acids from the original candidate mixture 
having the highest affinity to the target molecule. 
The basic SELEX.TM. method has been modified to achieve a number of 
specific objectives. For example, U.S. patent application Ser. No. 
07/960,093, filed Oct. 14, 1992, entitled "Method for Selecting Nucleic 
Acids on the Basis of Structure," now abandoned in favor of U.S. patent 
application Ser. No. 08/198,670, filed Feb. 22, 1994, now U.S. Pat. No. 
5,707,796, describes the use of the SELEX.TM. process in conjunction with 
gel electrophoresis to select nucleic acid molecules with specific 
structural characteristics, such as bent DNA. U.S. patent application Ser. 
No. 08/123,935, filed Sep. 17, 1993, entitled "Photoselection of Nucleic 
Acid Ligands," now abandoned in favor of U.S. patent application Ser. No. 
08/612,895, filed Mar. 8, 1996, now U.S. Pat. No. 5,763,177, describes a 
SELEX.TM. based method for selecting nucleic acid ligands containing 
photoreactive groups capable of binding and/or photocrosslinking to and/or 
photoinactivating a target molecule. U.S. patent application Ser. No. 
08/134,028, filed Oct. 7, 1993, entitled "High-Affinity Nucleic Acid 
Ligands That Discriminate Between Theophylline and Caffeine," now U.S. 
Pat. No. 5,580,737, describes a method for identifying highly specific 
nucleic acid ligands able to discriminate between closely related 
molecules, termed Counter-SELEX.TM.. U.S. patent application Ser. No. 
08/143,564, filed Oct. 25, 1993, entitled "Systematic Evolution of Ligands 
by Exponential Enrichment: Solution SELEX.TM.," abandoned in favor of U.S. 
patent application Ser. No. 08/461,069, now U.S. Pat. No. 5,567,588, 
describes a SELEX.TM.-based method which achieves highly efficient 
partitioning between oligonucleotides having high and low affinity for a 
target molecule. U.S. patent application Ser. No. 07/964,624, filed Oct. 
21, 1992, entitled "Nucleic Acid Ligands to HIV-RT and HIV-1 Rev," now 
U.S. Pat. No. 5,496,938, describes methods for obtaining improved nucleic 
acid ligands after SELEX.TM. has been performed. U.S. patent application 
Ser. No. 08/400,440, filed Mar. 8, 1995, entitled "Systematic Evolution of 
Ligands by Exponential Enrichment: Chemi-SELEX.TM.," now U.S. Pat. No. 
5,705,337, describes methods for covalently linking a ligand to its 
target. 
The SELEX.TM. method encompasses the identification of high-affinity 
nucleic acid ligands containing modified nucleotides conferring improved 
characteristics on the ligand, such as improved in vivo stability or 
improved delivery characteristics. Examples of such modifications include 
chemical substitutions at the ribose and/or phosphate and/or base 
positions. SELEX.TM. process-identified nucleic acid ligands containing 
modified nucleotides are described in U.S. patent application Ser. No. 
08/117,991, filed Sep. 8, 1993, entitled "High Affinity Nucleic Acid 
Ligands Containing Modified Nucleotides," now U.S. Pat. No. 5,660,985, 
that describes oligonucleotides containing nucleotide derivatives 
chemically modified at the 5- and 2'-positions of pyrimidines. U.S. patent 
application Ser. No. 08/134,028, supra, describes highly specific nucleic 
acid ligands containing one or more nucleotides modified with 2'-amino 
(2'-NH.sub.2), 2'-fluoro (2'-F), and/or 2'-O-methyl (2'-OMe). U.S. patent 
application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled "Novel 
Method of Preparation of Known and Novel 2' Modified Nucleosides by 
Intramolecular Nucleophilic Displacement," abandoned in favor of U.S. 
patent application Ser. No. 08/732,283, filed Oct. 30, 1996, describes 
oligonucleotides containing various 2'-modified pyrimidines. 
The SELEX.TM. method encompasses combining selected oligonucleotides with 
other selected oligonucleotides and non-oligonucleotide functional units 
as described in U.S. patent application Ser. No. 08/284,063, filed Aug. 2, 
1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment: 
Chimeric SELEX.TM.," now U.S. Pat. No. 5,637,459, and U.S. patent 
application Ser. No. 08/234,997, filed Apr. 28, 1994, entitled "Systematic 
Evolution of Ligands by Exponential Enrichment: Blended SELEX.TM.," now 
U.S. Pat. No. 5,683,867, respectively. These applications allow the 
combination of the broad array of shapes and other properties, and the 
efficient amplification and replication properties, of oligonucleotides 
with the desirable properties of other molecules. 
The SELEX.TM. process provides high affinity ligands of a target molecule. 
This represents a singular achievement that is unprecedented in the field 
of nucleic acids research. 
In co-pending and commonly assigned U.S. patent application Ser. No. 
07/964,624, filed Oct. 21, 1992, methods are described for obtaining 
improved nucleic acid ligands after the SELEX.TM. process has been 
performed (now U.S. Pat. No. 5,496,938). This patent, entitled "Nucleic 
Acid Ligands to HIV-RT and HIV-1 Rev," along with each of the patent 
applications discussed above, is specifically incorporated herein by 
reference. 
