WO2004085665A2

WO2004085665A2 - Nucleic acid ligand to b. anthracis protective antigen

Info

Publication number: WO2004085665A2
Application number: PCT/US2003/020844
Authority: WO
Inventors: Chareles Wilson; David Epstein; Sharon T. Cload; Nicholas Marsh; Nobuko Hamaguchi
Original assignee: Archemix Corp.
Priority date: 2002-06-19
Filing date: 2003-06-19
Publication date: 2004-10-07
Also published as: AU2003304015A1; WO2004085665A3

Abstract

Materials and methods are provided to treat subjects exposed to bacillus anthracis protective antigen to prevent virulence of anthrax disease caused by bacillus anthracis. Materials and methods are also provided to detect the presence of bacillus anthracis protective antigen in a sample.

Description

NUCLEIC ACID LIGANDS TO B. ANTHRACIS PROTECTIVE ANTIGEN

FIELD OF THE INVENTION [0001] The invention relates generally to peptides and nucleic acids molecules (aptamers) having high binding affinity and specificity against a non-nucleic acid target. The peptides and nucleic acid molecules of the present invention have therapeutic application against protective antigen of Bacillus anthracis and can inhibit the virulence of this organism and its toxins, and are thus useful as a therapeutic against anthrax. The nucleic acid molecules of the present invention also have diagnostic application in the detection of Bacillus anthracis and/or its toxins.

BACKGROUND OF THE INVENTION [0002] Anthrax is an acute infectious disease caused by the bacterium Bacillus anthracis. The inhalational form of the disease represents a particular concern for biodefense because of its high lethality (90-99% mortality) and the relative ease with which the disease may be deliberately spread using aerosolized spores. While antibiotics including penicillin, ciprofloxacin, and doxycycline can be administered prophylactically, these treatments are largely ineffective once symptoms of the disease have developed. Given the weaponization of anthrax by several nations/groups and recent events involving the intentional release of spores in the United States, a pressing need has emerged for development of a means for detecting of Bacillus anthracis and/or its toxins and for a therapeutic strategy for post- symptomatic inhalational anthrax.

[0003] Anthrax is an ancient disease that has existed throughout the world for centuries. While most commonly affecting herbivores such as cattle, goats, or sheep, anthrax sporadically affects humans who come in contact with infected animals or their tissues/byproducts (especially goat hair) (Lew, 1995). References cited herein by author and year of publication are given a full citation below, and each is herein incorporated by reference in its entirety. The disease presents in three clinical forms: [0004] Cutaneous anthrax results from exposure to open cuts or breaks in the skin. Following a 1-6 day incubation period, itching develops at the site of exposure and is rapidly followed by the development of a large, black lesion. While untreated cutaneous anthrax has 5-20% mortality, this form of the disease can be readily treated with antibiotics (Perm, 1998).

[0005] Gastrointestinal anthrax results from digestion of infected, incompletely cooked animal tissues and manifests as either an oral/esophageal ulcer that causes lymphadenopathy, edema, and sepsis or as an intestinal lesion that causes nausea, vomiting, diarrhea, and sepsis. While mortality rates are higher than those of cutaneous anthrax (25- 65%), this form of the disease is exceptionally rare.

[0006] Inhalational anthrax, the third form of the disease, provides the largest cause for concern because of its high mortality (90-99%), the ineffectiveness of antibiotics once symptoms have developed, and the ease with which the disease may be spread through the intentional release of processed spores.

[0007] Because of its lethality and relative ease of production, anthrax was a target for development as a bioweapon by several nations during the last century (Christopher, 1997). Following an accidental anthrax release in Sverdlovsk, Russia in 1979, at least 68 people are known to have died (Meselson, 1994). As part of its biological warfare programs, the Soviet Union is estimated to have produced tens of tons of weapons-grade anthrax. In 1995, Iraqi officials admitted they had produced 8,500 liters of anthrax cultures and prepared a variety of weapons carrying anthrax and other biowarfare agents (including Scud missile warheads, bombs and aerial dispensers) (Zilinskas, 1997). Projections by the CDC in 1997 suggested that a single terrorist attack (using a small airplane to release anthrax spores over a suburb) could expose 100,000 people, 50,000 of whom would likely develop the inhalational form of the disease, leading to over 30,000 deaths (Kaufman, 1997). The economic cost of such an attack (largely due to premature deaths) would be over $25 billion. In the fall of 2001, intentional distribution of spores through the US mail led to 23 confirmed cases of anthrax (11 inhalational) and five deaths. These recent events have clearly established the need to develop an effective treatment for inhalational anthrax as well as a means to detect the presence of Bacillus anthracis and/or its toxins. [0008] Spores represent the transmissible form of the disease and are generated from vegetative bacteria exposed to poor growth conditions (e.g., low nutrients, open to air) (Dragon, 1995). For spores to efficiently enter the lungs, they must be formulated as 1-5 μm particles (smaller particles are efficiently exhaled without being trapped and larger particles are trapped early in the respiratory tree or fall to the ground) (Druett, 1953). Spores embedded in the alveoli are digested by macrophages but, possibly as a result of bacterial hemolysins, a fraction survive and can be subsequently transported to the mediastinal lymph nodes (Lincoln, 1965). Germination of the spores in the lymph nodes can be delayed as much as 60 days following initial exposure but once initiated, leads to rapid progression of the disease (Friedlander, 1993). As the bacteria replicate, they release toxins causing hemorrhage, edema, and necrosis.

[0009] Clinically, inhalational anthrax presents as a 2-stage illness (Meselson, 1994). During stage 1, patients typically exhibit a variety of non-specific flu-like symptoms including coughing, fever, vomiting, chills, etc. These symptoms continue for hours to a few days before progression. Stage 2 symptoms develop rapidly thereafter and include sudden fever, dyspnea, diaphoresis, and shock. Roughly half of stage 2 patients develop hemorrhagic meningitis. Cyanosis and hypotension develop as the disease progresses and death can result in hours. In monkeys and humans, the average time between presentation with symptoms and death is 3 days (Friedlander, 1993).

[0010] Inhalational anthrax is not easily diagnosed in its earliest stages when treatment with antibiotics is most likely to be effective (Inglesby, 1999). ELISA-based tests for anthrax are available at national reference laboratories but are not generally available on-site at clinics and hospitals. In the absence of molecular diagnostics, anthrax could potentially be identified by radiological means (a widened mediastinum on a chest x-ray) or by microbiological testing (Gram staining of unspun blood). More likely, however, anthrax would be identified through a standard blood culture although definitive identification is likely to take 2-3 days.

[0011] Natural strains of anthrax are resistant to many antibiotics but most are sensitive to penicillin, the historically preferred therapy (Barnes, 1947). More recently, both doxycycline (a tetracyclme class antibiotic) and ciprofloxacin (a fluoroqumolone) have been shown effective in animal models and approved for treatment of pre-symptomatic, post- exposure anthrax (Kelly, 1992). Both antibiotics, however, have known safety issues for children and pregnant women (permanent or transient arthropathy, retarded skeletal growth, discolored teeth) (American Hospital Formulary Service, 1996). Soviet-era weapons programs were directed at the generation of anthrax strains with broad antibiotic resistance and strains resistant to both tetracycline and penicillin have been reported (Stepanov, 1996). [0012] A very short window exists between the earliest presentation of symptoms and the stage when antibiotics lose their effectiveness. Delay of antibiotic treatment by a matter of hours can substantially reduce the likelihood of survival (Lincoln, 1964). Once spores have germinated and the bacteria have started to release their toxin into the blood stream, antibiotics have little impact on the progression of the disease.

[0013] The virulence of Bacillus anthracis can be attributed to primarily two factors: a three-component protein exotoxin (expressed by genes on plasmid pXOl) and a glutamic acid capsule (generated by enzymes encoded on plasmid pX02). The three proteins of the exotoxin include protective antigen (PA, 83 kDa), lethal factor (LF, 90 kDa), and edema factor (EF, 89 kDa) (Dixon, 1999). The toxin is representative of the classic AB model in which PA (the ^ςB' component) acts to deliver the active A components, LF and EF, to the target cell. PA/LF forms the lethal toxin and PA/EF forms the edema toxin. Following initial binding to a cell surface receptor, PA83 is cleaved by proteases to generate a membrane associated 63 kDa fragment (PA63) which self-associates into a heptamer that is capable of binding LF or EF with high affinity (Bradley, 2001). Ffeptameric PA63 and associated LF and/or EF is internalized by receptor-mediated endocytosis. Acidification causes the PA63 heptamer to insert into the endosomal membrane and allows LF or EF translocation into the cytoplasm. Once in the cytoplasm, the catalytic activities of EF as an adenylate cyclase and LF as a MAPKK1 -targeting protease lead to death of the intoxicated cell.

[0014] In vaccination studies, generation of anti-PA antibodies correlates well with immune protection and PA is the primary immunogenic component of the approved AVA vaccine (Friedlander, 1997). Peptides (Mourez, 2001) and antibodies (Little, 1996) which bind to PA to block binding of the catalytic toxin subunits (EF, LF) or dominant mutant PAs that block oligomerization (Sellman, 2001) have been shown to promote survival in animal intoxication studies. Recent studies with recombinant antibody fragments show that protection against toxin correlates well with affinity (Maynard, 2002). [0015] Aptamers, like peptides generated by phage display or monoclonal antibodies (MAbs), are capable of specifically binding to selected targets and, through binding, block their target's ability to function. Created by an in vitro selection process from pools of random sequence oligonucleotides (Fig. 2), aptamers have been generated for over 100 proteins including growth factors, transcription factors, enzymes, immunoglobulins, and receptors. A typical aptamer is 10-15 kDa in size (30-45 nucleotides), binds its target with sub-nanomolar affinity, and discriminates against closely related targets (e.g., will typically not bind other proteins from the same gene family). A series of structural studies have shown that aptamers are capable of using the same types of binding interactions (hydrogen bonding, electrostatic complementarity, hydrophobic contacts, steric exclusion, etc.) that drive affinity and specificity in antibody-antigen complexes.

[0016] Aptamers have a number of desirable characteristics for use as therapeutics or diagnostic agents including high specificity and affinity, biological efficacy, and excellent pharmacokinetic properties. In addition, they offer specific competitive advantages over antibodies and other protein biologies, for example:

[0017] 1) Speed and control. Aptamers are produced by an entirely in vitro process, allowing for the rapid generation of initial leads. In vitro selection allows the specificity and affinity of the aptamer to be tightly controlled and allows the generation of leads against both toxic and non-immunoge ic targets.

[0018] 2) Toxicitv and Immunogenicity. Aptamers as a class have demonstrated little or no toxicity or immunogenicity. In chronic dosing of rats or woodchucks with high levels of aptamer (10 mg/kg daily for 90 days), no toxicity is observed by any clinical, cellular, or biochemical measure. Whereas the efficacy of many monoclonal antibodies can be severely limited by immune response to antibodies themselves, it is extremely difficult to elicit antibodies to aptamers (most likely because aptamers cannot be presented by T-cells via the MHC and the immune response is generally trained not to recognize nucleic acid fragments).

[0019] 3) Administration. Whereas all currently approved antibody therapeutics are administered by intravenous infusion (typically over 2-4 hours), aptamers can be administered by subcutaneous injection. This difference is primarily due to the comparatively low solubility and thus large volumes necessary for most therapeutic MAbs. With good solubility (>150 mg/ml) and comparatively low molecular weight (aptamer: 10- 50 KD; antibody: 150 KD), a weekly dose of aptamer may be delivered by injection in a volume of less than 0.5 ml. Aptamer bioavailabiliτy via subcutaneous administration is >80% in monkey studies (Tucker, 1999).

[0020] 4) Scalability and cost. Therapeutic aptamers are chemically synthesized and consequently can be readily scaled as needed to meet production demand. Whereas difficulties in scaling production are currently limiting the availability of some biologies (e.g., Enbrel, Remicade) and the capital cost of a large-scale protein production plant is enormous (e.g., $500 MM, Immunex), a single large-scale synthesizer can produce upwards of 100 kg oligonucleotide per year and requires a relatively modest initial investment (e.g., <$10 MM, Avecia). The current cost of goods for aptamer synthesis at the kilogram scale is estimated at $500/g, comparable to that for highly optimized antibodies. Continuing improvements in process development are expected to lower the cost of goods to < $100 / g in five years.

[0021] 5) Stability. Aptamers are chemically robust. They are intrinsically adapted to regain activity following exposure to heat, denaturants, etc. and can be stored for extended periods (>1 yr) at room temperature as lyophihzed powders. In contrast, antibodies must be stored refrigerated.

[0022] There is a need for a means of detecting the presence of Bacillus anthracis and a therapeutic agent that is specific against protective antigen (PA) that can protect against the disease in its virulent forms.

[0023] The present invention provides materials and methods of use thereof to treat post- symptomatic inhalational B. anthracis pathogenesis in subjects and to detect the presence of Bacillus anthracis and/or its toxins.

[0024] The references cited above by author and year of publication are given their full citation below and are herein incorporated by reference.

American Hospital Formulary Service. (1996) AHFS Drug Information. Bethesda, Md: American Society of Health System Pharmacists.

Barnes, J.M. (1947) "Penicillin and B anthracis." J Pathol Bacteriol. 194:113-125.

Bradley, K.A., Mogridge, J., Mourez, M., Collier, R.J., & Young, J.A. (2001) "Identification of the cellular receptor for anthrax toxin." Nature 414:225-9.

Christopher, G.W., Cieslak, T.J., Pavlin, J.A., Eitzen, E.M. (1997) "Biological warfare: a historical perspective". JAMA. 278:412-417.

Dixon TC, Meselson M, Guillemin J, Ha na PC. (1999) "Anthrax." N .Engl. J. Med. 341:815-26.

Dragon, D.C., Rennie, R.P.(1995) "The ecology of anthrax spores." Can VetJ. 36:295-301.

Druett, H.A., Henderson, D.W., Packman, L., Peacock, S. (1953) "Studies on respiratory infection." JHyg. 51:359-371.

Friedlander, A., Welkos, S.L., Pitt, M.L., et al. (1993) " Postexposure prophylaxis against experimental inhalation anthrax." J Infect Dis. 167:1239-1242.

Inglesby, Υ.Y., Henderson, D.A., Bartlett, J.G., Ascher, M.S., Eitzen, E., Friedlander, A.M., Hauer, J., McDade, J., Osterholm, M.T., O'Toole, T., Parker, G., Perl, T.M., Russell, P.K., Tonat K. (1999) "Anthrax as a biological weapon: medical and public health management. Working Group on Civilian Biodefense." JAMA 281 : 1735-45. Kaufmann AF, Meltzer MI, Schmid GP. (1997) "The economic impact of a bioterrorist attack: are prevention and postattack intervention programs justifiable?" Emerg. Infect. Dis. 3:83-94.

Lew O."Bacillus anthracis (anthrax)" In: Mandell GL, Bennett JE, Dolin R, eds. Principles and Practices of Infectious Disease.New York, NY: Churchill Livingstone Inc (1995), pp.1885-1889.

Lincoln RE, Hodges DR, Klein F, et al. (1965) "Role of the lymphatics in the pathogenesis of anthrax." J Infect Dis. 115:481-494.

Maynard, J.A., Maassen, C.B.M., Leppla, S.H., Brasky, K., Patterson, J.L., Iverson, B.L., & Georgiou, G., (2002) " Protection against anthrax toxin by recombinant antibody fragments correlates with antigen affinity," Nature Biotechnology, 20: 597-601.

Mourez, M., Kane, R.S., Mogridge, J., Metallo, S., Deschateles, P., Sellman, B.R., Whitesides, G.M., Collier, R.J., (2001) "Designing a polyvalent inhibitor of anthrax toxin," Nature Biotechnology, 19: 958-961

Penn, C, Klotz, S.A. (1998) "Anthrax" In: Gorbach SL, Bartlett JG, Blacklow NR, βdsJnfectious DiseasesΫhϊi&dβlp ia, Pa: WB Saunders Co, pp. 1575-1578.

Stepanov, AN., Marinin, L.I., Pomerantsev, A.P., Staritsin, Ν.A. (1996) "Development of novel vaccines against anthrax in man." J Biotechnol. 44:155-160.

Tucker, C.E., Chen, L.S., Judkins, M.B., Farmer J.A., Gill, S.G. & Drolet, D.W., (1999) "Detection and plasma pharmacokinetics of an anti- vascular endothelial growth factor oligonucleotide-aptamer (ΝX1838) in rhesus monkeys," J. Chromatography B, 732: 203-212.