One potential problem encountered in the diagnostic use of nucleic acids is 
that oligonucleotides in their phosphodiester form may be quickly degraded 
in bodily fluids by intracellular and extracellular enzymes such as 
endonucleases and exonucleases before the desired effect is manifest. 
Certain chemical modifications of the nucleic acid ligand can be made to 
increase the in vivo stability of the nucleic acid ligand or to enhance or 
to mediate the delivery of the nucleic acid ligand. See, e.g., U.S. patent 
application Ser. No. 08/117,991, filed Sep. 8, 1993, entitled "High 
Affinity Nucleic Acid Ligands Containing Modified Nucleotides," abandoned 
in favor of U.S. patent application Ser. No. 08/430,709, now U.S. Pat. No. 
5,660,985 which is specifically incorporated herein by reference. 
Modifications of the nucleic acid ligands contemplated in this invention 
include, but are not limited to, those which provide other chemical groups 
that incorporate additional charge, polarizability, hydrophobicity, 
hydrogen bonding, electrostatic interaction, and fluxionality to the 
nucleic acid ligand bases or to the nucleic acid ligand as a whole. Such 
modifications include, but are not limited to, 2'-position sugar 
modifications, 5-position pyrimidine modifications, 8-position purine 
modifications, modifications at exocyclic amines, substitution of 
4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone 
modifications, phosphorothioate or alkyl phosphate modifications, 
methylations, unusual base-pairing combinations such as the isobases 
isocytidine and isoguanidine and the like. Modifications can also include 
3' and 5' modifications such as capping. 
The modifications can be pre- or post-SELEX.TM. process modifications. 
PreSELEX.TM. process modifications yield nucleic acid ligands with both 
specificity for their SELEX.TM. target and improved in vivo stability. 
Post-SELEX.TM. process modifications made to 2'-OH nucleic acid ligands 
can result in improved in vivo stability without adversely affecting the 
binding capacity of the nucleic acid ligand. 
Other modifications are known to one of ordinary skill in the art. Such 
modifications may be made post-SELEX.TM. process (modification of 
previously identified unmodified ligands) or by incorporation into the 
SELEX.TM. process. 
Ligand beacons 
In one embodiment of the invention, a ligand beacon is used to detect a 
nucleic acid ligand that is or is not bound to its cognate target. The 
ligand beacon preferably consists of a single-stranded DNA molecule that 
assumes a stem-loop structure in solution. In this embodiment, the stem of 
the ligand beacon is formed by the intramolecular base-pairing of two 
antiparallel strands of nucleic acid. The 5' terminus of one strand is 
linked to the 3' terminus of the other strand with a loop of single 
stranded DNA. These nucleic acid molecules can be rapidly synthesized as 
single-stranded oligonucleotides with the general structure: 
5' AAA--------------A'A'A' 3' 
wherein sequence A' is both complementary in sequence and reversed in 
orientation relative to A. When heat-denatured and slowly cooled, this 
oligonucleotide will form a stem-loop structure wherein the dashed line 
forms the loop, and wherein A and A' pair to form the stem. 
The loop domain comprises sequences that are at least partially 
complementary to a region of the nucleic acid ligand. In preferred 
embodiments, the sequences are chosen such that they can only hybridize to 
one another when the nucleic acid ligand is not bound to its cognate 
target (FIG. 2). Furthermore, when the ligand beacon hybridizes to the 
nucleic acid ligand, the nucleic acid ligand can no longer bind to its 
cognate target. In particularly preferred embodiments, the loop of the 
ligand beacon binds to a sequence in the nucleic acid ligand that is at 
least about 20 nucleotides long; the stem region of the ligand beacon is 
preferably shorter. 
The formation of the intermolecular duplex between the loop of the ligand 
beacon and the target-free nucleic acid ligand is energetically favored 
because the resulting duplex is longer, and hence more stable, than the 
intramolecular duplex. As the loop sequence and the nucleic acid ligand 
form a duplex, torsional forces arc developed in the ligand beacon. These 
forces are transmitted to the stem region which unwinds in response, 
usually starting at the base of the stem where the termini are located. 
One base pair in the stem is unwound for each new base pair that is made 
between the ligand beacon and the nucleic acid ligand. Thus nucleotide 
positions that were adjacent to one another on opposite sides of the stem 
become separated. In particular, because unwinding begins at the base of 
the stem, the termini of the ligand beacon become widely separated (FIG. 
2). 
In some embodiments, nucleotide positions in the ligand beacon that become 
separated from one another arc labeled with an energy transfer pair. The 
preferred energy transfer pair of the instant invention comprises a 
quenching group and a fluorescent group. In preferred embodiments, the 
nucleotide positions on the ligand beacon that are labeled with the 
quenching group and the fluorescent group are chosen from those that form 
the intramolecular stem. In especially preferred embodiments, the 5' and 
3' termini of the ligand beacon are labeled with these groups, as the 
termini become widely separated upon hybridization to the nucleic acid 
ligand (FIG. 2). 
The fluorescent group and the adjacent quenching group take part in energy 
transfer. In some instances, the energy transfer occurs through 
fluorescence resonance energy transfer (FRET). FRET takes place when 
fluorescence emission from a fluorescent group is transferred to an 
adjacent group that somehow modifies the signal (in this case, quenching 
the signal). This effect is strongly dependent on the distance between the 
two groups, such that when separated by a critical distance, FRET does not 
take place, and the fluorescence emission is unmodified. FRET also 
requires that the emission spectrum of the fluorescent group overlaps with 
the absorbance spectrum of the modifying group. 
In the instant invention, the preferred fluorescent groups are fluorescein, 
tetramethylrhodamine, and 5-[(2-aminoethyl)amino]napthalene-1-sulfonic 
acid (EDANS). The preferred quencher is (4-dimethylaminophenylazo)benozoic 
acid (DABCYL). When DABCYL and fluorescein or EDANS are close enough 
together for FRET to occur, DABCYL absorbs light emitted from the 
fluorescein or EDANS, and dissipates the absorbed energy as heat. As 
mentioned above, this effect is strongly dependent on the distance between 
the group. For example, at separations greater than 60 Angstroms, DABCYL 
is unable to quench the fluorescence from EDANS. DABCYL itself is 
non-fluorescent at the wavelengths used to excite EDANS or fluorescein. 
In other embodiments, the fluorescent group and the quenching group take 
place in a form of energy transfer termed direct energy transfer. Direct 
energy transfer occurs when the fluorescent group and the quenching group 
directly perturb each others electronic structure. When a direct transfer 
takes place, it is possible for a quenching group to quench at a much 
higher efficiency and over a broader spectrum than in FRET. Indeed, it has 
been reported that paired-groups that do not even display FRET, such as 
Texas Red and DABCYL, can be made to undergo direct energy transfer, 
leading to the efficient quenching of the fluorescence group by the other 
group. For example, it has been reported that under such circumstances, 
the quenching group DABCYL can quench almost all fluorophores (with 
emission spectra ranging from 475 nm-615 nm) with close to 100% efficiency 
(Tyagi, et al. 1998. Nature Biotechnology 16: 49-53). 
In one preferred embodiment, fluorescein and DABCYL function as a direct 
energy transfer pair when present at the 5' and 3' termini, even though 
they are not an efficient FRET Pair. In other embodiments, nucleotide 
positions that form an individual base pair in the stem are labeled with 
the fluorescent group and the quenching group. Labeling at these positions 
also allows direct energy transfer to take place. It is even possible to 
get fluorescence quenching when two identical fluorescent groups, such as 
two fluorescein groups, are sufficiently close together. 
There is no limitation in the present invention as to the nature of the 
energy transfer pair, and there is no limitation as to the exact mechanism 
by which they function together. All that is required is that the spectral 
properties of the energy transfer pair change in some measurable way as 
the distance between the individual members of the energy transfer pair is 
varied. 
It is possible to label the ligand beacon with more than one of each member 
of an energy transfer pair. For example, in some embodiments, two or more 
nucleotides are labeled with fluorescent groups and the same number of 
nucleotides are labeled with quenching groups. In preferred embodiments, 
all of the fluorescent groups are attached to the nucleotides that 
comprise one strand of the stem, and all of the quenching groups are 
attached to the nucleotides that comprises the other strand. In these 
embodiments, more than one base pair in the stem is labeled with both a 
fluorescent group and a quenching group. Such ligand beacons may give an 
increased signal relative to singly-labeled ligand beacons upon unwinding 
of the stem. 
Where more than one fluorescent group or more than one quenching group is 
used, it is not required that there be an equal number of the two groups. 
For example, the ligand beacon can be labeled with one fluorescent group 
and two quenching groups. If the sites of labeling are sufficiently close 
to one another, then more efficient quenching of the fluorescent group 
would be expected to result. Alternatively, if a given quenching group is 
capable of quenching more than one fluorescent group, then the separation 
of a single effective quenching group from multiple fluorescent groups 
would be expected to give an increased signal relative to separation from 
a single fluorescent group. 
Labeling the ligand beacons with energy transfer pairs can be accomplished 
easily by standard methods well known in the art. For example, it is 
possible to incorporate the fluorescent group fluorescein into the ligand 
beacon at the 5' end during automated oligonucleotide synthesis of the 
sequence. The quenching group DABCYL can be attached to the ligand beacon 
by first incorporating an amino group at the 3' end during oligonucleotide 
synthesis, and then reacting the amino group after synthesis with the 
succinimidyl ester of DABCYL in anhydrous N,N, dimethyl formamide. 
Alternatively, DABCYL can be incorporated directly into the ligand beacon 
during oligonucleotide synthesis. It is important to note that these 
methods can be adapted to place the members of the energy transfer pair at 
any location desired in the ligand beacon. In some embodiments it may not 
be useful to have the labels at the termini. In some instances, for 
example, it may be preferable to label the stem of the ligand beacon at 
positions other than the 5' and 3' termini. This is because under certain 
conditions, the termini of the ligand beacon may temporarily unwind in the 
absence of free nucleic acid ligand; which can lead to background 
fluorescence. 
It is possible to use fluorescent groups with molecules other than 
quenching groups. For example, a fluorescent group can be placed next to a 
modifying group that shifts the emission wavelength, polarizes the 
emission, or even enhances it. All of these effects result from FRET. 