Zilinskas RA. (1997) "Iraq's biological weapons: the past as future?" JAMA. 278:418-424.

SUMMARY OF THE INVENTION [0025] The Protective Antigen (PA) is a target for the therapeutic aptamer and peptide materials of the present invention to treat anthrax in subjects exposed to B. anthracis in its virulent forms.

[0026] Aptamers, like peptides generated by phage display or monoclonal antibodies (MAbs), are capable of specifically binding to selected targets and, through binding, block their target's ability to function. Thus, the present invention provides aptamers with high affinity and specificity for the protective antigen of B. anthracis and thus cost-effective, stable, low cost, and easily administered therapeutics against early stages of anthrax. [0027] In one embodiment, the present invention provides methods of use of aptamer therapeutics capable of specifically binding with high affinity to protective antigen of B. anthracis having a neutralizing and therapeutic effect in subjects or patients having been exposed to anthrax toxins and showing post-symptomatic inhalational anthrax pathogenesis. [0028] In one embodiment, the present invention provides methods of use of peptide therapeutics capable of specifically binding with high affinity to protective antigen of B. anthracis having a neutralizing and therapeutic effect in subjects or patients having been exposed to anthrax toxins and showing post-symptomatic inhalational anthrax pathogenesis. [0029] In one embodiment, the present invention provides methods to obtain aptamers having high specific binding affinity to B. anthracis protective antigen. [0030] In one embodiment, the present invention provides materials to detect the presence of B. anthracis protective antigen in a sample.

[0031] In one embodiment, the materials of the present invention are useful as therapeutics in subjects exposed to anthrax in its various virulent forms. In one embodiment, those subjects are humans . In another embodiment, the subjects are animals exposed to Bacillus anthracis and or anthrax toxins in their various virulent forms.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 shows the pathogenesis mechanism of B. anthracis protective antigen. [0033] Figure 2 shows the in vitro aptamer selection process from pools of random sequence oligonucleotides. [0034] Figure 3 shows the steps required to generate a therapeutic aptamer. The process can be approximately considered in four phases: (i) set up, (ii) lead generation, (iii) lead minimization, and (iv) lead optimization for stability and distribution.

[0035] Figure 4 shows run off transcription with 2'-fluoropyrimidine NTPs. All transcriptions included 4 mM natural ATP, GTP and 4 mM 2'-F-CTP, 2'-F-UTP. 1.

Control fixed sequence template, wild type T7 RNA polymerase; 2. control template;

Y639F T7 RNA polymerase; 3. N40 pool template, Y639F T7 RNA polymerase.

[0036] Figure 5 shows the enrichment of a functional aptamer diluted into a background of mutant, non-binding aptamers.

[0037] Figure 6 shows improvement in plasma pharmacokinetics as 2'-ribonucleotides are progressively replaced with either 2'-fluoro or 2'-0-methyl nucleotides

[0038] Figure 7 shows A) 20K or 40K PEGs attached to the 5 '-end of the VEGF aptamer have a minimal effect on binding affinity; and B) PEGylation of the VEGF aptamer substantially increases plasma half-life in Sprague Dawley Rats (n=3-6).

[0039] Figure 8 shows pool RNA (42A, 42B, and 42C) incubated in binding buffer with 0,

250 or 500 nM PA for 30 minutes at room temperature followed by partitioning with a nitrocellulose/nylon sandwich filter. RNA:protein complex is captured on the nitrocellulose membrane and unbound RNA is captured on the nylon membrane.

[0040] Figure 9 shows a schematic of binding curves for aptamers of the present invention.

[0041] Figure 10 shows the Anti-PA aptamers of the present invention protect RAW 264.7 cells from PA/LF-induced cell death.

[0042] Figure 11 shows the hammerhead nucleic acid sensor molecule selection methodology.

[0043] Figure 12 shows a schematic diagram in which the oligonucleotide population is screened for a nucleic acid sensor molecule which comprises a target molecule activatable ligase activity.

[0044] Figure 13 shows a schematic diagram in which an oligonucleotide population is screened for a nucleic acid sensor molecule which comprises a target molecule having activatable self-cleaving activity.

[0045] Figure 14 shows the arrangement of various fluorophore-quencher pairs.

[0046] Figures 15A, B, and C, show a schematic diagram of a self-cleaving ribozyme such as the hammerhead (in this case attached to a solid support via a linker molecule is shown) labeled with a fluorophore. [0047] Figure 16 shows a schematic diagram of core hammerhead NASMs modified to contain a donor fluorophore (D) covalently attached to the 3 '-end of the NASM.

[0048] Figure 17 shows a schematic diagram of the 3 '-terminus that contains one of the dye modifications separated and dissociated away from the core NASM upon effector-mediated cleavage of the hammerhead NASM.

[0049] Figures 18A and 18B show an exemplary embodiment of a non-isotopic proximity assay based on nucleic acid sensor molecules.

[0050] Figure 19A is a schematic representation of an example of a self-ligating nucleic acid sensor molecule bound to a solid support when used in a TIR-illuminated detection scheme where there is a signal increase upon target binding. Figure 19B is a schematic representation of the same sensor in an epi-illuminated configuration, where target binding is detected by monitoring changes of the fluorophore bound to the substrate at the surface of the array. Figure 19C is a schematic representation of the same epi-illuminated configuration, where target binding is detected by monitoring changes in the fluorescence polarization.

[0051] Figure 20 is a schematic representation of a NASM of a ligase ribozyme tethered to a chip by a capture oligonucleotide.

[0052] Figure 21 shows a nitrocellulose filter binding assay.

[0053] Figure 22 shows the alignment of the consensus sequences for each of the 3 protective antigen binding groups, the underlined regions originate from the random region of the pool RNA.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0054] In order to more clearly and concisely describe and point out the subject matter of the claimed invention, the following definitions are provided for specific terms which are used in the following written description and the appended claims. [0055] As defined herein, "nucleic acid" means either DNA, RNA, single-stranded or double-stranded, and any chemical modifications thereof. Modifications include, but are 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.

[0056] As defined herein, "oligonucleotide" is used interchangeably with the term "nucleic acid" and includes RNA or DNA (or RNA/DNA) sequences of more than one nucleotide in either single strand or double-stranded form. A "modified oligonucleotide" includes at least one nucleotide residue with any of: an altered internucleotide linkage(s), altered sugar(s), altered base(s), or combinations thereof.

[0057] As defined herein, "target" means any compound or molecule of interest for which a nucleic acid ligand exists or can be generated. A target molecule can be naturally occurring or artificially created, including 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. [0058] As defined herein, a nucleic acid sensor molecule which "recognizes a target molecule" is a nucleic acid molecule whose activity is modulated upon binding of a target molecule to the target modulation domain to a greater extent than it is by the binding of any non-target molecule or in the absence of the target molecule. The recognition event between the nucleic acid sensor molecule and the target molecule need not be permanent during the time in which the resulting allosteric modulation occurs. Thus, the recognition event can be transient with respect to the ensuing allosteric modulation (e.g., conformational change) of the nucleic acid sensor molecule.

[0059] As defined herein, a molecule which "naturally binds to DNA or RNA" is one which is found within a cell in an organism found in nature. [0060] As defined herein, a "random sequence" or a "randomized sequence" is a segment of a nucleic acid having one or more regions of fully or partially random sequences. A fully random sequence is a sequence in which there is an approximately equal probability of each base (A, T, C, and G) being present at each position in the sequence. In a partially random sequence, instead of a 25% chance that an A, T, C, or G base is present at each position, there are unequal probabilities. [0061] As defined herein, an "aptamer" is a nucleic acid which binds to a non-nucleic acid target molecule or a nucleic acid target through non-Watson-Crick base pairing.

[0062] As defined herein, an aptamer nucleic acid molecule which "recognizes a target molecule" is a nucleic acid molecule which specifically binds to a target molecule.

[0063] As defined herein, a "nucleic acid sensor molecule" or "NASM" refers to either or both of a catalytic nucleic acid sensor molecule and an optical nucleic acid sensor molecule.

[0064] As defined herein, a "nucleic acid ligand" refers to either or both an aptamer or a

NASM.

[0065] As defined herein, a "biosensor" comprises a plurality of nucleic acid ligands.

[0066] As defined herein, "substrate" means any physical supporting surface, whether rigid, flexible, solid, porous, gel-based, or of any other material or composition. A substrate includes a 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 as spherical surfaces and grooved surfaces.

[0067] As defined herein, an "array" or "microarray" refers to a biosensor comprising a plurality of nucleic acid sensor molecules immobilized on a substrate.

[0068] As defined herein, "specificity" refers to the ability of a nucleic acid of the present invention to recognize and discriminate among competing or closely-related targets or ligands. The degree of specificity of a given nucleic acid is not necessarily limited to, or directly correlated with, the binding affinity of a given molecule. For example, hydrophobic interaction between molecule A and molecule B has a high binding affinity, but a low degree of specificity. A nucleic acid that is 100 times more specific for target A relative to target B will preferentially recognize and discriminate for target A 100 times better than it recognizes and discriminates for target B.

[0069] As defined herein, "selective" refers to a molecule that has a high degree of specificity for a target molecule.

[0070] As defined herein, a "fixed region" is a nucleic acid sequence which is known.

[0071] As defined herein, a "signal" is a detectable physical quantity, impulse or object. [0072] As defined herein, an "optical signal" is a signal the optical properties of which can be detected.

[0073] As defined herein, a "biological agent" is a substance produced by or found within a living organism.

[0074] As defined 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.

[0075] As defined 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.

[0076] As defined 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-ammoethyl)amino]napthalene-l-sulfonic acid (EDANS), and Lucifer yellow. Fluorescent groups may also be referred to as "fluorophores". [0077] As defined 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. [0078] As defined 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.

[0079] As defined 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.

[0080] "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.

[0081] As defined 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). [0082] As defined 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. [0083] As defined 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.

[0084] 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.

[0085] As defined herein, an "aptamer" is a nucleic acid which binds to a non-nucleic acid target molecule or a nucleic acid target through non-Watson-Crick base pairing. [0086] As defined herein, an aptamer nucleic acid molecule which "recognizes a target molecule" is a nucleic acid molecule which specifically binds to a target molecule. [0087] As defined herein, a "nucleic acid sensor molecule" or "NASM" refers to either or both of a catalytic nucleic acid sensor molecule and an optical nucleic acid sensor molecule.

[0088] As defined herein, a "catalytic nucleic acid sensor molecule" is a nucleic acid sensor molecule comprising a target modulation domain, a linker region, and a catalytic domain.

[0089] As defined herein, an "optical nucleic acid sensor molecule" is a catalytic nucleic acid sensor molecule wherein the catalytic domain has been modified to emit an optical signal as a result of and/or in lieu of catalysis by the inclusion of an optical signal generating unit.

[0090] As defined herein, a "nucleic acid ligand" refers to either or both an aptamer or

NASM.

[0091] As defined herein, a "target modulation domain" (TMD) is the portion of a nucleic acid sensor molecule which recognizes a target molecule. The target modulation domain is also sometimes referred to herein as the "target activation site" or "effector modulation domain".

[0092] As defined herein, a "catalytic domain" is the portion of a nucleic acid sensor molecule possessing catalytic activity which is modulated in response to binding of a target molecule to the target modulation domain.

[0093] As defined herein, a "linker region" or "linker domain" is the portion of a nucleic acid sensor molecule by or at which the "target modulation domain" and "catalytic domain" are joined. Linker regions include, but are not limited to, oligonucleotides of varying length, base pairing phosphodiester, phosphothiolate, and other covalent bonds, chemical moieties (e.g., PEG), PNA, formacetal, bismaleimide, disulfide, and other bifunctional linker reagents. The linker domain is also sometimes referred to herein as a

"connector" or "stem".

[0094] As defined herein, an "optical signal generating unit" is a portion of a nucleic acid sensor molecule comprising one or more nucleic acid sequences and/or non-nucleic acid molecular entities, which change optical or electrochemical properties or which change the optical or electrochemical properties of molecules in close proximity to them in response to a change in the conformation or the activity of the nucleic acid sensor molecule following recognition of a target molecule by the target modulation domain.

[0095] As defined herein, a nucleic acid sensor molecule which "recognizes a target molecule" is a nucleic acid molecule whose activity is modulated upon binding of a target molecule to the target modulation domain to a greater extent than it is by the binding of any non-target molecule or in the absence of the target molecule. The recognition event between the nucleic acid sensor molecule and the target molecule need not be permanent during the time in which the resulting allosteric modulation occurs. Thus, the recognition event can be transient with respect to the ensuing allosteric modulation (e.g., conformational change) of the nucleic acid sensor molecule. [0096] As defined herein, a "cleavage substrate" is an oligonucleotide or portion of an oligonucleotide cleaved upon target molecule recognition by a target modulation domain of an endonucleolytic nucleic acid sensor molecule.

[0097] As defined herein, an "oligonucleotide substrate" is an oligonucleotide that is acted upon by the catalytic domain of a nucleic acid sensor molecule with ligase activity.

[0098] As defined herein, an "effector oligonucleotide" is an oligonucleotide that base pairs with the effector oligonucleotide binding domain of a nucleic acid sensor molecule with ligase activity.

[0099] As defined herein, an "effector oligonucleotide binding domain" is the portion of the nucleic acid sensor molecule with ligase activity which is complementary to the effector oligonucleotide.

[00100] As defined herein, a "capture oligonucleotide" is an oligonucleotide that is used to attach a nucleic acid sensor molecule to a substrate by complementarity and or hybridization.

[00101] As defined herein, an "oligonucleotide substrate binding domain" is the portion on the nucleic acid sensor molecule with ligase activity that is complementary to and can base pair with an oligonucleotide substrate.

[00102] As defined herein, a "oligonucleotide supersubstrate" is an oligonucleotide substrate that is complementary to and can base pair with the oligonucleotide substrate binding domain and to the effector oligonucleotide binding domain of a nucleic acid sensor molecule with ligase activity. The oligonucleotide supersubstrate may or may not carry an affinity tag.

[00103] As defined herein, a "oligonucleotide supersubstrate binding domain" is the region of a nucleic acid sensor molecule with ligase activity that is complementary to and can base pair with the oligonucleotide supersubstrate.

[00104] As defined herein, "switch factor" is the enhancement observed in the catalytic activity and/or catalytic initial rate of a nucleic acid sensor molecule upon recognition of a target molecule by the target modulation domain.

[00105] As defined herein, an "amplicon" is the sequence of a nucleic acid sensor molecule with ligase activity covalently ligated to an oligonucleotide substrate.

[00106] As defined herein, "amplicon dependent nucleic acid amplification" refers to a technique by which one can amplify the signal of a nucleic acid sensor molecule by use of standard RT/PCR or Real-Time RT-PCR methods." [00107] As defined herein, a "3-piece ligase" is a 3-component trans-ligase ribozyme. The first component consists of the catalytic domain, the linker, the target modulation domain, the substrate binding domain and the effector oligonucleotide binding domain. The second component is the effector oligonucleotide that is complementary to the effector oligonucleotide binding domain. The third component is the oligonucleotide substrate that is complementary to the substrate binding domain.

[00108] As defined herein, a "cis-ligase ribozyme" is a ligase ribozyme that ligates its 3' end to its 5' end. The cis-ligase ribozyme is also referred herein as "1-piece ligase" and is a 1 -component system where oligonucleotide substrate, oligonucleotide substrate binding domain, catalytic domain, effector oligonucleotide and effector oligonucleotide binding domains are fused.

[00109] As defined herein, a "trans-ligase ribozyme" is a ligase ribozyme that ligates its 5' end to the 3' end of an oligonucleotide substrate.

[00110] As defined herein, a "2-piece ligase" is a 2-component trans-ligase ribozyme. The first component consists of the catalytic domain, the linker region, the target modulation domain, the substrate binding domain and the effector oligonucleotide binding domain. The second component is the oligonucleotide substrate that is complementary to the substrate binding domain and the effector oligonucleotide binding domain. [00111] As defined herein, "stem selection" refers to a process performed on a pool of nucleic molecules comprising a target modulation domain, a catalytic domain and an oligonucleotide linker region wherein the linker region is fully or partially randomized. [00112] As defined herein, "rational design/engineering" refers to a technique used to construct nucleic acid sensor molecules in which a non-conserved region of a ribozyme is replaced with a target modulation domain and joined to the catalytic domain of the ribozyme by an oligonucleotide linker region.

Nucleic Acid Compositions

[00113] In addition to carrying genetic information, nucleic acids can adopt complex three-dimensional structures. These three-dimensional structures are capable of specific recognition of target molecules and, furthermore, of catalyzing chemical reactions. Nucleic acids will thus provide candidate detection molecules for diverse target molecules, including those which do not naturally recognize or bind to DNA or RNA. [00114] In aptamer selection, combinatorial libraries of oligonucleotides are screened in vitro to identify oligonucleotides which bind with high affinity to pre-selected targets. In NASM selection, on the other hand, combinational libraries of oligonucleotides are screened in vitro to identify oligonucleotides which exhibit increased catalytic activity in the presence of targets. Possible target molecules for both aptamers and NASMS include natural and synthetic polymers, including proteins, polysaccharides, glycoproteins, hormones, receptors, and cell surfaces, and small molecules such as drugs, metabolites, transition state analogs, specific phosphorylation states, and toxins. Small biomolecules, e.g., amino acids, nucleotides, NAD, S-adenosyl methionine, chloramphenicol, and large biomolecules, e.g., thrombin, Ku, DNA polymerases, are effective targets for aptamers, catalytic RNAs (ribozymes) discussed herein (e.g., hammerhead RNAs, hairpin RNAs) as well as NASMs.

[00115] In preferred embodiments, the aptamers and NASMs of the invention specifically recognize Bacillus anthracis protective antigen. The nucleic acids of the invention are therefore useful in the detection of Bacillus anthracis protective antigen as indication of the presence of Bacillus anthracis or its pathological pathological components. [00116] While the aptamer selection processes described identifies aptamers through affinity-based (binding) selections, the selection processes as described for NASMs identifies nucleic acid sensor molecules through target modulation of the catalytic core of a ribozyme. In NASM selection, selective pressure on the starting population of NASMs (starting pool size is as high as 10 to 10¹⁷ molecules) results in nucleic acid sensor molecules with enhanced catalytic properties, but not necessarily in enhanced binding properties. Specifically, the NASM selection procedures place selective pressure on catalytic effectiveness of potential NASMS by modulating both target concentration and reaction time-dependence. Either parameter, when optimized throughout the selection, can lead to nucleic acid molecular sensor molecules which have custom-designed catalytic properties, e.g, NASMs that have high switch factors, and or NASMs that have high specificity.

Selection and Generation of a Target Specific Nucleic Acid Aptamer

[00117] Systematic Evolution of Ligands by Exponential Enrichment, "SELEX™," is a method for making a nucleic acid ligand for any desired target, as described, e.g., in U.S. Pat. Nos. 5,475,096; 5,670,637; 5,696,249; 5,270,163; 5,707,796; 5,595,877; 5,660,985; 5,567,588; 5,683,867; 5,637,459; 5,705,337; 6,011,020; 5,789,157; 6,261,774; EP 0 553 838 and PCT/US91/04078, each of which is specifically incorporated herein by reference.

[00118] SELEX™ technology is based on the fact 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 (i.e., form specific binding pairs) with virtually any chemical compound, whether large or small in size. [00119] The method involves selection from a mixture of candidates and step-wise iterations of structural improvement, using the same general selection theme, 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™ 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 to target molecules, dissociating the nucleic acid-target pairs, amplifying the nucleic acids dissociated from the nucleic acid-target pairs 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.

[00120] Within a nucleic acid mixture containing a large number of possible sequences and structures, there is a wide range of binding affinities for a given target. A nucleic acid mixture comprising, for example a 20 nucleotide randomized segment can have 4²⁰ candidate possibilities. Those which have the higher affinity constants for the target are most likely to bind to the target. After partitioning, dissociation and amplification, a second nucleic acid mixture is generated, enriched for the higher binding affinity candidates. Additional rounds of selection progressively favor the best ligands until the resulting nucleic acid mixture is predominantly composed of only one or a few sequences. These can then be cloned, sequenced and individually tested for binding affinity as pure ligands. [00121] Cycles of selection and amplification are repeated until a desired goal is achieved. In the most general case, selection/amplification is continued until no significant improvement in binding strength is achieved on repetition of the cycle. The method may be used to sample as many as about 10¹⁸ different nucleic acid species. The nucleic acids of the test mixture preferably include a randomized sequence portion as well as conserved sequences necessary for efficient amplification. Nucleic acid sequence variants can be produced in a number of ways including synthesis of randomized nucleic acid sequences and size selection from randomly cleaved cellular nucleic acids. The variable sequence portion may contain fully or partially random sequence; it may also contain subportions of conserved sequence incorporated with randomized sequence. Sequence variation in test nucleic acids can be introduced or increased by mutagenesis before or during the selection/amplification iterations.

[00122] In one embodiment of SELEX™, the selection process is so efficient at isolating those nucleic acid ligands that bind most strongly to the selected target, that only one cycle of selection and amplification is required. Such an efficient selection may occur, for example, in a chromatographic-type process wherein the ability of nucleic acids to associate with targets bound on a column operates in such a manner that the column is sufficiently able to allow separation and isolation of the highest affinity nucleic acid ligands.

[00123] In many cases, it is not necessarily desirable to perform the iterative steps of

SELEX™ until a single nucleic acid ligand is identified. The target-specific nucleic acid ligand solution may include a family of nucleic acid structures or motifs that have a number of conserved sequences and a number of sequences which can be substituted or added without significantly affecting the affinity of the nucleic acid ligands to the target. By terminating the SELEX™ process prior to completion, it is possible to determine the sequence of a number of members of the nucleic acid ligand solution family. [00124] A variety of nucleic acid primary, secondary and tertiary structures are known to exist. The structures or motifs that have been shown most commonly to be involved in non- Watson-Crick type interactions are referred to as hairpin loops, symmetric and asymmetric bulges, pseudoknots and myriad combinations of the same. Almost all known cases of such motifs suggest that they can be formed in a nucleic acid sequence of no more than 30 nucleotides. For this reason, it is often preferred that SELEX™ procedures with contiguous randomized segments be initiated with nucleic acid sequences containing a randomized segment of between about 20-50 nucleotides.

[00125] The basic SELEX™ method has been modified to achieve a number of specific objectives. For example, U.S. Patent No. 5,707,796 describes the use of SELEX™ in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. Patent No. 5,763,177 describes a SELEX™ 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 No. 5,567,588 and U.S. Application No. 08/792,075, filed January 31, 1997, entitled "Flow Cell SELEX", describe SELEX™ based methods which achieve highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule. U.S. Patent No. 5,496,938 describes methods for obtaining improved nucleic acid ligands after the SELEX™ process has been performed. U.S. Patent No. 5,705,337 describes methods for covalently linking a ligand to its target. Each of these patents and applications is specifically incorporated herein by reference.

[00126] SELEX™ can also be used to obtain nucleic acid ligands that bind to more than one site on the target molecule, and to nucleic acid ligands that include non-nucleic acid species that bind to specific sites on the target.

[00127] Counter-SELEX™ is a method for improving the specificity of nucleic acid ligands to a target molecule by eliminating nucleic acid ligand sequences with cross- reactivity to one or more non-target molecules. Counter-SELEX™ is comprised of the steps of a) preparing a candidate mixture of nucleic acids; b) 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; c) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; d) contacting the increased affinity nucleic acids with one or more non- target molecules such that nucleic acid ligands with specific affinity for the non-target molecule(s) are removed; and e) amplifying the nucleic acids with specific affinity to the target molecule to yield a mixture of nucleic acids enriched for nucleic acid sequences with a relatively higher affinity and specificity for binding to the target molecule. [00128] The random sequence portion of the oligonucleotide is flanked by at least one fixed sequence which comprises a sequence shared by all the molecules of the oligonucleotide population. Fixed sequences include sequences such as hybridization sites for PCR primers, promoter sequences for RNA polymerases (e.g., T3, T4, T7, SP6, and the like), restriction sites, or homopolymeric sequences, such as poly A or poly T tracts, catalytic cores (described further below), sites for selective binding to affinity columns, and other sequences to facilitate cloning and/or sequencing of an oligonucleotide of interest. [00129] In one embodiment, the random sequence portion of the oligonucleotide is about 15-70 (e.g., about 30-40) nucleotides in length and can comprise ribonucleotides and/or deoxyribonucleotides. Random oligonucleotides can be synthesized from phosphodiester-linked nucleotides using solid phase oligonucleotide synthesis techniques well known in the art (Froehler et al, Nucl. Acid Res. 14:5399-5467 (1986); Froehler et al, Tet. Lett. 27:5575-5578 (1986)). Oligonucleotides can also be synthesized using solution phase methods such as triester synthesis methods (Sood et al, Nucl. Acid Res. 4:2557 (1977); Hirose et al, Tet. Lett., 28:2449 (1978)). Typical syntheses carried out on automated DNA synthesis equipment yield 10 -10 molecules. Sufficiently large regions of random sequence in the sequence design increases the likelihood that each synthesized molecule is likely to represent a unique sequence.

[00130] To synthesize randomized sequences, mixtures of all four nucleotides are added at each nucleotide addition step during the synthesis process, allowing for random incorporation of nucleotides. In one embodiment, random oligonucleotides comprise entirely random sequences; however, in other embodiments, random oligonucleotides can comprise stretches of nonrandom or partially random sequences. Partially random sequences can be created by adding the four nucleotides in different molar ratios at each addition step.

[00131] The SELEX™ 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™-identified nucleic acid ligands containing modified nucleotides are described in U.S. Patent No. 5,660,985, which describes oligonucleotides containing nucleotide derivatives chemically modified at the 5' and 2' positions of pyrimidines. U.S. Patent No. 5,756,703 describes oligonucleotides containing various 2 '-modified pyrimidines. U.S. Patent No. 5,580,737 describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2'-amino (2'-NH₂), 2'-fluoro (2'-F), and/or 2'-0-methyl (2'-OMe) substituents.

[00132] The SELEX™ method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. Patent No. 5,637,459 and U.S. Patent No. 5,683,867. The SELEX™ method further encompasses combining selected nucleic acid ligands with lipophilic or non-immunogenic high molecular weight compounds in a diagnostic or therapeutic complex, as described in U.S. Patent No. 6,011,020.

[00133] SELEX™ identified 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 No. 5,859,228. 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 polyalkylene glycol are further described in U.S. Patent No. 6,051,698. See also PCT Publication No. WO 98/18480. These patents and applications allow the combination of a broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules.

[00134] The identification of nucleic acid ligands to small, flexible peptides via the

SELEX™ method has been explored. Small peptides have flexible structures and usually exist in solution in an equilibrium of multiple conformers, and thus it was initially thought that binding affinities may be limited by the conformational entropy lost upon binding a flexible peptide. However, the feasibility of identifying nucleic acid ligands to small peptides in solution was demonstrated in U.S. Patent No. 5,648,214. In this patent, high affinity RNA nucleic acid ligands to substance P, an 11 amino acid peptide, were identified. [00135] To generate oligonucleotide populations which are resistant to nucleases and hydrolysis, modified oligonucleotides can be used and can include one or more substitute internucleotide linkages, altered sugars, altered bases, or combinations thereof. In one embodiment, oligonucleotides are provided in which the P(0)0 group is replaced by P(0)S ("thioate"), P(S)S ("dithioate"), P(0)NR₂ ("amidate"), P(0)R, P(0)OR', CO or CH₂ ("formacetal") or 3 '-amine (-NH-CH₂-CH₂-), wherein each R or R' is independently H or substituted or unsubstituted alkyl. Linkage groups can be attached to adjacent nucleotide through an -0-, -N-, or -S- linkage. Not all linkages in the oligonucleotide are required to be identical.

[00136] In further embodiments, the oligonucleotides comprise modified sugar groups, for example, one or more of the hydroxyl groups is replaced with halogen, aliphatic groups, or functionalized as ethers or amines. In one embodiment, the 2 '-position of the furanose residue is substituted by any of an O-methyl, O-alkyl, O-allyl, S-alkyl, S-allyl, or halo group. Methods of synthesis of 2'-modified sugars are described in Sproat, et al, Nucl. Acid Res. 19:733-738 (1991); Gotten, et al, Nucl. Acid Res. 19:2629-2635 (1991); and Hobbs, et al, Biochemistry 12:5138-5145 (1973). The use of 2-fluoro-ribonucleotide oligomer molecules can increase the sensitivity of an aptamer for a target molecule by ten- to- one hundred-fold over those generated using unsubstituted ribo- or deoxyribooligonucleotides (Pagratis, et al, Nat. Biotechnol. 15:68-73 (1997)), providing additional binding interactions with a target molecule and increasing the stability of the secondary structure(s) of the aptamer (Kraus, et al, Journal of Immunology 160:5209-5212 (1998); Pieken, et al, Science 253:314-317 (1991); Lin, et al, Nucl. Acids Res. 22:5529- 5234 (1994); Jellinek, et al Biochemistry 34:11363-11372 (1995); Pagratis, et al, Nat. Biotechnol 15:68-73 (1997)).

[00137] Nucleic acid aptamer molecules are generally selected in a 5 to 20 cycle procedure. In one embodiment, heterogeneity is introduced only in the initial selection stages and does not occur throughout the replicating process.

[00138] The starting library of DNA sequences is generated by automated chemical synthesis on a DNA synthesizer. This library of sequences is transcribed in vitro into RNA using T7 RNA polymerase and purified. In one example, the 5'-fixed:random:3'-fixed sequence is separated by a random sequence having 30 to 50 nucleotides. Alternatively, the starting library can also be random RNA sequences synthesized on an RNA synthesizer. [00139] Sorting among the billions of aptamer candidates to find the desired molecules starts from the complex sequence pool, whereby desired aptamers are isolated through an iterative in vitro selection process. The selection process removes both nonspecific and non-binding types of contaminants. In a following amplification stage, thousands of copies of the surviving sequences are generated to enable the next round of selection. During amplification, random mutations can be introduced into the copied molecules — this 'genetic noise' allows functional nucleic acid aptamer molecules to continuously evolve and become even better adapted. The entire experiment reduces the

17 pool complexity from 10 molecules down to around 100 aptamer candidates that require detailed characterization.

[00140] Aptamer selection is accomplished by passing a solution of oligonucleotides through a column containing the target molecule. The flow-through, containing molecules which are incapable of binding target, is discarded. The column is washed, and the wash solution is discarded. Oligonucleotides which bound to the column are then specifically eluted, reverse transcribed, amplified by PCR (or other suitable amplification techniques), transcribed into RNA, and then reapplied to the selection column. Successive rounds of column application are performed until a pool of aptamers enriched in target binders is obtained.

[00141] Negative selection steps can also be performed during the selection process.

Addition of such selection steps is useful to remove aptamers which bind to a target in addition to the desired target. Additionally, where the target column is known to contain an impurity, negative selection steps can be performed to remove from the binding pool those aptamers which bind selectively to the impurity, or to both the impurity and the desired target. For example, where the desired target is knovra , care must be taken so as to remove aptamers which bind to closely related molecules or ananlogs. Examples of negative selection steps include, for example, incorporating column washing steps with analogs in the buffer, or the addition of an analog column before the target selection column (e.g., the flow through from the ananlog column will contain aptamers which do not bind the analog). [00142] After the completion of selection, the target-specific aptamers were reverse transcribed into DNA, cloned and amplified.

[00143] The typical process by which compounds present in a test mixture are identified is a high throughput screen. A high throughput screen is typically an assay configured to produce a detectible signal that is correlated to the presence or concentration of a component of the mixture. Samples whose detectible signal is unchanged relative to control samples without target do not contain the assayed compound and are called "misses". Samples whose detectible signal is significantly changed relative to control samples without target, contain the assayed compound and are called "hits". [00144] Because the process of high throughput screening requires thousands to millions of assays, each assay will ideally be very reliable to prevent both false hits and false misses. The assay should also require minimal manipulation and additional reagents to keep the cost per assay as low as possible.

[00145] To facilitate use of the aptamers in high throughput screening assays, an aptamer can be generated with a 3' sequence tag which specifically hybridizes with a biotinylated capture oligo. Such a capture oligo then can be used to immobilize the aptamer on a streptavidin coated substrate through the biotin-streptavidin binding. When such a streptavidin coated substrate is a flash plate (e.g., a plate containing a scintillant imbedded therein), surface immobilized aptamer RNA that binds to ³H-target will concentrate the tritiated nucleotide on the surface of the flash plate and generate a detectable scintillation proximity signal.