Using the instant methods, it is possible to simultaneously detect multiple 
target molecules in a test solution using ligand beacons. In this method, 
each target molecule is recognized by a distinct nucleic acid ligand and 
each nucleic acid ligand can hybridize to a different ligand beacon. Each 
ligand beacon in the assay has at least a different loop sequence, 
specific for a particular nucleic acid ligand. However, it is not 
necessary that each ligand beacon has a different stem sequence: the stem 
sequence does not impart the specificity of the ligand beacon, so it is 
possible to use a common stem for every ligand beacon. In addition, each 
ligand beacon is labeled with at least a different fluorescent group. For 
example, to detect two different targets, two different nucleic acid 
ligands and two different ligand beacons are required. For example, one 
ligand beacon may be labeled with fluorescein and DABCYL, and the second 
labeled with rhodamine and DABCYL. Therefore, the concentration of the two 
targets can be determined in the test solution by monitoring the increase 
in both fluorescein and rhodamine emission. 
It is important to note that it is not necessary to have any structural 
information about a nucleic acid ligand when designing its cognate ligand 
beacon. Given the rapidity with which one can synthesize the ligand 
beacons, only simple, routine experimentation is required to design 
several different ligand beacons for each nucleic acid ligand, each ligand 
beacon recognizing a sequence that it at least partially unique. The 
candidate ligand beacons can be quickly tested to determine which one has 
the desired activity. 
As described above, preferred embodiments use ligand beacons that can bind 
to nucleic acid ligands only when the nucleic acid ligand is not bound to 
its target. However, the invention also includes ligand beacons that 
function in the converse manner. Specifically, the invention also includes 
ligand beacons that can only hybridize to nucleic acid ligands that are 
bound to their cognate targets. For example, it is possible to obtain 
nucleic acid ligands that adopt a primary conformation in the absence of 
target, but undergo a conformational change upon target binding. Such a 
conformational change may cause regions of the nucleic acid ligand that 
are initially double-stranded to become single-stranded. The ligand beacon 
can hybridize to these single-stranded regions, but not when they are 
double-stranded. As a result, the increase in fluorescence intensity that 
occurs upon mixing the nucleic acid ligand, the ligand beacon and the 
target is directly proportional to the amount of the target. 
In other embodiments, the ligand beacon has a structure in which nucleotide 
positions that are initially separated become adjacent upon hybridizing to 
the nucleic acid ligand. If these nucleotide positions are labeled as 
described above with a fluorescent group and a quenching group, then 
hybridization to the nucleic acid ligand results in a decrease in the 
ligand beacon's fluorescence emission. 
Although the preferred ligand beacons of the invention have a stem-loop 
architecture, there is no limitation on the structure of ligand beacons. 
Any nucleic acid structure that undergoes a change in configuration upon 
hybridizing to a nucleic acid ligand wherein individual nucleotides move 
relative to one another in a reproducible manner is contemplated herein. 
It is possible to stack more than one G-quartet on top of each other under 
appropriate ionic conditions. In this embodiment of the invention, the 
nucleotides that are located between the G-quartet residues comprise the 
nucleic acid sequences complementary to the nucleic acid ligand. The 
G-quartet residues are labeled with the energy transfer pair(s); upon 
hybridization of the ligand beacon to the nucleic acid ligand, the 
G-quartet is disrupted, and the energy transfer pair(s) are separated. 
In order to determine the concentration of a target molecule in a test 
mixture, it is preferable to first obtain reference data in which constant 
amounts of ligand beacon and nucleic acid ligand are contacted with 
varying amounts of target. The fluorescence emission of each of the 
reference mixtures is used to derive a graph or table in which target 
concentration is compared to fluorescence emission. For example, a ligand 
beacon that a) hybridizes to a target-free nucleic acid ligand; and b) has 
a stem-loop architecture with the 5' and 3' termini being the sites of 
fluorescent group and quenching group labeling, could be used to obtain 
such reference data. Such a ligand beacon would give a characteristic 
emission profile in which the fluorescence emission decreases as the 
target concentration increases in the presence of a constant amount of 
ligand beacon and nucleic acid ligand. Then, a test mixture with an 
unknown amount of target would be contacted with the same amount of first 
nucleic acid ligand and second ligand beacon, and the fluorescence 
emission would be determined. The value of the fluorescence emission would 
then be compared with the reference data to obtain the concentration of 
the target in the test mixture. 
In some embodiments, the nucleic acid ligand becomes covalently attached to 
its target molecule in the assay. Methods for obtaining nucleic acid 
ligands with this capability are described in U.S. patent application Ser. 
No. 08/123,935, filed Sep. 17, 1993, entitled "Photoselection of Nucleic 
Acid Ligands", now abandoned, and in U.S. patent application Ser. No. 
08/612,895 filed Mar. 8 1996, now U.S. Pat. No. 5,763,177, both of which 
are specifically incorporated herein in their entirety. 
The assays that are possible using ligand beacons are far simpler than 
conventional techniques for detecting non-nucleic acid target molecules. 
The assays require only three manipulations: a) addition of the nucleic 
acid ligand(s); b) addition of the ligand beacons; and c) measurement of 
the fluorescence. In many embodiments, there is no need to perform any 
washing steps to remove background signal, unlike the ELISA assays known 
in the art. Therefore, the present invention provides a single common 
method that can be applied to virtually any target molecule. Because of 
the simplicity of the assay, it is particularly well suited to 
high-throughout automated analysis for medical diagnostic purposes. 
In some embodiments, the ligand beacons are used in assays in which nucleic 
acid ligands are attached to the surface of a solid support. Methods for 
attaching nucleic acids to solid supports are well known in the art. In 
these assays, the fluorescence emission from the solid support is 
monitored after the solid support is contacted with the test mixture 
suspected of containing the target, and the ligand beacon. It is also 
possible to use multiple ligand beacons in assays in which a plurality of 
different nucleic acid ligands are attached to spatially discrete 
addresses on a solid support, forming an array. Nucleic acid ligand arrays 
are described in co-pending and commonly assigned U.S. patent application 
Ser. No. 08/990,436, filed Dec. 15, 1997 entitled "Nucleic Acid Ligand 
Diagnostic Biochip", specifically incorporated herein by reference in its 
entirety. These assays require that each nucleic acid ligand is recognized 
by a different ligand beacon with at least a unique loop sequence and a 
unique fluorescent group, as described above. Measuring the fluorescence 
emission profile of each address on the array reveals the concentration of 
each target molecule. 
In still further embodiments, one or more ligand beacons are attached to 
the solid support. Each ligand beacon can be attached via one of its 
termini by a spacer molecule to allow the ligand beacon to adopt the 
appropriate conformations without hindrance from the underlying solid 
support. A test mixture is contacted with one or more nucleic acid 
ligands, and the mixture is contacted with the solid support. Again, 
measurement of the fluorescent emission profile at each address of the 
array reveals the concentration of each target molecule in the test 
mixture. 
The present invention also provides kits for the detection of particular 
targets in test mixtures. The kit comprises separate containers containing 
solutions of a nucleic acid ligand to the particular target, and 
containing solutions of the appropriate ligand beacon. In some 
embodiments, the kit comprises a solid support to which is attached the 
nucleic acid ligand to the particular target. In further embodiments, the 
kit further comprises a container containing a standardized solution of 
the target. With this solution, it is possible for the user of the kit to 
prepare a graph or table of fluorescence units vs. target concentration; 
this table or graph is then used to determine the concentration of the 
target in the test mixture. 
EXAMPLES 
Example 1 
Ligand Beacon for Use with PDGF Nucleic Acid Ligand 
A nucleic acid ligand to human platelet derived growth factor (PDGF) with 
the following sequence was obtained by the SELEX.TM. process: 
SEQ ID NO: 1 5'-tgggagggcgcgttcttcgtggttacttttagtcccgt-3' 
The sequence in bold above was used to design a ligand beacon with the 
following sequence: 
SEQ ID NO: 2 5'gcgagaaagtaaccacgaagaagaacgcgcccctcgc3' 
wherein the bold sequence in the ligand beacon is complementary to the bold 
sequence in the nucleic acid ligand, and the underlined sequences form the 
stem. 
The PDGF ligand beacon was synthesized by standard oligonucleotide 
chemistry with an amino linker at the 3' terminus and a fluorescein at the 
5' terminus. After deprotection, the oligonucleotide was resuspended in 
100 mM sodium borate buffer (pH 9.3) at 4 mg/ml, and mixed with an equal 
volume of the succinimidyl ester of (4-dimethylaminophenylazo)benozoic 
acid (DABCYL) in anhydrous N,N, dimethyl formamide (5 mg/100 .mu.L). The 
reaction was allowed to proceed for 30 minutes at room temperature. 
Unreacted DABCYL was removed from the derivatized oligonucleotide by 
passing the reaction mixture through a 5000 MW cutoff Centricon filter. 
Subsequently, derivatized oligonucleotide was purified by gel 
electrophoresis under denaturing conditions. The ligand beacon was heated 
to 80.degree. C. in PBSM buffer [10.1 mM Na.sub.2 HPO.sub.4, 1.8 mM 
KH.sub.2 PO.sub.4, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl.sub.2, (pH 7.4)] and 
slowly cooled to room temperature before use. 
The PDGF ligand beacon was used in an assay in which 100 nM PDGF nucleic 
acid ligand above was mixed with an increasing concentration of PDGF for 
10 minutes at 30.degree. C. polymerase in PBSM buffer containing 4 .mu.M 
tRNA. Then, 100 nM PDGF ligand beacon was added and the mixture was 
incubated for 10 minutes at 30.degree. C. A measurement of fluorescein 
emission at 530 nm was made for each concentration of PDGF using 488 nm 
monochromatic laser light for excitation in a 96 well format Vistra 
Fluorimager SI. The results are displayed in FIG. 3, wherein the X axis 
displays the concentration of PDGF in nanomoles and the Y axis displays 
fluorescein emission in arbitrary units. The results show that as the 
concentration of PDGF increases, the fluorescence signal decreases. This 
is the expected result, because as the concentration of PDGF increases, 
the concentration of PDGF nucleic acid ligand that is not bound to PDGF 
decreases. Thus, there is a smaller pool of PDGF nucleic acid ligand for 
the PDGF ligand beacon to hybridize to. 