[00146] Using this methodology, aptamers can be analyzed for the ability to yield target-mediated signal in the scintillation proximity assay (SPA). Additionally, the aptamers can be analyzed for the ability to discriminate between target and closely related structural analogs. Aptamer target optimization

[00147] Natural RNAs/DNAs have exceptionally short half-lives in blood, primarily due to nuclease degradation and clearance by the kidneys. Nucleases responsible for natural RNA degradation are blocked by appropriate modifications to the 2 '-hydroxyl position of potential substrates. As such, therapeutic aptamer SELEX™ can be carried out using pools of nucleic acids in which natural nucleotides have been substituted for modified nucleotides with higher nuclease resistance. 2'-fluoropyrimidines are efficiently incorporated into RNA transcripts using a mutant form of T7 polymerase (Sousa, 1999) in which active site residue Tyr-639 is replaced with phenylalanine. The Y639F mutant can be prepared using the Strategene QuikChange^® kit, beginning with plasmid pT7-911 which encodes the wild type T7 polymerase appended with an N-terminal his tag (gift from Tom Shrader). Mutant polymerase was expressed in E. coli (BL21 DE3) and purified using under standard Ni- NTA chromatography conditions. Sufficient quantities of the protein have been prepared to perform 360 mL of in vitro transcription reactions (one 200 μL transcription is required per round of selection). The ability of this mutant polymerase to incorporate 2'- fluoropyrimidines into RNA transcripts is shown in Fig. 4.

[00148] The gene encoding protective antigen was cloned and engineered into conventional E. coli expression vectors. The protein can be easily expressed and purified as previously described (e.g. Miller, 1999). Our studies were carried out using protein prepared in this way and provided by the laboratory of Dr. John Collier (Harvard Medical School).

[00149] In a typical aptamer selection, 10¹³-10¹⁴ RNA molecules are contacted with the target of choice and the RNA molecules that specifically recognize the target are physically partitioned from those that do not. The bound RNA is next amplified by reverse transcription, PCR (RT-PCR), and in vitro transcription to yield an enriched pool of RNA which can serve as the starting point for a second round of selection. A complete SELEX™ experiment typically requires between 5 and 10 rounds of iterative selection / amplification. Once more than 10% of the applied pool binds specifically to the target protein, the pool is cloned and individual members are assayed for binding affinity, specificity, and other desired characteristics.

[00150] Manual selection techniques are used to identify high affinity aptamers for the B. anthracis protective antigen (PA). This method has been demonstrated successfully for a range of other targets in our laboratory (including metabolites, peptides, and proteins). When manual selection under typical selection conditions fails to yield aptamers to PA with the desired characteristics, selections are carried out under a variety of conditions, varying, for example, salt, pH, and pool sequence composition. Automated procedures were used as needed to increase the capacity for screening.

[00151] A semi-automated SELEX™ process has been developed at Archemix. As currently configured, the process is carried out using a 96-well plate format (allowing up to 96 different targets or selection conditions to be carried out in parallel) and human intervention is required only to move plates from one instrument to another. Nunc Maxisorp™ hydrophobic plates are used to capture approximately 5 μg of protein target / well. After dispensing an RNA pool into the target well using an automated liquid handler, non-bound species are stringently washed away using a BioTek plate washer. A reverse transcription cocktail is pipeted directly into the target well and the resulting cDNA is amplified by PCR. PCR products are purified sequentially on the basis of size using the Transgenomic WAVE system to reduce the likelihood of size artifacts arising during amplifications. Purified DNA is combined with a transcription cocktail to produce RNA for the next round of selection. A protein-binding aptamer was combined with a non-binding mutant in ratios ranging from 1 : 10 to 1:10000. Following the automated selection procedure described above, these RNA mixtures were subjected to one round of selection for target binding. The presence of a unique restriction site in the aptamer sequence allowed quantization of the ratio of binding to non-binding sequences in the resulting template pool. An approximately 100-fold enrichment of the correct binding sequence was observed in one round of selection. As shown in Figure 5, a functional aptamer diluted into a background of mutant, non-binding aptamer, can be easily enriched using this procedure. [00152] SELEX™ typically yields RNA molecules 70 to 90 nucleotides long.

Minimizing aptamer length facilitates chemical synthesis of aptamer candidates and can increase the affinity of the aptamer-ligand complex by eliminating alternative, non-binding structures. Once individual aptamers are identified from the original pool, the minimal sequence element required for high affinity binding can be identified through two parallel approaches: (1) truncation analysis by limited alkaline hydrolysis, and (2) doped reselection (methods reviewed in Fitzwater, 1996).

[00153] Truncation analysis. A combination of deletion and affinity selection may be used to map the 5'- and 3 '-boundaries of the minimal binding element. In this method, the aptamer is separately prepared with a radioactive label either the 5'- or the 3 '-end. Partial hydrolysis by incubation at high pH and temperature produce a labeled ladder of aptamer fragments. The hydrolysis products are contacted with their target and the pool partitioned on the basis of function into active and inactive pools. A high resolution denaturing polyacrylamide gel is used to resolve the labeled ladders on the basis of size. The smallest radiolabeled fragment that retains high affinity for the target defines the boundary of the binding element. Combining results from the 5'- and 3 '-boundary experiments, a minimal contiguous binding element can be identified. It is worth noting that sequences internal to this element may be non-essential - doped reselection described below makes it possible to identify these sequences such that they may be engineered out of the aptamer. [00154] Doped reselection. In the preferred minimization method, the sequence of the highest affinity aptamer sequence is synthesized as a pool in which all residues are mutated at a frequency of 15-30%. SELEX™ is performed using this "doped" pool followed by cloning and sequence analysis of individual clones. Highly conserved residues are likely to be critical for binding, and residues that co-vary are likely to be found in stem regions.

[00155] Optimization for stability and distribution. Natural RNAs/DNAs have poor pharmacokinetics, primarily due to nuclease degradation and clearance via the kidneys. Both limitations can be addressed with appropriate chemical modifications as described below. Following optimization, aptamers typically exhibit half-lives on the order of 6 hours in rats and 9-12 hours in monkeys (data not shown).

[00156] Nucleic acids are degraded in serum by a combination of endonucleases and

5'- 3' and 3'- 5' exonucleases. Appropriate chemical modifications block each activity (Pieken, 1991; Cummins, 1995; Jellinek, 1995; Dougan, 2000).

[00157] Endonucleases are blocked by modifications to the 2 '-hydroxyl position as noted in section III.A.i. Figure 6 shows the dramatic improvement in plasma pharmacokinetics as 2'-ribonucleotides are progressively replaced with either 2'-fluoro or 2'-0-methyl nucleotides. In this study, the pharmacokinetics of four variants of the human P-selectin aptamer were evaluated in Sprague-Dawley rats following an intravenous bolus administration. All variants contained a 3'-3'-thymidine cap and a 5'-40K PEG (discussed below). The parent molecule derived from the SELEX™ process was a 38-mer consisting of 2'-hydroxy purines and 2'-fluoro pyrimidines. Modification of the parent molecule was achieved by substitution of purines with 2'-Omethyl purines and substitution of the 2' fluoro pyrimidines with 2' O-methyl pyrimidines. These substitutions resulted in increased plasma residence times with the all 2'-0-mefhyl oligonucleotide being cleared approximately 15-fold slower than the parent molecule. Similar results have been observed with other aptamers.

[00158] While the 2'-hydroxyl clearly reduces the plasma half-life of an aptamer, it often plays a key role in aptamer folding/structure and global substitution generally leads to a loss of function. By carrying out selection using random sequence pools containing 2'- fluoropyrimidines instead of natural pyrimidines, isolated aptamers are both partially stabilized and functional. To further improve their stability, however, the purine nucleotides must be individually tested for the ability to accommodate stabilizing 2'-0- methyl modifications. While most purines can generally be substituted, a handful of positions are often required for function. For example, the lead clinical aptamer, NX1838, contains two natural adenosines which are required to maintain binding affinity to the VEGF protein.

[00159] Exonucleases can be blocked by appropriate modifications to the 5'- and 3'- ends of an aptamer. Addition of a 3 '-3 '-linked thymidine cap prevents 3 '->5' exonuclease degradation from the 3 '-terminus (Dougan, 2000). Similarly, 5 '-caps (such as PEG adducts described below) prevent exonuclease degradation from the 5 '-terminus to increase aptamer residence times in the blood.

[00160] Clearance. Even with extensive modification to block nuclease degradation, stabilized molecules must also exhibit molecular weights of greater than 40 kD to remain in circulation for extended periods of time. A variety of studies have shown that complexation to form high molecular weight conjugates dramatically increases the serum half-life of aptamers. While several strategies have been enabled (including protein-aptamer complexation, tagging with lipids such as cholesterol, and attachment to liposomes), most efforts have been concentrated on PEGylation.

[00161] As shown in Fig. 8, high molecular weight polyethylene glycol polymers

(PEGs) can be covalently attached to aptamers without substantially altering their ability to tightly bind to targets. At the same time, these modifications have a profound effect on aptamer half-life in animals, extending aptamer half-life from 24 min. (no PEG) to 6 hours (40 K PEG).

[00162] As shown in Fig. 1, high molecular weight polyethylene glycol polymers

[00163] Assaying anti-PA agents for activity. Previous efforts to develop assays for protective antigen activity and anthrax infection in connection with vaccine development have established reliable assays for anthrax human disease (Mourez, 2001; Sellman, 2001; Maynard, 2002). Good cellular and animal models have been enabled and a body of data indicates they provide a useful measure of the human disease.

[00164] In vitro binding assays. Binding of selected aptamers to PA is assayed using nitrocellose filter partitioning. Such assays are standard in the field for monitoring the progress of protein aptamer selections and analyzing individual clone sequences (Gold, 1990; Conrad, 1996). Nitrocellulose filter binding assays have been enabled at Archemix and are in general use for detecting proteimnucleic acid complex formation. [00165] Cellular assays for cytotoxicity. Additional assays to evaluate an aptamer's ability to inhibit PA toxicity are established using in vitro cytotoxicity assays as described previously (Mourez et al., 2001; Maynard et al., 2002). In these assays, cultured mouse macrophages are challenged by addition of protective antigen and lethal factor. Cytotoxicity is assessed using MTT, a chromogenic substrate that monitors mitochondrial activity. This assay is used routinely at Archemix to monitor cell viability and proliferation (Fig. 8).

[00166] Animal models for intoxication. Previous work has shown that potential

PA anti-toxins can be evaluated for biological efficacy using a rat protection from intoxication model. In these studies, protective antigen and lethal factor are either co- administered with the anti-toxin (Mourez, 2001; Sellman, 2001) or administered shortly prior to anti-toxin administration (Maynard, 2002) (all administrations via intravenous injection). In the absence of anti-toxin, rats typically die within 60-90 minutes whereas active anti-toxins prolong survival to five hours (Maynard, 2002) or longer (Mourez, 2001).

[00167] Animal models for infection. Pathological changes observed in rhesus macaques exposed to anthrax have been reported to be similar to those seen in humans (Fritz et al., 1995). A number of studies have examined the efficacy of human anthrax vaccine (Anthrax Vaccine Adsorbed (AVA)) against aerosol anthrax challenge in rhesus monkeys (Ivins et al., 1996; Pitt et al., 1996; Ivins et al., 1998). The anthrax inhalation rhesus monkey model has been well validated in evaluating the response to antibiotics and has been established as highly representative of the human disease. [00168] Once identified, PA specific aptamers can be used for therapeutic purposes as well as diagnostic purposes. Pharmaceutical Compositions

[00169] The invention also includes pharmaceutical compositions containing PA specific aptamer molecules. In some embodiments, the compositions are suitable for internal use and include an effective amount of a pharmacologically active compound of the invention, alone or in combination, with one or more pharmaceutically acceptable carriers. The compounds are especially useful in that they have very low, if any toxicity. [00170] In practice, the compounds or their pharmaceutically acceptable salts, are administered in amounts which will be sufficient to prevent or reduce formation of PA multimers.

[00171] For instance, for oral administration in the form of a tablet or capsule (e.g. , a gelatin capsule), the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include starch, magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone, natural sugars such as glucose or beta- lactose, com sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum starches, agar, alginic acid or its sodium salt, or effervescent mixtures, and the like. Diluents, include, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine.

[00172] Injectable compositions are preferably aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions. The compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances. The compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1 to 75%, preferably about 1 to 50%, of the active ingredient.

[00173] The compounds of the invention can also be administered in such oral dosage forms as timed release and sustained release tablets or capsules, pills, powders, granules, elixers, tinctures, suspensions, syrups and emulsions.

[00174] Liquid, particularly injectable compositions can, for example, be prepared by dissolving, dispersing, etc. The active compound is dissolved in or mixed with a pharmaceutically pure solvent such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form the injectable solution or suspension. Additionally, solid forms suitable for dissolving in liquid prior to injection can be formulated. Injectable compositions are preferably aqueous isotonic solutions or suspensions. The compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances.

[00175] The compounds of the present invention can be administered in intravenous

(both bolus and infusion), intraperitoneal, subcutaneous or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions. [00176] Parental injectable administration is generally used for subcutaneous, intramuscular or intravenous injections and infusions. Additionally, one approach for parenteral administration employs the implantation of a slow-release or sustained-released systems, which assures that a constant level of dosage is maintained, according to U.S. Pat. No. 3,710,795, incorporated herein by reference.

[00177] Furthermore, preferred compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen. Other preferred topical preparations include creams, ointments, lotions, aerosol sprays and gels, wherein the concentration of active ingredient would range from 0.01% to 15%, w/w or w/v. [00178] For solid compositions, excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like may be used. The active compound defined above, may be also formulated as suppositories using for example, polyalkylene glycols, for example, propylene glycol, as the carrier. In some embodiments, suppositories are advantageously prepared from fatty emulsions or suspensions.

[00179] The compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, containing cholesterol, stearylamine or phosphatidylchohnes. In some embodiments, a film of lipid components is hydrated with an aqueous solution of drug to a form lipid layer encapsulating the drug, as described in U.S. Pat. No. 5,262,564. For example, the aptamer- toxin and/or riboreporter molecules described herein can be provided as a complex with a lipophilic compound or non-immunogenic, high molecular weight compound constructed using methods known in the art. An example of nucleic-acid associated complexes is provided in US Patent No. 6,011,020.

[00180] The compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropyl-methacrylamide-phenol, polyhydroxyethylaspanamidephenol, or polyethyleneoxidepolylysine substituted with palmitoyl residues. Furthermore, the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.

[00181] If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and other substances such as for example, sodium acetate, triethanolamine oleate, etc.

[00182] The dosage regimen utilizing the compounds is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition. [00183] Oral dosages of the present invention, when used for the indicated effects, will range between about 0.05 to 1000 mg/day orally. The compositions are preferably provided in the form of scored tablets containing 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100.0, 250.0, 500.0 and 1000.0 mg of active ingredient. Effective plasma levels of the compounds of the present invention range from 0.002 mg to 50 mg per kg of body weight per day.

[00184] Compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily.

[00185] In addition, the PA specific aptamers can be used to generate PA specific nucleic acid sensor molecules (NASMs) as described below.

SELECTION AND GENERATION OF A TARGET SPECIFIC-NUCLEIC ACID SENSOR MOLECULE 1) GENERATION AND SELECTION OF NASMS

[00186] Nucleic acid-based detection schemes have exploited the ligand-sensitive catalytic properties of some nucleic acids, e.g., such as ribozymes. Ribozyme-based nucleic acid sensor molecules have been designed both by engineering and by in vitro selection methods. Some engineering methods exploit the apparently modular nature of nucleic acid structures by coupling molecular recognition to signaling by simply joining individual target-modulation and catalytic domains using, e.g., a double-stranded or partially double- stranded linker. ATP sensors, for example, have been created by appending the previously- selected, ATP-selective sequences (see, e.g., Sassanfar et αl, Nature 363:550-553 (1993)) to either the self-cleaving hammerhead ribozyme (see, e.g., Tang et αl., Chem. Biol. 4:453-459 (1997)) as a hammerhead-derived sensor, or the LI self-ligating ribozyme (see, e.g., Robertson et αl, Nucleic Acids Res. 28:1751-1759 (2000)) as a ligase-derived sensor. Hairpin-derived sensors are also contemplated. In general, the target modulation domain is defined by the minimum number of nucleotides sufficient to create a three-dimensional structure which recognizes a target molecule.