Example 2 
Ligand Beacon for use with Nucleic Acid Ligand to TAQ Polymerase 
A nucleic acid ligand to Thermophilus aqualicus (TAQ) DNA Polymerase with 
the following sequence was obtained through the SELEX.TM. methodology: 
SEQ ID NO:3 5'-tggcggagcgatcatctcagagcattcttagcgttttgttcttgtgtatga-3' 
The sequence in bold above was used to design a ligand beacon with the 
following sequence: 
SEQ ID NO:4 5'-gcgagcaagaacaaaacgtaagaatgctctcgc-3' 
wherein the bold sequence in the ligand beacon is complementary to the bold 
sequence in the nucleic acid ligand, and the underlined sequences form the 
stem. The ligand beacon was labeled with fluorescein at the 5' terminus 
and DABCYL at the 3' terminus as described in Example 1. 
An assay using constant concentrations of TAQ nucleic acid ligand and TAQ 
ligand beacon, and varying concentrations of TAQ DNA Polymerase, was 
carried out according to the method of Example 1. Again, the fluorescence 
emission decreased with increasing amounts of the ligand TAQ Polymerase. 
The results are shown in FIG. 4. 
Example 3 
Specificity of Ligand Beacon Interaction with Nucleic Acid Ligand 
In order to test the specificity of the interactions of the ligand beacons 
with their cognate nucleic acid ligands, the TAQ ligand beacon and the 
PDGF ligand beacon were contacted with either their cognate nucleic acid 
ligand, or a twenty nucleotides-long linear template oligonucleotide 
sequence that is complementary to the ligand beacon, or a non-cognate 
nucleic acid ligand. The results are shown in FIGS. 5 and 6. In the 
example shown in FIG. 5, 200 nM PDGF-ligand beacon was mixed with 
increasing concentration of PDGF nucleic acid ligand (closed circles), 
20-nt linear PDGF template (open circle) or TAQ nucleic acid ligand 
(asterisks) in PBSM buffer containing 4 PM tRNA and incubated at 
37.degree. C. for 10 min before fluorescence was measured. Fluorescence 
was measured at 530 nm after exiting at 488 nm using monochromatic laser 
light in 96-well format Vistra fluorimager SI. Each experiment was done in 
duplicate. 
In the example shown in FIG. 6, 200 nM TAQ-ligand beacon was mixed with 
increasing concentration of TAQ nucleic acid ligand (closed circles), 
20-nt linear TAQ template (open circle) and PDGF nucleic acid ligand 
(asterisks) in PBSM buffer containing 4 .mu.M tRNA and incubated at 
30.degree. C. for 10 min before fluorescence was measured. Each experiment 
was done in duplicate. 
In both FIG. 5 and FIG. 6, open circles depict the signal when the ligand 
beacon met with the linear 20-nt template, whereas closed circles show the 
signal generated in the presence of nucleic acid ligands. In both systems 
the signal generated in the presence of nucleic acid ligands was somewhat 
lower but not all that different from the signal generated in the presence 
of 20-nt template. This observation suggests that ligand beacons can 
effectively hybridized with nucleic acid ligands at temperatures under 
which we typically carry out affinity selections (30.degree. C.). When 
wrong combinations of nucleic acid ligand-ligand beacon pairs were mixed, 
virtually no fluorescence signal is generated, indicating the high degree 
of specificity in signal generation. This demonstrates that a multiplexed 
assay involving multiple nucleic acid ligand and ligand beacons is 
feasible. 
Moreover, these experiments were carried out in the presence of a vast 
excess of tRNA, further indicating the lack of interference on signal 
generation by the presence of non-20 specific nucleic acids. 
Example 4 
Specificity of Nucleic Acid Ligand Interaction with Target in the Presence 
of Ligand Beacon 
In the previous examples, a concentration-dependent signal reduction was 
observed when the target was added. This should not be observed when the 
wrong target protein is added. This point is illustrated in FIG. 7, where 
TAQ-nucleic acid ligand (100 nM) was mixed with increasing concentrations 
of PDGF in PBSM buffer containing 4 .mu.M tRNA at 37.degree. C. for 10 
min. Then 110 nM TAQ-ligand beacon was added, incubated for an additional 
10 min at the same temperature and fluorescence was measured. 
The results of FIGS. 5, 6, and 7 illustrate that signal generation is 
specific for the target protein. 
Example 5 
Use of ligand beacons and nucleic acid ligands to Selectins 
Ligand beacons were synthesized for P-Selectin and L-Selectin nucleic acid 
ligands. The sequences of the appropriate nucleic acid ligands and their 
cognate ligand beacons are given below: 
SEQ ID NO: 5 
5'-tagccaaggt aaccagtacaa ggtgctaaac gtaatggcttc ggcttac-3': L-Selectin 
nucleic acid ligand 
SEQ ID NO: 6 
5'-Fgcgagtgtac tggttacctt ggctactcg cD-3' L-Selectin ligand beacon 
SEQ ID NO: 7 
5'-cucaacgagc caggaacauc gaggucagca aacgcgag-3' P-Selectin nucleic acid 
ligand 
SEQ ID NO: 8 
5'-Fgcgagctcgc gtttgctgac gtcgactcg cD-3' P-Selectin ligand beacon 
wherein the L-Selectin nucleic acid ligand is a 49-mer single-stranded DNA, 
and the P-Selectin nucleic acid ligand is a 38-mer RNA molecule containing 
2' F-substituted pyrimidines. The F represents fluorescein, and D 
represents DABCYL. As in the previous examples, the ligand beacons were 
synthesized with fluorescein at the 5' end, and a free amino group at the 
3' end. The free amino group was reacted with the succinimidyl ester of 
DABCYL in order to position DABCYL at the 3' end of the ligand beacon. 