[00187] Catalytic nucleic acid sensor molecules (NASMs) are selected which have a target molecule-sensitive catalytic activity (e.g., self-cleavage) from a pool of randomized or partially randomized oligonucleotides. The catalytic NASMs have a target modulation domain which recognizes the target molecule and a catalytic domain for mediating a catalytic reaction induced by the target modulation domain's recognition of the target molecule. Recognition of a target molecule by the target modulation domain triggers a conformational change and/or change in catalytic activity in the nucleic acid sensor molecule. In one embodiment, by modifying (e.g., removing) at least a portion of the catalytic domain and coupling it to an optical signal generating unit, an optical nucleic acid sensor molecule is generated whose optical properties change upon recognition of the target molecule by the target modulation domain. In one embodiment, the pool of randomized oligonucleotides comprises the catalytic site of a ribozyme.

[00188] A heterogeneous population of oligonucleotide molecules comprising randomized sequences is screened to identify a nucleic acid sensor molecule having a catalytic activity which is modified (e.g., activated) upon interaction with a target molecule. As with the aptamer nucleic acids, the oligonucleotide can be RNA, DNA, or mixed RNA/DNA, and can include modified or nonnatural nucleotides or nucleotide analogs. [00189] Each oligonucleotide in the population comprises a random sequence and at least one fixed sequence at its 5' and/or 3' end. In one embodiment, the population comprises oligonucleotides which include as fixed sequences an aptamer known to specifically bind a particular target and a catalytic ribozyme or the catalytic site of a ribozyme, linked by a randomized oligonucleotide sequence. In a preferred embodiment, the fixed sequence comprises at least a portion of a catalytic site of an oligonucleotide molecule (e.g., a ribozyme) capable of catalyzing a chemical reaction. [00190] Catalytic sites are well known in the art and include, e.g., the catalytic core of a hammerhead ribozyme (see, e.g., U.S. Patent Number 5,767,263; U.S. Patent Number 5,700,923) or a hairpin ribozyme (see, e.g., U.S. Patent Number 5, 631,359). Other catalytic sites are disclosed in U.S. Patent Number 6,063,566; Koizumi et al, FEBS Lett. 239: 285-288 (1988); Haseloff and Gerlach, Nature 334: 585-59 (1988); Hampel and Tritz, Biochemistry 28: 4929-4933 (1989); Uhlenbeck, Nature 328: 596-600 (1987); and Fedor andUhlenbeck, Proc. Natl. Acad. Sci. USA 87: 1668-1672 (1990).

[00191] In some embodiments, a population of partially randomized oligonucleotides is generated from known aptamer and ribozyme sequences joined by the randomized oligonucleotides. Most molecules in this pool are non-functional, but a handful will respond to a given target and be useful as nucleic acid sensor molecules. Catalytic NASMs are isolated by the iterative process described above. In all embodiments, during amplification, random mutations can be introduced into the copied molecules — this 'genetic noise' allows functional NASMs to continuously evolve and become even better adapted as target-activated molecules.

[00192] In another embodiment, the population comprises oligonucleotides which include a randomized oligonucleotide linked to a fixed sequence which is a catalytic ribozyme, the catalytic site of a ribozyme or at least a portion of a catalytic site of an oligonucleotide molecule (e.g., a ribozyme) capable of catalyzing a chemical reaction. The starting population of oligonucleotides is then screened in multiple rounds (or cycles) of selection for those molecules exhibiting catalytic activity or enhanced catalytic activity upon recognition of the target molecule as compared to the activity in the presence of other molecules, or in the absence of the target.

[00193] The nucleic acid sensor molecules identified through in vitro selection, e.g., as described above, comprise a catalytic domain (i.e., a signal generating moiety), coupled to a target modulation domain, (i.e., a domain which recognizes a target molecule and which transduces that molecular recognition event into the generation of a detectable signal). In addition, the nucleic acid sensor molecules of the present invention use the energy of molecular recognition to modulate the catalytic or conformational properties of the nucleic acid sensor molecule.

[00194] Nucleic acid sensor molecules are generally selected in a 5 to 20 cycle procedure. In one embodiment, heterogeneity is introduced only in the initial selection stages and does not occur throughout the replicating process. Figure 12 shows a schematic diagram in which the oligonucleotide population is screened for a nucleic acid sensor molecule which comprises a target molecule activatable ligase activity. Figure 11 shows the hammerhead nucleic acid sensor molecule selection methodology. Each of these methods are readily modified for the selection of NASMs with other catalytic activities. [00195] Additional procedures may be incorporated in the various selection schemes, including: pre-screening, negative selection, etc. For example, individual clones isolated from selection experiments are tested early for allosteric activation in the presence of target- depleted extracts as a pre-screen, and molecules that respond to endogenous non-specific activators are eliminated from further consideration as target-modulated NASMs; to the extent that all isolated NASMs are activated by target-depleted extracts, depleted extracts are included in a negative selection step of the selection process; commercially available RNase inhibitors and competing RNAse substrates (e.g., tRNA) may be added to test samples to inhibit nucleases; or by carrying out selection in the presence of nucleases (e.g., by including depleted extracts during a negative selection step) the experiment intrinsically favors those molecules that are resistant to degradation; covalent modifications to RNA that can render it highly nuclease-resistant can be performed (e.g., 2'-0-methylation) to minimize non-specific cleavage in the presence of biological samples (see, e.g., Usman et al). Clin. Invest. 106:1197-202 (2000).

[00196] In one embodiment, nucleic acid sensor molecules are selected which are activated by target molecules comprising molecules having an identified biological activity (e.g., a known enzymatic activity, receptor activity, or a known structural role); however, in another embodiment, the biological activity of at least one of the target molecules is unknown (e.g., the target molecule is a polypeptide expressed from the open reading frame of an EST sequence, or is an uncharacterized polypeptide synthesized based on a predicted open reading frame, or is a purified or semi-purified protein whose function is unknown). [00197] Although in one embodiment the target molecule does not naturally bind to nucleic acids, in another embodiment, the target molecule does bind in a sequence specific or non-specific manner to a nucleic acid ligand. In a further embodiment, a plurality of target molecules binds to the nucleic acid sensor molecule. Selection for NASMs specifically responsive to a plurality of target molecules (i.e., not activated by single targets within the plurality) may be achieved by including at least two negative selection steps in which subsets of the target molecules are provided. Nucleic acid sensor molecules can be selected which bind specifically to a modified target molecule but which do not bind to closely related target molecules. Stereochemically distinct species of a molecules can also be targeted.

A. TARGET MODULATION DOMAIN WITH ENDONUCLEOLYTIC ACTIVITY

[00198] Figure 11 shows the hammerhead nucleic acid sensor molecule selection methodology. As shown in Figure 11, selection of an endonucleolytic nucleic acid sensor molecule (e.g., a hammerhead-derived NASM) begins with the synthesis of a ribozyme sequence on a DNA synthesizer. Alternatively, synthesis occurs on a RNA synthesizer. Random nucleotides are incorporated generating pools of roughly 10¹⁶ molecules. Most molecules in this pool are non-functional, but a handful will respond to a given target and be useful as nucleic acid sensor molecules. Sorting among the billions of species to find the desired molecules starts from the complex sequence pool. Nucleic acid sensor molecule are isolated by an iterative process: in addition to the target-activated ribozymes that one desires, the starting pool is usually dominated by either constitutively active or completely inactive ribozymes. The selection process removes both types of contaminants by incorporating both negative and positive selection incubation steps. In the following amplification stage, thousands of copies of the surviving sequences are generated to enable the next round of selection. During amplification, random mutations can be introduced into the copied molecules — this 'genetic noise' allows functional NASMs to continuously evolve and become even better adapted as target-activated molecules. The entire experiment reduces the pool complexity from 10¹⁶ down to < 100. [00199] The starting library of DNA sequences (the "pool") is generated by automated chemical synthesis on a DNA synthesizer. This library of sequences is transcribed in vitro into RNA using T7 RNA polymerase and subsequently purified. Alternatively, the pool is generated in an RNA synthesizer. In the absence of the desired target molecule of interest, the RNA library is incubated together with the binding buffer alone as a negative selection incubation. During this incubation, non-allosteric (or non- target activated) ribozymes are expected to undergo a catalytic reaction, in this case, cleavage. Undesired members of the hammerhead pool, those that are constitutively active in the absence of the target molecule, are removed from the unreacted members by size- based purification, e.g., by PAGE-chromatography; 7 M Urea, 8-10% acrylamide, IX TBE. Higher molecular weight species are eluted as a single broad band from the gel matrix into TBE buffer, then purified for subsequent steps in the selection cycle. The remaining RNA pool is then incubated under identical conditions but now in the presence of the target molecule of interest in binding buffer, as a positive selection incubation. In another size- based purification, desired members of the hammerhead pool, those that are only active in the presence of the target molecule, are removed from the remaining unreacted members by PAGE-chromatography; 7 M Urea, 8-10% acrylamide, IX TBE. In this step, lower molecular weight species are eluted as a single broad band from the gel matrix into TBE buffer, then purified for subsequent steps in the selection cycle. RT-PCR amplified DNA is then purified and transcribed to yield an enriched pool for a subsequent round of reselection. Rounds of selection and amplification are repeated until functional members sufficiently dominate the resultant library.

B. TARGET MODULATION DOMAIN WITH LIGASE ACTIVITY

[00200] Figure 12 shows a schematic diagram in which the oligonucleotide population is screened for a nucleic acid sensor molecule which comprises a target molecule activatable ligase activity. In the embodiment shown in Figure 12, the ligation reaction involves covalent attachment of an oligonucleotide substrate to the 5 '-end of the NASM through formation of a phosphodiester linkage. Other ligation chemistries can form the basis for selection of NASMs (e.g., oligonucleotide ligation to the 3'-end, alkylations (see, e.g., Wilson et al, Nature 374 (6525):777-782 (1995)), peptide bond formation (see, e.g., Zhang etal, Nature 390 (6655):96-100 (1997)), Diels- Alder reactions to couple alkenes and dienes (see, e.g., Seelig et al, Chemistry and Biology 3:167-176 (1999)). For some chemistries, the chemical functional groups that constitute the reactants in the ligation reaction may not naturally appear within nucleic acids. Thus, it may be necessary to synthesize an RNA pool in which one of the ligation reactants is covalently attached to each member of the pool (e.g., attaching a primary amine to the 5'-end of an RNA to enable selection for peptide bond formation).

[00201] In this embodiment, the oligonucleotide population from which the NASMs are selected is initially screened in a negative selection procedure to eliminate any molecules which have ligase activity even in the absence of target molecule binding. A solution of oligonucleotides (e.g., 100 pM) comprising a 5' and 3' fixed sequence ("5'- fixed: random: 3 '-fixed") is denatured with a 3' primer sequence ("3' prime") (e.g., 200 pM) which binds to at least a portion of the 3' fixed sequence. Ligation buffer (e.g., 30 mM Tris HCl, pH 7.4, 600 mM NaCl, 1 mM EDTA, 1% NP-40, 60 mM MgCl₂) and a tagged oligonucleotide substrate sequence ("tag-substrate") (e.g., Tag-UGCCACU) are added and the mixture is incubated for about 16 to about 24 hours at 25 °C in the absence of target molecule (STEP 1). Tags encompassed within the scope include, e.g., radioactive labels, fluorescent labels, a chemically reactive species such as thiophosphate, the first member of a binding pair comprising a first and second binding member, each member bindable to the other (e.g., biotin, an antigen recognized by an antibody, or a tag nucleic acid sequence). The reaction is stopped by the addition of EDTA. Alternatively, the reaction can be terminated by removal of the substrate or addition of denaturants (e.g., urea or formamide). [00202] Ligated molecules are removed from pool of selectable molecules (STEP 2), generating a population of oligonucleotides substantially free of ligated molecules (as measured by absence of the tag sequence in the solution). In the embodiment shown in Figure 4, the tag is the first member of a binding pair (e.g., biotin) and the ligated molecules ("biotin-oligonucleotide substrate:5'-fixed:random:3'-fixed") are physically removed from the solution by contacting the sample to a solid support to which the second member of the binding pair is bound ("S") (e.g., streptavidin). The eluant collected comprises a population of oligonucleotides enriched for non-ligated molecules (5'-fιxed:random:3'-fixed). This step can be repeated multiple times until the oligonucleotide population is substantially free of molecules having target-insensitive ligase activity.

[00203] This step allows for suppression of the ability of constitutively active molecules to be carried through to the next cycle of selection. Physical separation of ligated and unligated molecules is one mechanism by which this can be achieved. Alternatively, the negative selection step can be configured such that catalysis converts active molecules to a form that blocks their ability to be either retained during the subsequent positive selection step or to be amplified for the next cycle of selection. For example, the oligonucleotide substrate used for ligation in the negative selection step can be synthesized without a capture tag. Target-independent ligases covalently self-attach the untagged oligonucleotide substrate during the negative selection step and are then unable to accept a tagged form of the oligonucleotide substrate provided during the positive selection step that follows. In another embodiment, the oligonucleotide substrate provided during the negative selection step has a different sequence from that provided during the positive selection step. When PCR is carried out using a primer complementary to the positive selection oligonucleotide substrate, only target-activated ligases were capable of amplification. [00204] A positive selection phase follows. In this phase, more 3' primer and tagged oligonucleotide substrate are added to the pool resulting from the negative selection step. Target molecules are then added to form a reacted solution and the reacted solution is incubated at 25 °C for about 2 hours (STEP 3). Target molecules encompassed within the scope include, e.g., proteins or portions thereof (e.g., receptors, antigen, antibodies, enzymes, growth factors), peptides, enzyme inhibitors, hormones, carbohydrates, polysaccharides, glycoproteins, lipids, phospholipids, metabolites, metal ions, cofactors, inhibitors, drugs, dyes, vitamins, nucleic acids, membrane structures, receptors, organelles, and viruses. Target molecules can be free in solution or can be part of a larger cellular structure (e.g., such as a receptor embedded in a cell membrane). In one embodiment, a target molecule is one which does not naturally bind to nucleic acids. [00205] The reacted solution is enriched for ligated molecules (biotin-oligonucleotide substrate: 5'-fixed:random:3'-fixed) by removing non-tagged molecules (5'- fixed:random:3 '-fixed) from the solution. For example, in one embodiment, the tagged oligonucleotide substrate comprises a biotin tag and ligated molecules are isolated by passing the reacted solution over a solid support to which streptavidin (S) is bound (STEP 4). Eluant containing non-bound, non-ligated molecules (5'-fixed:random:3'-fixed) is discarded and bound, ligated molecules (biotin-oligonucleotide substrate: 5'- fixed:random:3 '-fixed) are identified as nucleic acid sensor molecules and released from the support by disrupting the binding pair interaction which enabled capture of the catalytically active molecules. For example, heating to 95° C in the presence of 10 mM biotin allows release of biotin-tagged catalysts from an immobilized streptavidin support. In another embodiment, the captured catalysts remain attached to a solid support and are directly amplified (described below) while immobilized. Multiple positive selection phases can be performed (STEPS 3 and 4). In one embodiment, the stringency of each positive selection phase is increased by decreasing the incubation time by one half. [00206] Physically removing inactive species from the pool adds stringency to the selection process. However, to the extent that the ligation reaction increases the amplification potential of the NASMs, this step may be omitted. In the illustrated embodiment, for example, ligation of an oligonucleotide to the active species provides a primer binding site that enables subsequent PCR amplification using an oligonucleotide substrate complementary to the original oligonucleotide substrate. Unligated species do not necessarily need to be physically separated from other species because they are less likely to amplify in the absence of a covalently tethered primer binding site. Selected nucleic acid sensor molecules are amplified (or in the case of RNA molecules, first reverse transcribed, then amplified) using an oligonucleotide substrate primer ("S primer") which specifically binds to the ligated oligonucleotide substrate sequence (STEP 5). In one embodiment, amplified molecules are further amplified with a nested PCR primer that regenerates a T7 promoter ("T7 Primer") from the 5' fixed and the litigated oligonucleotide substrate sequence (STEP 6). Following transcription with T7 RNA polymerase (STEP 7), the oligonucleotide pool may be further selected and amplified to eliminate any remaining unligated sequences (5'-frxed:random:3'-fixed) by repeating STEPS 3-7. It should be obvious to those of skill in the art that in addition to PCR, and RT-PCR, any number of amplification methods can be used (either enzymatic, chemical, or replication-based, e.g., such as by cloning), either singly, or in combination. Exemplary amplification methods are disclosed in Saiki, et al, Science 230:1350-1354 (1985); Saiki, et al, Science 239:481-491 (1988); Kwoh, et al, Proc. Natl. Acad. Sci. 86:1173 (1989); Joyce, Molecular Biology of RNA: UCLA Symposia on Molecular and Cellular Biology, T. R. Cech (ed.) pp. 361-371 (1989); and Guatelli, et al, Proc. Natl. Acad. Sci. 87:1874 (1990).