As shown in FIG. 8, L-Selectin nucleic acid ligand (200 nM) was mixed with 
increasing concentrations of L-Selectin in SHMCK buffer containing 4 .mu.M 
tRNA at 37.degree. C. for 15 min. Then 220 nM L-Selectin-Beacon was added, 
incubated for an additional 10 min at the same temperature and 
fluorescence was measured. The observed fluorescence signal was plotted 
against the corresponding concentration of L-Selectin. As depicted in FIG. 
9, P-Selectin nucleic acid ligand (200 nM) was mixed with increasing 
concentration of P-Selectin in SHMCK buffer containing 4 .mu.M tRNA at 
37.degree. C. for 15 min. Then 220 nM P-Selectin-Beacon was added, 
incubated for an additional 10 min. at the same temperature and 
fluorescence was measured. The observed fluorescence signal was plotted 
against the corresponding concentration of P-Selectin. In both 
experiments, the intensity of the fluorescence signal decreased with the 
increase in Selectin concentration. 
L-Selectin nucleic acid ligand (800 nM) was mixed with increasing 
concentration of L-Selectin in SHMCK buffer containing 4 .mu.M tRNA at 
37.degree. C. for 15 min. The results are shown in FIG. 10. The 
concentrations used here were higher than those used in the example shown 
in FIG. 9. Then 800 nM L-Selectin-ligand beacon was added, incubated for 
an additional 10 min at the same temperature and fluorescence was 
measured. The observed fluorescence signal was plotted against the 
corresponding concentration of L-Selectin. It can be seen that the dynamic 
range of the assay can be easily varied by changing the concentration of 
the nucleic acid ligand/ligand beacon pair. 
Example 6 
Interaction of the Ligand Beacon with Nucleic Acid Ligands to Homologous 
Proteins 
In order to demonstrate the specificity of the ligand beacon/nucleic acid 
ligand interaction, an assay was performed in which the P-Selectin nucleic 
acid ligand was mixed with its cognate ligand beacon and L-Selectin. 
Specifically, L-Selectin nucleic acid ligand (200 nM) was mixed with 
increasing concentrations of P-Selectin in SHMCK buffer containing 4 .mu.M 
tRNA. The mixture was incubated at 37.degree. C. for 15 min. Then 220 
.mu.M L-Selectin-Beacon was added, incubated additional 10 min at the same 
temperature and fluorescence was measured (FIG. 11 closed circles). Then, 
P-Selectin nucleic acid ligand was mixed with its cognate ligand beacon 
and L-Selectin. Specifically, P-Selectin nucleic acid ligand (200 nM) was 
mixed with increasing concentrations of L-Selectin in SHMCK buffer 
containing 4 .mu.M tRNA at 37.degree. C. for 15 min. Then 220 nM 
P-Selectin-Beacon was added, incubated for an additional 10 min at the 
same temperature and fluorescence was measured (FIG. 11; open circles). 
As can be seen from the results shown in FIG. 1, there is a little or no 
change in the fluorescence intensity when the wrong Selectin is added. In 
the presence of the wrong target protein the nucleic acid ligand is 
available for binding to the ligand beacon resulting in high fluorescence. 
This result indicates that the change in fluorescence is dependent on the 
presence of the specific target. 
Example 7 
Use of Ligand Beacons in Plasma 
The results of a ligand beacon assay for detecting PDGF in human plasma arc 
illustrated in FIG. 12. In this assay, PDGF nucleic acid ligand (100 nM) 
was mixed with increasing concentrations of PDGF in human plasma 
containing 4 .mu.M tRNA. The mixture was incubated at 37.degree. C. for 15 
min. Then 110 nM PDGF-Beacon was added, incubated for an additional 10 min 
at the same temperature and fluorescence was measured. The observed 
fluorescence signal was plotted against the corresponding concentration of 
PDGF. 
The results of a ligand beacon assay for detecting L-Selectin in human 
plasma are illustrated in FIG. 13. In this assay, L-Selectin nucleic acid 
ligand (800 nM) was mixed with increasing concentrations of L-Selectin in 
human plasma containing 4 .mu.M tRNA. The mixture was incubated at 
37.degree. C. for 15 min. Then 800 nM L-Selectin-Beacon was added, 
incubated for an additional 10 min at the same temperature and 
fluorescence was measured. The observed fluorescence signal was plotted 
against the corresponding concentration of L-Selectin. 
The results of a ligand beacon assay for detecting P-Selectin in human 
plasma are illustrated in FIG. 14. L-Selectin nucleic acid ligand (100 nM) 
was mixed with increasing concentrations of P-Selectin in human plasma 
containing 4 .mu.M tRNA. The mixture was incubated at 37.degree. C. for 15 
min. Then 100 nM P-Selectin-Beacon was added, incubated for an additional 
10 min at the same temperature and fluorescence was measured. The observed 
fluorescence signal was plotted against the corresponding concentration of 
P-Selectin. 
These three examples demonstrate that ligand beacons can be successfully 
used with plasma. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- &lt;160&gt; NUMBER OF SEQ ID NOS: 8 
- &lt;210&gt; SEQ ID NO 1 
&lt;211&gt; LENGTH: 38 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
#Sequence:Nucleic AcidN: Description of Artificial 
#Factor (PDGF)o Platelet Derived Growth 
- &lt;400&gt; SEQUENCE: 1 
# 38 tcgt ggttactttt agtcccgt 
- &lt;210&gt; SEQ ID NO 2 
&lt;211&gt; LENGTH: 37 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
#Sequence:LigandRMATION: Description of Artificial 
Beacon to PDGF Nucleic Acid Ligan - #d of SEQ ID NO:1 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: misc.sub.-- feature 
&lt;222&gt; LOCATION: (1) 
&lt;223&gt; OTHER INFORMATION: 5' Fluorescein 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: misc.sub.-- feature 
&lt;222&gt; LOCATION: (37) 
&lt;223&gt; OTHER INFORMATION: 3' (4-dimethylaminophenylazo - #) benzoic acid 
(DABCYL) 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: stem.sub.-- loop 
&lt;222&gt; LOCATION: (1)..(37) 
- &lt;400&gt; SEQUENCE: 2 
# 37 gaag aagaacgcgc ccctcgc 
- &lt;210&gt; SEQ ID NO 3 
&lt;211&gt; LENGTH: 51 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
#Sequence:Nucleic AcidN: Description of Artificial 
Ligand to Thermophilus aquaticus (TA - #Q) DNA 
Polymerase 
- &lt;400&gt; SEQUENCE: 3 
# 51atctcag agcattctta gcgttttgtt cttgtgtatg a 
- &lt;210&gt; SEQ ID NO 4 
&lt;211&gt; LENGTH: 34 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
#Sequence:LigandRMATION: Description of Artificial 
Beacon to TAQ Polymerase Nucleic - # Acid Ligand of SEQ ID NO:3 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: misc.sub.-- feature 
&lt;222&gt; LOCATION: (1) 
&lt;223&gt; OTHER INFORMATION: 5' Fluorescein 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: misc.sub.-- feature 
&lt;222&gt; LOCATION: (34) 
&lt;223&gt; OTHER INFORMATION: 3' (4-dimethylaminophenylazo - #) benzoic acid 
(DABCYL) 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: stem.sub.-- loop 
&lt;222&gt; LOCATION: (1)..(34) 
- &lt;400&gt; SEQUENCE: 4 
# 34 cgct aagaatgctc tcgc 
- &lt;210&gt; SEQ ID NO 5 
&lt;211&gt; LENGTH: 49 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
#Sequence:Nucleic AcidN: Description of Artificial 
Ligand to L-Selectin 
- &lt;400&gt; SEQUENCE: 5 
# 49gtaca aggtgctaaa cgtaatggct tcggcttac 
- &lt;210&gt; SEQ ID NO 6 
&lt;211&gt; LENGTH: 30 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
#Sequence:LigandRMATION: Description of Artificial 
#Ligand of SEQ ID NO:5ctin Nucleic Acid 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: misc.sub.-- feature 
&lt;222&gt; LOCATION: (1) 
&lt;223&gt; OTHER INFORMATION: 5' Fluorescein 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: misc.sub.-- feature 
&lt;222&gt; LOCATION: (30) 
&lt;223&gt; OTHER INFORMATION: 3' (4-dimethylaminophenylazo - #) benzoic acid 
(DABCYL) 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: stem.sub.-- loop 
&lt;222&gt; LOCATION: (1)..(30) 
- &lt;400&gt; SEQUENCE: 6 
# 30 cctt ggctactcgc 
- &lt;210&gt; SEQ ID NO 7 
&lt;211&gt; LENGTH: 38 
&lt;212&gt; TYPE: RNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
#Sequence:Nucleic AcidN: Description of Artificial 
Ligand to P-Selectin 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: modified.sub.-- base 
&lt;222&gt; LOCATION: (1)..(38) 
&lt;223&gt; OTHER INFORMATION: C, U are 2' F 
- &lt;400&gt; SEQUENCE: 7 
# 38 cauc gaggucagca aacgcgag 
- &lt;210&gt; SEQ ID NO 8 
&lt;211&gt; LENGTH: 30 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
#Sequence:LigandRMATION: Description of Artificial 
#Ligand of SEQ ID NO:7ctin Nucleic Acid 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: misc.sub.-- feature 
&lt;222&gt; LOCATION: (30) 
&lt;223&gt; OTHER INFORMATION: 3' (4-dimethylaminophenylazo - #) benzoic acid 
(DABCYL) 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: stem.sub.-- loop 
&lt;222&gt; LOCATION: (1)..(30) 
- &lt;400&gt; SEQUENCE: 8 
# 30 tgac gtcgactcgc 
__________________________________________________________________________