[00207] Because the 3' primer (3' prime) (see STEP 3 in Figure 12) is included in the ligation mixture, selected nucleic acid sensor molecules may require this sequence for activation. In cases where this is undesirable, the 3' primer may be omitted from the mix. Alternatively, the final nucleic acid sensor molecule can be modified by attaching the 3' primer via a short sequence loop or a chemical linker to the 3' end of the nucleic acid sensor molecule, thereby eliminating the requirement for added primer, allowing 3' primer sequence to self-prime the molecule.

C. TARGET MODULATION DOMAIN WITH SELF-CLEAVING ACTIVITY [00208] In another embodiment, as shown in Figure 13, an oligonucleotide population is screened for a nucleic acid sensor molecule which comprises a target molecule having activatable self-cleaving activity. In this embodiment, the starting population of oligonucleotide molecules comprises 5' and 3' fixed regions ("5 '-fixed and 3' fixed A- 3'fixed B") and at least one of the fixed regions, in this example, the 3' fixed region, comprises a ribozyme catalytic core including a self cleavage site (the junction between 3' fixed A-3 'fixed B). The population of oligonucleotide molecules comprising random oligonucleotides flanked by fixed 5' and 3' sequences (5'-fιxed:random:3'-fixed A: 3' fixed B) are negatively selected to remove oligonucleotides which self-cleave (i.e., 5'- fιxed:random:3'-fixed-A molecules) even in the absence of target molecules. The oligonucleotide pool is incubated in reaction buffer (e.g., 50 mM Tris HCl, pH 7.5, 20 mM MgCl₂) for 5 hours at 25 °C, punctuated at one hour intervals by incubation at 60 °C for one minute (STEP 1). In one embodiment, the uncleaved fraction of the oligonucleotide population (containing 5'-fixed and 3' fixed A-3'-fixed B molecules) is purified by denaturing 10% polyacrylamide gel electrophoresis (PAGE) (STEP 2). Target molecule dependent cleavage activity is then selected in the presence of target molecules in the presence of reaction buffer by incubation at 23 °C for about 30 seconds to about five minutes (STEP 3). Cleaved molecules (5'-fixed:random:3'fixed-A molecules) are identified as nucleic acid sensor molecules and are purified by PAGE (STEP 4). [00209] Amplification of the cleaved molecule is performed using primers which specifically bind the 5 '-fixed and the 3 '-fixed A sequences, regenerating the T7 promoter and the 3 '-fixed B site (STEP 5), and the molecule is further amplified further by RNA transcription using T7 polymerase (STEP 6). In one embodiment, the process (STEPS 1-6) is repeated until the starting population is reduced to about one to five unique sequences. [00210] Alternative methods for separating cleaved from uncleaved RNAs can be used. Tags can be attached to the 3'-fixed B sequence and separation can be based upon separating tagged sequences from non-tagged sequences at STEP 4. Chromatographic procedures that separate molecules on the basis of size (e.g., gel filtration) can be used in place of electrophoresis. One end of each molecule in the RNA pool can be attached to a solid support and catalytically active molecules isolated upon release from the support as a result of cleavage. Alternate catalytic cores may be used. These alternate catalytic cores and methods using these cores are also are encompassed within the scope of the invention.

D. OTHER TARGET MODULATION DOMAINS

[00211] Nucleic acid sensor molecules which utilize other catalytic actions or which combine both cleavage and ligase activities in a single molecule can be isolated by using one or a combination of both of the selection strategies outlined independently above for ligases and endonucleases. For example, the hairpin ribozyme is known to catalyze cleavage followed by ligation of a second oligonucleotide substrate (Berzal-Herranz et al, Genes and Development 1:129-134 (1992)). Target activated sensor molecules based on the hairpin activity can be isolated from a pool of randomized sequence RNAs. Hairpin-based NASMs can be isolated on the basis of target molecule dependent release of the fragment in the same way that hammerhead-based NASMs are isolated (e.g., target molecule dependent increase in electrophoretic mobility or target molecule dependent release from a solid support). Alternatively, nucleic acid sensor molecules can be selected on the basis of their ability to substitute the 3 '-sequence released upon cleavage for another sequence as described in an target molecule independent manner by Berzal-Herranz et al, Genes and Development 1:129-134 (1992). In this scheme, the original 3'-end of the NASM is released in an initial cleavage event and an exogenously provided oligonucleotide substrate with a free 5 '-hydroxyl is ligated back on. The newly attached 3 '-end provides a primer binding site that can form the basis for preferential amplification of catalytically active molecules. Constitutively active molecules that are not activated by a provided target molecule can be removed from the pool by (1) separating away molecules that exhibit increased electrophoretic mobility in the absence of an exogenous oligonucleotide substrate or in the absence of target molecule, or (2) capturing molecules that acquire an exogenous oligonucleotide substrate (e.g., using a 3 '-biotinylated substrate and captured re-ligated species on an avidin column).

[00212] Like the hairpin ribozyme, the group I intron self-splicing ribozymes combine cleavage and ligation activities to promote ligation of the exons that flank it. In the first step of group I intron-catalyzed splicing, an exogenous guanosine cofactor attacks the 5 '-splice site. As a result of an intron-mediated phosphodiester exchange reaction, the 5'- exon is released coincident with attachment of the guanosine cofactor to the ribozyme. In a second chemical step, the 3 '-hydroxyl at the end of the 5 '-exon attacks the phosphodiester linkage between the intron and the 3 '-exon, leading to ligation of the two exons and release of the intron. Group I intron-derived NASMs can be isolated from degenerate sequence pools by selecting molecules on the basis of either one or both chemical steps, operating in either a forward or reverse direction. NASMs can be isolated by specifically enriching those molecules that fail to promote catalysis in the absence of target molecule but which are catalytically active in its presence. Specific examples of selection schemes follow. In each case, a pool of RNAs related in sequence to a representative group I intron (e.g., the Tetrahymena thermophila pre-rRNA intron or the phage T4 td intron) serves as the starting point for selection. Random sequence regions can be embedded within the intron at sites known to be important for proper folding and activity (e.g., substituting the P5abc domain of the Tetrahymena intron, Williams et al, Nucl. Acid Res. 22(11):2003-2009 (1994)). Intron nucleic acid sensor molecules, in this case, sensitive to thio-GMP can be generated as follows.

[00213] In the first step, forward direction, the intron is synthesized with a short 5'- exon. In the negative selection step, a guanosine cofactor is provided and constitutively active molecules undergo splicing. In the positive selection step, the target molecule is provided together with thio-GMP. Molecules responsive to the target undergo activated splicing and as a result acquire a unique thiophosphate at their 5 '-termini. Thio-tagged NASMs can be separated from untagged ribozymes by their specific retention on mercury gels or activated thiol agarose columns.

[00214] The first step, reverse direction method is performed as described in Green &

Szostak. An intron is synthesized with a 5 '-guanosine and no 5 '-exon. An oligonucleotide substrate complementary to the 5 '-internal guide sequence is provided during the negative selection step and constitutively active molecules ligate the substrate to their 5 '-ends, releasing the original terminal guanosine. A second oligonucleotide substrate with a different 5 '-sequence is provided together with target in the positive selection step. NASMs specifically activated by the target molecule ligate the second oligonucleotide substrate to their 5'-ends. PCR amplification using a primer corresponding to the second substrate can be carried out to preferentially amplify target molecule sensitive nucleic acid sensor molecules.

[00215] The second step, reverse direction method is performed as described in

Nature 344:467-468 (1990). The intron is synthesized with no flanking exons. During the negative selection step, pool RNAs are incubated together with a short oligonucleotide substrate under conditions which allow catalysis to proceed. During the positive selection step, a second oligonucleotide substrate with a different 3 '-sequence is provided together with the sensor target. NASMs are activated and catalyze ligation of the 3 '-end of the second substrate. Reverse transcription carried out using a primer complementary to the 3'- end of the second substrate specifically selects NASMs for subsequent amplification. [00216] In generating a PA specific NASM from a PA aptamer, it is helpful to determine the minimal secondary structure elements of the aptamer as the core ligand binding element of the aptamer can be appended directly to the randomized stem used in the catalytic NASM stem selection. This can be done in a number of ways known to one skilled in the art, including 3' end mapping and doped RNA reselection. [00217] A combination of deletion and affinity selection may be used to map the 5 '- and 3 '-boundaries of the minimal binding element of an aptamer. In this method, the aptamer is separately prepared with a radioactive label either the 5'- or the 3 '-end. Partial hydrolysis by incubation at high pH and temperature produce a labeled ladder of aptamer fragments. The hydrolysis products are contacted with their target and the pool partitioned on the basis of function into active and inactive pools. A high resolution denaturing polyacrylamide gel is used to resolve the labeled ladders on the basis of size. The smallest radiolabeled fragment that retains high affinity for the target defines the boundary of the binding element. Combining results from the 5'- and 3 '-boundary experiments, a minimal contiguous binding element can be identified. It is worth noting that sequences internal to this element may be non-essential - doped reselection described below makes it possible to identify these sequences such that they may be engineered out of the aptamer. [00218] In the preferred minimization method, the sequence of the highest affinity aptamer sequence is synthesized as a pool in which all residues are mutated at a frequency of 15-30%). SELEX is performed using this "doped" pool followed by cloning and sequence analysis of individual clones. Highly conserved residues are likely to be critical for binding, and residues that co-vary are likely to be found in stem regions.

2) CHARACTERIZATION OF NASMs

[00219] Once particular aptamers or nucleic acid sensor molecules have been selected, they can be isolated, cloned, sequenced, and/or resynthesized using natural or modified nucleotides. Accordingly, synthesis intermediates of nucleic acid compositions are also encompassed within the scope of the invention, as are replicatable sequences (e.g., plasmids) comprising the nucleic acid compositions of the invention.

[00220] The pool of NASMs is cloned into various plasmids transformed, e.g., into

E. coli. Individual NASM encoded DNA clones are isolated, PCR amplified and to generate NASM RNA. The NASM RNAs are then tested in target modulation assays which determine the rate or extent of ribozyme modulation. For hammerhead NASMs, the extent of target dependent and independent reaction is determined by quantifying the extent of endonucleolytic cleavage of an oligonucleotide substrate. The extent of reaction can be followed by electrophoresing the reaction products on a denaturing PAGE gel, and subsequently analyzed by standard radiometric methods. For ligase NASMs, the extent of target dependent and independent reaction is determined by quantifying the extent of ligation of an oligonucleotide substrate, resulting in an increase in NASM molecular weight, as determined in denaturing PAGE gel electrophoresis.

[00221] Individual NASM clones which display high target dependent switch factor values, or high k_act rate values are subsequently chosen for further modification and evaluation.

[00222] Hammerhead-derived NASM clones are then further modified to render them suitable for the optical detection applications that are described in detail below. These

NASMs are used as fluorescent biosensors affixed to solid supports, as fluorescent biosensors in homogeneous (solution) FRET-based assays, and as biosensors in SPA applications.

[00223] Ligase and intron-derived NASM clones are further modified to render them suitable for a number of detection platforms and applications, including, but not limited to,

PCR and nucleotide amplification detection methods; fluorescent-based biosensors detectable in solution and chip formats; and as in vivo, intracellular detection biosensors.

[00224] An important kinetic consideration in NASM characterization is the fact that

RNAse-mediated degradation of the nucleic acid sensor molecule proceeds at a rate in competition with the rate of nucleic acid sensor molecule catalysis. As such, nucleic acid sensor molecules with fast turnover rates can be assayed for shorter times and are thus less susceptible to RNAse problems. Nucleic acid sensor molecules with fast turnover can be obtained by (1) reducing the length of the incubation during the positive selection step, and/or (2) choosing fast nucleic acid sensor molecules (potentially with less favorable allosteric activation ratios) when screening individual clones emerging from the selection experiment.

[00225] The relative stabilities of the activated and unactivated forms of the nucleic acid sensor molecules can be optimized to achieve the highest sensitivity of detection of target molecule. In one embodiment, the nucleic acid sensor molecule is further engineered to enhance the stability of one form over another, such as favoring the formation of the target molecule activated form. As in the case where certain bases do not form base pairs when the nucleic acid sensor molecule is unactivated, the unactivated form is not stabilized. [00226] A number of methods can be used to evaluate the relative stability of different conformations of the nucleic acid sensor molecule. In one embodiment, the free energy of the structures formed by the nucleic acid sensor molecule is determined using software programs such as mfold®, which can be found on the Rensselaer Polytechnic Institute (RPI) web site (www.rpi.edu/dept.).

[00227] In another embodiment, a gel assay is performed which permits detection of different conformations of the nucleic acid sensor molecule. In this embodiment, the nucleic acid sensor molecule is allowed to come to equilibrium at room temperature or the temperature at which the nucleic acid sensor molecule were used. The molecule is then cooled to 4 °C and electrophoresed on a native (non-denaturing) gel at 4 °C. Each of the conformations formed by the nucleic acid sensor molecule will run at a different position on the gel, allowing visualization of the relative concentration of each conformation. Similarly, the conformation of nucleic acid sensor molecules which form in the presence of target molecule is then determined by a method such as circular dichroism (CD). By comparing the conformation of the nucleic acid sensor molecule formed in the presence of target molecule with the conformations formed in the absence of target molecule, the conformation which corresponds to the activated conformation can be identified in a sample in which there is no target molecule. The nucleic acid sensor molecule can then be engineered to minimize the formation of the activated conformation in the absence of target molecule. The sensitivity and specificity of nucleic acid sensor molecule can be further tested using target molecule modulation assays with known amounts of target molecules. [00228] Modifications to stabilize one conformation of the nucleic sensor molecule over another may be identified using the mfold program or native gel assays discussed above. A labeled nucleic acid sensor molecule is generated by coupling a first signaling moiety (F) to a first nucleotide and a second signaling moiety (D) to a second nucleotide as discussed above. As above, the sensitivity and specificity of the nucleic acid sensor molecule can be further assayed by using target molecule modulation assays with known amounts of target molecules.

3) CONVERTING A CATALYTIC NASM TO AN OPTICAL NASM

[00229] During or after synthesis of the NASM, an optical signal generating unit is either added or inserted into the oligonucleotide sequence comprising the derived nucleic acid sensor molecule. In one embodiment, in order to convert a catalytic nucleic acid sensor molecule into an optical nucleic acid sensor molecule, at least a portion of the catalytic domain is modified (e.g., deleted). In one embodiment, the deletion enhances the conformational stability of the optical nucleic acid sensor molecule in either the bound or unbound forms. In one embodiment, deletion of the entire catalytic domain of the catalytic NASM stabilizes the unbound form of the nucleic acid sensor molecule. In another embodiment, the deletion may be chosen so as to take advantage of the inherent fluorescence-quenching properties of unpaired guanosine (G) residues (Walter and Burke, RNA 3:392 (1997)).

[00230] In another embodiment, the target modulation domain from a previously identified nucleic acid sensor molecule is incorporated into an oligonucleotide sequence that changes conformation upon target recognition. Nucleic acid sensor molecules of this type can be derived from allosteric ribozymes, such as those derived from the hammerhead, hairpin, LI ligase, or group 1 intron ribozymes and the like, all of which transduce molecular recognition into a detectable signal. For example, 3', 5 '-cyclic nucleotide monophosphate (cNMP)-dependent hammerhead ribozymes were reengineered into RNA molecules which specifically bound to cNMP (Soukup et al, RNA 7:524 (2001)). The catalytic cores for hammerhead ribozymes were removed and replaced with 5-base duplex forming sequences. The binding of these reengineered RNA sensor molecules to cNMP was then confirmed experimentally. By adjusting the duplex length, sensor molecules can be redesigned to undergo significant conformational changes. The conformational changes can then be coupled to detection via FRET or simply changes in fluorescence intensity (as in the case of a molecular beacon). For example, by adding an appropriate probe on each end of the duplex, the stabilization of duplex by target binding can be monitored with the change in fluorescence.

[00231] While the above experimental example is performed in solution and utilizes a cuvette-based fluorescence spectrometer, in alternative embodiments the methods are performed in microwell multiplate readers (e.g., the Packard Fusion, or the Tecan Ultra) for high-throughput solution phase measurements.

[00232] In one embodiment, after deletion of at least a portion of the catalytic site from a catalytic nucleic acid sensor molecule, an optical signaling unit is either added to, or inserted within, the nucleic sensor molecule, generating a sensor molecule whose optical properties change in response to binding of the target molecule to the target modulation domain. In one embodiment, the optical signaling unit is added by exposing at least a 5' or 3' nucleotide that was not previously exposed. The 5' nucleotide or a 5' subterminal nucleotide (e.g., an internal nucleotide) of the molecule is couplable to a first signaling moiety while the 3' nucleotide or 3' subterminal nucleotide is couplable to a second signaling moiety. Target molecule recognition by the optical nucleic acid sensor molecule alters the proximity of the 5' and 3' nucleotide (or subterminal nucleotides) with respect to each other, and when the first and second signaling moieties are coupled to their respective nucleotides, this change in proximity results in a target sensitive change in the optical properties of the nucleic acid sensor molecule. Detection of changes in the optical properties of the nucleic acid sensor molecule can therefore be correlated with the presence and/or quantity of a target molecule in a sample.

[00233] In another embodiment, optical NASMs are generated by adding first and second signaling moieties, that are coupled to the 5' terminal or subterminal sequences, and 3'-l terminal and subterminal sequences respectively, of the catalytic NASM. Signaling molecules can be coupled to nucleotides which are already part of the nucleic acid sensor molecule or may be coupled to nucleotides which are inserted into the nucleic acid sensor molecule, or can be added to a nucleic acid sensor molecule as it is synthesized. Coupling chemistries to attach signaling molecules are well known in the art (see, e.g., The Molecular Probes Handbook, R. Haughland). Suitable chemistries include, e.g., derivatization of the 5-position of pyrimidine bases (e.g., using 5'-amino allyl precursors), derivatization of the 5'-end (e.g., phosphoroamidites that add a primary amine to the 5'-end of chemically- synthesized oligonucleotide) or the 3 '-end (e.g., periodate treatment of RNA to convert the 3'-ribose into a dialdehyde which can subsequently react with hydrazide-bearing signaling molecules).

[00234] In another embodiment, a single signaling moiety is either added to, or inserted within, the catalytic nucleic sensor molecule. In this embodiment, binding of the target molecule results in changes in both the conformation and physical aspect (e.g., molecular volume, and thus rotational diffusion rate, etc.) of the optical nucleic acid sensor molecule. Conformational changes in the optical nucleic acid sensor molecule upon target recognition will modify the chemical environment of the signaling moiety, while changes in the physical aspect of the nucleic acid sensor molecule will alter the kinetic properties of the signaling moiety. In both cases, the result were a detectable change in the optical properties of the nucleic acid sensor molecule.

[00235] In one embodiment, the optical nucleic acid sensor molecule is prepared without a quencher group. Instead of a quencher group, a moiety with a free amine group can be added. This free amine group allows the sensor molecule to be attached to an aldehyde-derivatized glass surface via standard protocols for Schiff base formation and reduction. The nucleic acid sensor molecules can be bound in discrete regions or spots to form an array, or uniformly distributed to cover an extended area. In the absence of target, the optical nucleic acid sensor molecule will diffusionally rotate about its point of attachment to the surface at a rate characteristic of its molecular volume and mass. After target recognition and modulation of the structure of the NASM, the optical NASM-target complex will have a correspondingly larger volume and mass. This change in molecular volume (mass) will slow the rate of rotational diffusion, and result in a measurable change in the polarization state of the fluorescence emission from the fluorophore. [00236] In one embodiment of the invention, a single signaling moiety is attached to a portion of a catalytic NASM that is released as a result of catalysis (e.g., either end of a self-cleaving ribozyme or the pyrophosphate at the 5 '-end of a ligase). Target molecule- activated catalysis leads to release of the signaling moiety from the optical NASM to generate a signal correlated with the presence of the target. Release can be detected by either (1) changes in the intrinsic optical properties of the signaling moiety (e.g., decreased fluorescence polarization as the released moiety is able to tumble more freely in solution), or (2) changes in the partitioning of the signaling moiety (e.g., release of a fluorophore from a chip containing immobilized ribozymes such that the total fluorescence of the chip is reduced following washing).

[00237] In another embodiment of the invention, the catalytic nucleic acid sensor molecule is unmodified and the optical signaling unit is provided as a substrate for the NASM. One example of this embodiment includes a fluorescently tagged oligonucleotide substrate which can be joined to a NASM with ligase activity. In a heterogeneous assay using the ligase as a sensor molecule, analyte-containing samples are incubated with the fluorescent oligonucleotide substrate and the ligase under conditions that allow the ligase to function. Following an incubation period, the ligase is separated from free oligonucleotide substrate (e.g., by capturing ligases onto a solid support on the basis of hybridization to ligase-specific sequences or by pre-immobilizing the ligases on a solid support and washing extensively).

[00238] Quantitation of the captured fluorescence signal provides a means for inferring the concentration of analyte in the sample. In a second example of this embodiment, catalytic activity alters the fluorescence properties of an oligonucleotide substrate without leading to its own modification. Fluorophore pairs or fluorophore/quencher pairs can be attached to nucleotides flanking either side of the cleavage site of an oligonucleotide substrate for a trans-acting endonuclease ribozyme (Jenne et al, Nature Biotechnology 19(1):56-61 (2001)). Target activated cleavage of the substrate leads to separation of the pair and a change in its optical properties. [00239] In another embodiment of the invention, the ligase catalytic NASM and its oligonucleotide substrates are unmodified and detection relies on catalytically-coupled changes in the ability of the NASM to be enzymatically amplified. In one example, a target-activated ligase is incubated together with oligonucleotide substrate and an analyte- containing sample under conditions which allow the ligase to function. Following an incubation period, the reaction is quenched and the mixture subjected to RT/PCR amplification using a primer pair that includes the oligo sequence corresponding to the ligation substrate. Amplification products can be detected by a variety of generally practiced methods (e.g. Taqman®). Only those ribozymes that have self-ligated an oligonucleotide substrate are capable of amplification under these conditions and will generate a signal that can be coupled to the concentration of the sensor target. 4) DETECTION OF OPTICAL NASMs

DProximity Dependent Signaling Moieties [00240] Many proximity dependent signaling moieties are known in the art and are encompassed within the scope of the present invention (Morrison, Nomsotopic DNA Probe Techniques, Kricka, ed., Academic Press, Inc., San Diego, Calif., chapter 13; Heller et al, Academic Press, Inc. pp. 245-256 (1985)). Systems using these signaling moieties rely on the change in fluorescence that occurs when the moieties are brought into close proximity. Such systems are described in the literature as fluorescence energy transfer (FET), fluorescence resonance energy transfer (FRET), nonradiative energy transfer, long-range energy transfer, dipole-coupled energy transfer, or Forster energy transfer (U.S. Patent Number 5,491,063, Wu et al, Anal. Biochem. 218:1 (1994)). The arrangement of various fluorophore-quencher pairs is shown in Figure 14. (See Jenne et al, Nature Biotechnology 1:56-61 (2001); Singh et al, RNA 5:1348 (1999); Frauendorf et al, Bioorg Med. Chem. 10:2521-2524 (2001); Perkins et al, Biochemistry 35(50):16370-16377 (1996)), and WO 99/47704 for discussion of various FRET formats.

[00241] Suitable fluorescent labels are known in the art and commercially available from, for example, Molecular Probes (Eugene, Oreg.). These include, e.g., donor/acceptor (i.e., first and second signaling moieties) molecules such as: fluorescein isothiocyanate (FITC) /tetramethylrhodamine isothiocyanate (TRITC), FITC/Texas Red), FITC/N- hydroxysuccinimidyl 1-pyrenebutyrate (PYB), FITC/eosin isothiocyanate (EITC), N- hydroxysuccinimidyl 1-ρyrenesulfonate (PYS)/FITC, FITC/Rhodamine X (ROX), FITC/tetramethylrhodamine (TAMRA), and others. In addition to the organic fluorophores already mentioned, various types of nonorganic fluorescent labels are known in the art and are commercially available from, for example, Quantum Dot Corporation, Inc. (Hayward, CA). These include, e.g., donor/ acceptor (i.e., first and second signaling moieties) semiconductor nanocrystals (i.e., 'quantum dots') whose absorption and emission spectra can be precisely controlled through the selection of nanoparticle material, size, and composition (see, e.g., Bruchez et al, Science 281 :2013 (1998); Chan et al, J. Colloid and Interface Sci. 203:197 (1998), Han et al, Nature Biotechnol 19:631 (2001)). [00242] The selection of a particular donor/acceptor pair is not critical to practicing the invention provided that energy can be transferred between the donor and the acceptor. P- (dimethyl aminophenylazo) benzoic acid (DABCYL) is one example of a non-fluorescent acceptor dye which effectively quenches fluorescence from an adjacent fluorophore, e.g., fluorescein or 5-(2'-aminoethyι) aminonaphthalene (EDANS).

[00243] The first and second signaling moieties can be attached to terminal or to nonterminal sequences. The position of the non-terminal sequences coupled to signaling moieties is limited to a maximal distance from the 5' or 3' nucleotide which still permits proximity dependent changes in the optical properties of the molecule. Coupling chemistries are routinely practiced in the art, and oligonucleotide synthesis services provided commercially (e.g., Integrated DNA Technologies, Coralville, IA) can also be used to generate labeled molecules. In a further embodiment, the nucleic acid sensor molecule is used, either tethered to a solid support or free in solution, to detect the presence and concentration of target molecules in a complex biological fluid. [00244] For example, the first signaling moiety (F) can be fluorescein molecule coupled to the 5' end and the second signaling molecule (D) can be a DABCYL molecule (a quenching group) coupled to the 3' end. When the nucleic acid sensor molecule is not activated by target molecule, the fluorescent group and the quenching group are in close proximity and little fluorescence is detectable from the fluorescent group. Addition of target molecule causes a change in the conformation of the optical nucleic acid sensor molecule. When the molecule is activated by target recognition, and the first and second signaling moieties (F and D, respectively) are no longer in sufficient proximity for the quenching group to quench the fluorescence of the fluorescent group, the result is a detectable fluorescent signal being produced upon recognition of the target molecule. [00245] One general method for implementing a FRET-based (fluorescence resonance energy transfer) assay utilizing nucleic acid sensor molecules is described for a hammerhead nucleic acid sensor molecule, wherein the nucleic acid sensor molecule is immobilized on a solid substrate, e.g., within a microtiter plate well, on a membrane, on a glass or plastic microscope slide, etc. In the embodiment shown in Figures 15A, B, and C, a self-cleaving ribozyme such as the hammerhead (in this case attached to a solid support via a linker molecule is shown) is labeled with a fluorophore. In Figure 15A, the labeled NASM in the unactivated state comprises two oligonucleotides including a transacting cleavage substrate which bears a first and second fluorescent label. In the unactivated state, i.e., in the absence of target molecule, the donor fluorophore and the acceptor fluorophore are in sufficiently close proximity for FRET to occur; thus, minimal fluorescent emission is detected from the donor fluorophore at wavelength 3, λ3, upon epi-illumination excitation at the excitation wavelength, λ_EX. Upon target molecule recognition, the cleavage fragment of the cleavage substrate bearing the acceptor fluorophore dissociates from the NASM- target complex. Once separated from the acceptor fluorophore, the donor fluorophore can no longer undergo de-excitation via FRET, resulting in a detectable increase in its fluorescent emission at wavelength, λ_EM (see, e.g., Singh, et al, RNA 5:1348 (1999); Wu et al, Anal. Biochem. 218:1 (1994); Walter et al, RNA 3:392 (1997); Walter et al, The EMBO Journal 17(8):2378 (1998)). In a further embodiment, the change in the polarization state of the fluorescent emission from the donor fluorophore (due to the increased diffusional rotation rate of the smaller cleavage fragment) can be detected/monitored in addition to changes in fluorescent emission intensity (see, e.g., Singh et al, Biotechniques 29:344 (2000)). In a further embodiment, the NASMs are free in solution. [00246] In another embodiment, shown in Figure 15B, the acceptor fluorophore attached to the cleavage substrate is replaced by a quencher group. This replacement will also result in minimal fluorescent donor emission at wavelength λ_EXwhen the NASM is in the unbound state under epi-illumination excitation at wavelength λ_EX. Upon target molecule recognition, the cleavage fragments of the cleavage substrate bearing the donor and quencher groups dissociate from the NASM-target molecule complex. Once separated from the quencher, the donor fluorophore will exhibit a detectable increase in its fluorescent emission at wavelength λ_EM. In a further embodiment, the change in the polarization state of the fluorescent emission from the donor fluorophore (due to the increased diffusional rotation rate of the smaller cleavage fragment) can be detected/monitored in addition to changes in fluorescent emission intensity. In a further embodiment, NASMs are free in solution.

[00247] In a different embodiment, the optical configuration is designed to provide excitation via total internal reflection (TIR)-illumination, as shown in Figure 15C. Also, the donor fluorophore is attached to the NASM body while the quencher is attached to the cleavage substrate. In this configuration, with the surface-immobilized NASM in the unbound state, the fluorescent donor emission at wavelength λ_EM were minimal. Upon target module recognition, the cleavage fragment of the cleavage substrate bearing the quencher group dissociates from the NASM-target module complex. Once separated from the quencher, the donor fluorophore will exhibit a detectable increase in its fluorescent emission at wavelength λ_EM. In an alternative embodiment to that shown in shown in Figure 15C, the quencher group can be replaced with an acceptor fluorophore. In yet another alternative embodiment to those shown in Figures 15 A, B, and C, the donor fluorophore is coupled to the cleavage fragment of the cleavage substrate and the acceptor fluorophore or quencher group is deleted. Upon target molecule recognition and dissociation of the cleavage fragment, the polarization state of the fluorescent emission from the donor fluorophore will undergo a detectable change due to the difference in the diffusional rotation rates of the surface-bound NASM target complex and the free cleavage fragment. [00248] In one embodiment, a universal FRET trans-substrate is synthesized for all

NASMs derived from self-cleaving allosteric ribozymes. This substrate would have complementary optical signaling units (i.e., donor and acceptor groups) coupled to opposite ends of the synthetic oligonucleotide sequence. Such a universal substrate would obvzαte the need for coupling optical signaling units to the sensor (i.e., ribozyme) molecule itself. [00249] In addition to the herein described methods, any additional proximity dependent signaling system known in the art can be used to practice the method according to the invention, and are encompassed within the scope.

[00250] In one specific embodiment described here, a first oligonucleotide of the nucleotide sensor molecule is 3 '-labeled with an acceptor or quencher fluorophore, such as TAMRA, AlexaFluor 568, or DABCYL, via specific periodate oxidation. A second oligonucleotide of the nucleic acid sensor molecule, complementary to at least part of the first oligo portion of the NASM, is labeled with a 3' biotin and a 5' donor fluorophore, such as fluorescein (FAM, FITC, etc.). These two nucleic oligonucleotides are heat-denatured in solution and allowed to anneal hybridize during cooling to room temperature. After hybridization, the NASM solution is applied to a surface which has been coated with some type of avidin (streptavidin, neutravidin, avidin, etc.). This surface could include a microtiter plate well, a streptavidin-impregnated membrane, a glass or plastic microscope slide, etc. In any case, the ribozyme-oligo complex is specifically immobilized via the 3' biotin on the donor oligo, leaving the binding domain free to interact with the target effector molecule.

[00251] The donor and acceptor fluorophores form an efficient FRET-pair; that is, upon excitation of the donor fluorophore near its spectral absorption maxima, the incident electromagnetic energy is efficiently transferred (nonradiatively) via resonant electric dipole coupling from the donor fluorophore to the acceptor fluorophore. The efficiency of this resonant energy transfer is strongly dependent on the separation between the donor and acceptor fluorophores, the transfer rate being proportional to 1 R⁶, where R is the intermolecular separation. Therefore, when the donor and acceptor are in close proximity, i.e., a few bond-lengths or roughly 10-50 Angstroms, the fluorescent emission from donor species were reduced relative to its output in an isolated configuration, while the emission from the acceptor species, through indirect excitation by the donor, were detectable. Upon separation of the donor and acceptor, the donor fluorescence emission signal will increase strongly, while the acceptor emission signal will show a commensurate decrease in intensity. After effector-mediated cleavage at room temperature, the cleavage fragment will rapidly dissociate from the ribozyme body and diffuse away into solution. [00252] This target-activated nucleic acid sensor molecule system constitutes a highly sensitive real-time sensor for detecting and quantitating the concentration of the target molecule present in an unknown sample solution. The ultimate limit of detection (LOD) for this system is determined by the switch factor, defined as the ratio of the catalytic rate (in this example, the rate of cleavage) of the ribozyme sensor in the presence of its target to that of the ribozyme in the absence of its target. The dynamic range of the ribozyme sensor were determined by the switch factor and the dissociation constant, Ka, for the interaction of the ribozyme binding domain with the target molecule. In theory, the effective dynamic range over which the rate-response of the NASM is linear in the target concentration has K_d as an upper bound.

[00253] In practice, concentration measurements up to 1 mM are possible with this sensor in solution-phase measurements. The absolute precision of measurements made with this NASM will depend on the amount of background catalytic activity (i.e., in the absence of target) and baseline drift of the fluorescence signals from both sample and controls due to physical factors, such as liquid handling errors, reagent adhesion, evaporation, or mixing. After some optimization, run-to-run CVs of a few percent are possible with FRET-based NASMs measured in solution. Immobilization of the NASM does not degrade its catalytic activity, although it may limit the effective availability of the target-binding domain for interaction with target molecules. The locally high concentration of surface-immobilized NASM will tend to offset this effect by driving the equilibrium for the association (and subsequent catalytic) reactions toward formation of ribozyme-target complex. Detection of the fluorescent signals can be accomplished by a microplate fluorescence reader equipped with the appropriate lamps, optics, filters, and optical detectors (PMT) manufactured by Packard Instrument Co. [00254] Such a sensor array could be used to detect and quantify the presence of an arbitrary target molecule in a complex solution, e.g., crude cell extract or biological fluid, in real time. In addition, this general NASM strategy could be extended to accomplish multiplexed detection of multiple analytes in a sample simultaneously, by using NASMs labeled with fluorophores having different emission wavelengths. In all of these scenarios, optical detection of the FRET signals could be accomplished using a commercially available microarray imager or scanning fluorescence microscope.

[00255] For example, fluorescence energy resonance transfer (FRET) can be used as a general detection method for hammerhead ribozyme or effector-dependent hammerhead ribozyme activity. Hammerhead NASMs typically consist of a catalytic domain responsible for RNA phosphodiester cleavage activity, plus a target modulation domain which, upon binding of an analyte molecule, triggers a structural change within the NASM and leads to the cleavage reaction. In one specific embodiment, described herein, such core hammerhead NASMs are modified to contain a donor fluorophore (D) covalently attached to the 3 '-end of the NASM. In addition, a sequence domain to which a fluorescence quencher/acceptor dye (Q/A) containing auxiliary oligonucleotide can be hybridized is attached adjacent to either stem I or stem III (Figure 16). The fluorophores are chosen to form an efficient FRET-pair; that is, upon excitation of the first, or donor fluorophore near its spectral absorption maxima, the incident electromagnetic energy is efficiently transferred (nonradiatively) via resonant electric dipole coupling from the donor fluorophore to the second, or acceptor fluorophore. The efficiency of this resonant energy transfer is strongly dependent on the separation between the donor and acceptor fluorophores, the transfer rate being proportional to 1/R⁶, where R is the intermolecular separation. Therefore, when the donor and acceptor are in close proximity, i.e., a few bond-lengths or roughly 10-50 Angstroms, the fluorescent emission from donor species were reduced relative to its output in an isolated configuration, while the emission from the acceptor species, through indirect excitation by the donor, were detectable. Therefore the relative positioning of the fluorescence-labeled NASM 3 '-terminus and the second fluorophore should be in close proximity to allow for such an energy transfer.

[00256] One example of FRET pairs are fluorescein as donor and TAMRA as acceptor. Alternatively, the acceptor can be replaced by a so-called dark quencher, such as DABCYL or QSY-7. Either relative orientation of the fluorophores (donor/acceptor and NASM/auxiliary oligo) can be chosen. The exact distance is governed by the number of unpaired nucleotides connecting stem I or III and the hybridization domain for the second oligo, and preferably is between 2 and 4 nucleotides long. The stem involving the 3'- terminus must be long enough to ensure proper folding into a hammerhead structure, but not too long to prevent rapid dissociation after hammerhead cleavage, and is preferably between 5 and 8 nucleotides. The attachment of the first fluorophore to the NASM 3'-terminus can be done by a variety of methods such as enzymatic ligation of a fluorescent nucleotide using terminal transferase or RNA ligase, or by oxidizing the terminal ribonucleotide with sodium periodate, followed by reaction with a fluorophore amine in the presence of sodium borohydride/cyanoborohydride, or a fluorophore hydrazide, semicarbazide or thiocarbazide (Agrawal in Protocols for Oligonucleotide Conjugates, Humana Press, Totowa, 1994, 26, 93; Wu et al, Nucleic Acids Research 24(17):3472 (1996)). Notably, apart from the 3'- modifications, the NASMs can be synthesized entirely through in simple vitro transcription reactions and do not have to contain any other internal or 5' chemical modifications that are potentially difficult to introduce. The auxiliary oligonucleotide can be of any nucleotide sequence or composition (e.g., DNA, RNA, 2'-OMe-RNA, 2'-F-RNA or combination thereof), with a length ensuring tight hybridization to the complementary NASM domain, preferably between 20 to 30 nucleotides. Conversely the length and sequence of the corresponding NASM domain can be freely chosen to accommodate the auxiliary oligonucleotide.

[00257] An example of a stem I-modified FRET hammerhead NASM is illustrated in

Figure 9. In addition, the NASM can be immobilized on a solid support via its auxiliary oligonucleotide, for example through incorporation of a biotin and capture on a streptavidin surface (Figure 10). This surface could include a microtiter plate well, a streptavidin- impregnated membrane, a glass or plastic microscope slide, etc. Preferably immobilization takes place though the remote end of the auxiliary oligo, exposing the NASM core to the solution and not restricting it's accessibility or activity. The generalization of this application of surface-immobilized ribozyme sensors with FRET detection to a micro- or macro-arrayed format on an extended substrate such as glass or plastic is easily envisioned. Such a sensor array could be used to detect and quantify the presence of an arbitrary target molecule in a complex solution, e.g., crude cell extract or biological fluid, in real time. In this scenario, optical detection of the FRET signals could be accomplished using a commercially available microarray imager or scanning fluorescence microscope. [00258] Upon effector-mediated cleavage of the hammerhead NASM, the 3 '- terminus that contains one of the dye modifications is separated and dissociates away from the core NASM (Figure 17). Thereby the donor and acceptor fluorophores are separated, leading to a strong increase in the donor fluorescence emission signal, while the acceptor emission signal will show a commensurate decrease in intensity. The increase or decrease in fluorescence can be recorded as a function of reaction time. Since the hammerhead NASM construct described herein exerts cis-cleavage activity, they follow a first-order cleavage kinetic model which allows the calculation of reaction rates after analysis of the resulting fluorescence vs. time curves (Figures 11 A and 1 IB). Typically, within a certain range, the catalytic rate is a function of the effector concentration and can therefore be used to calculate an unknown effector concentration based on a measured rate value. This type of 1st order kinetic analysis in completely independent on the absolute fluorescent signal values, but relies only on their relative change over time. This makes this system particularly robust against signal fluctuations due to pipetting errors etc. compared to other, trans-reacting systems (i.e., hammerhead ribozymes acting on a separate substrate molecule).

[00259] To perform fluorescence resonance energy transfer (FRET) measurements, fluorescein-labeled RNA and quencher oligo are mixed to form the nucleic acid sensor cleavage solution. Cleavage reactions are performed in black 96-well microplates, and are started by mixing the nucleic acid sensor solution with target molecule in assay buffer. The fluorescence signals are monitored in a Fusion™ a-FP plate reader and the obtained fluorescence (rfu) values are plotted against time. The apparent reaction rates can be calculated assuming the 1st order kinetic model equation y = A(l-e^"kt)+NS (A: signal amplitude; k: observed catalytic rate; NS: nonspecific background signal) using a curve fit algorithm (KaleidaGraph, Synergy Software, Reading, PA), as shown in Figure 11. Dose- response curves are generated by plotting the calculated rates vs. the corresponding target concentrations. ii) Indirect Energy Transfer [00260] Other proximity-dependent signaling systems that do not rely on direct energy transfer between signaling moieties are also known in the art and can be used in the methods described herein. These include, e.g., systems in which a signaling moiety is stimulated to fluoresce or luminesce upon activation by the target molecule. This activation may be direct (e.g., as in the case of scintillation proximity assays (SPA), via a photon or radionucleide decay product emitted by the bound target), or indirect (e.g., as in the case of AlphaScreen™ assays, via reaction with singlet oxygen released from a photosensitized donor bead upon illumination). In both scenarios, the activation of detected signaling moiety is dependent on close proximity of the signaling moiety and the activating species. In general, for both fluorescence, fluorescence polarization, and scintillation-proximity-type assays, the nucleic acid sensor molecule may be utilized in either solution-phase or solid- phase formats. That is, in functional form, the nucleic acid sensor molecule may be tethered (directly, or via a linker) to a solid support or free in solution.

[00261] In one embodiment of an SPA assay, nucleic acid sensor molecules which ligate an oligonucleotide substrate in the presence of a target molecule (PA), are bound to a scintillant-impregnated microwell plate (e.g., FlashPlates, NEN Life Sciences Products , Boston, MA) coated with, for example, streptavidin via a (biotin) linker attached to the 5' end of a capture oligonucleotide sequence. The various plate-sensor coupling chemistries are determined by the type and manufacturer of the plates, and are well-known in the art. Upon the addition of a solution containing target molecule and excess radiolabeled (e.g.,

35

S) oligonucleotide substrate in ligation buffer, the NASMs hybridize and ligate the substrate oligonucleotide. Some fraction of the radiolabeled oligonucleotide substrate were ligated to surface-immobilized NASMs on the plate, while unligated oligonucleotide substrate were free in solution. Only those oligonucleotide substrates ligated to surface- immobilized NASMs on the plate were in close enough proximity to the scintillant molecules embedded in the plate to excite them, thereby stimulating luminescence which can be easily detected using a luminometer (e.g., the TopCount luminescence plate reader, Packard Biosciences, Meriden, CT). This type of homogeneous assay format provides straightforward, real-time detection, quantification, and kinetic properties of target molecule binding.

[00262] In another embodiment, a similar SPA assay format is performed using scintillant-impregnated beads (e.g., Amersham Pharmacia Biotech, Inc., Piscataway, NJ). In this embodiment, NASMs which ligate on an oligonucleotide substrate in the presence of a target molecule are coupled to scintillant-impregnated beads which are suspended in solution in, for example, a microwell plate. The various bead-sensor coupling chemistries are determined by the type and manufacturer of the beads, and are well-known in the art. Upon the addition of a solution containing target molecule and excess radiolabeled (e.g.,

35

S) oligonucleotide substrate in ligation buffer, the NASMs hybridize and ligate the oligonucleotide substrate. Some fraction of the radiolabeled substrate were ligated to surface-immobilized NASMs on the beads, while unligated substrate were free in solution. Only those substrates ligated to surface-immobilized NASMs on the beads were in close enough proximity to the scintillant molecules embedded in the beads to excite them, thereby stimulating luminescence which can be easily detected using a luminometer (e.g., the TopCount luminescence plate reader, Packard Biosciences, Meriden, CT). In addition to enabling real-time target detection and quantification, this type of homogeneous assay format can be used to investigate cellular processes in situ in real time. This could be done by culturing cells directly onto a microwell plate and allowing uptake of scintillant beads and radioisotope by cells. Biosynthesis, proliferation, drug uptake, cell motility, etc. can then be monitored via the luminescence signal generated by beads in presence of selected target molecules (see, e.g., Cook et al, Pharmaceutical Manufacturing International pp. 49- 53 (1992) or Heath et al, Cell Signaling: Experimental Strategies pp. 193-194 (1992)). [00263] Figures 18A and 18B show an exemplary embodiment of a non-isotopic proximity assay based on nucleic acid sensor molecules used in conjunction with AlphaScreen™ beads (Packard Biosciences, Meriden, CT). In this embodiment, the nucleic acid sensor molecules, which ligate an oligonucleotide substrate in the presence of a target molecule, are bound to a chemiluminescent compound-impregnated acceptor bead coated with, for example, streptavidin, via a (biotin) linker attached to the 5' end of the effector oligonucleotide sequence. The various bead-sensor coupling chemistries are determined by the type and manufacturer of the beads, and are well-known in the art. The oligonucleotide substrate is coupled to a photosensitizer-impregnated donor bead coated with, for example, streptavidin, via a (biotin) linker attached to the 3' end of the substrate. The donor (substrate) and acceptor (ribozyme) beads and target molecules are then combined in solution in a microwell plate, some of the NASMs hybridize and ligate the oligonucleotide substrate, bringing the donor and acceptor beads into close proximity (< 200 nm). Upon illumination at 680 nm, the photosensitizer in the donor bead converts ambient oxygen into the singlet state at a rate of approximately 60,000/second per bead. The singlet oxygen will diffuse a maximum distance of approximately 200 nm in solution; if an acceptor bead containing a chemiluminescent compound is within this range, i.e., if ligation has occurred in the presence of the target molecule, chemiluminescence at 370 nm is generated. This radiation is immediately converted within the acceptor bead to visible luminescence at 520- 620 nm with a decay half-life of 0.3 sec. The visible luminescence at 520-620 nm is detected using a time-resolved fluorescence/luminescence plate reader (e.g., the Fusion multifunction plate reader, Packard Biosciences, Meriden, CT). This type of nomsotopic homogeneous proximity assay format provides highly sensitive detection and quantification of target molecule concentrations in volumes < 25 microliters for high throughput screening (see, e.g., Beaudet et al, Genome Res. 11:600 (2001)).

[00264] SPA assays can be performed with any type of NASM (i.e. , endonucleases as well as ligases). This type of assay can also be used with the aptamers of the invention to monitor the presence or concentration of target in a solution. iii) Optical Signal Generating Units With Single Signaling Moieties [00265] In one embodiment, the optical nucleic acid sensor molecule comprises an optical signaling unit with a single signaling moiety introduced at either an internal or terminal position within the nucleic acid sensor molecule. In this embodiment, binding of the target molecule results in changes in both the conformation and physical aspect (e.g., molecular volume or mass, rotational diffusion rate, etc.) of the nucleic acid sensor molecule. Conformational changes in the nucleic acid sensor molecule upon target recognition will modify the chemical environment of the signaling moiety. Such a change in chemical environment will in general change the optical properties of the signaling moiety. Suitable signaling moieties are described in Jhaveri et al, Am. Chem. Soc. 122:2469-2473 (2000), and include, e.g., fluorescein, acridine, and other organic and nonorganic fluorophores.

[00266] In one embodiment, a signaling moiety is introduced at a position in the catalytic nucleic acid molecule near the target activation site (identifiable by footprinting studies, for example). Binding of the target molecule will (via a change in conformation of the nucleic acid molecule) alter the chemical environment and thus affect the optical properties of the signaling moiety in a detectable manner.

[00267] Recognition of the target molecule by the NASM will result in changes in the conformation and physical aspect of the nucleic acid sensor molecule, and will thus alter the kinetic properties of the signaling moiety. In particular, the changes in conformation and mass of the sensor-target complex will reduce the rotational diffusion rate for the sensor- target complex, resulting in a detectable change in the observed steady state fluorescence polarization (FP) from the signaling moiety. The expected change in FP signal with target concentration can be derived using a modified form of the well-known Michaelis-Menten model for ligand binding kinetics (see, e.g., Lakowicz, J.R., Principles of Fluorescence Spectioscopy, Second Edition, 1999, Kluwer Academic/Plenum Publishers, New York). FP is therefore a highly sensitive means of detecting and quantitatively determining the concentration of target molecules in a sample solution (Jameson et al, Methods in Enzymology 246:283 (1995); Jameson et al, METHODS 19:222 (1999); Jolley, Comb. Chem. High Throughput Screen 2(4):177 (1999); Singh, et al, BioTechniques 29:344 (2000); Owicki et al, Genetic Engineering News 17(19) (1997)). FP methods are capable of functioning in both solution- and solid-phase implementations.

[00268] Numerous additional methods can be used that, e.g. , make use of a single fluorescent label and an unpaired guanosine residue (instead of a quencher group), to enable the use of FRET in target detection and quantitation as described in the embodiments above (see, e.g, Walter et al, RNA 3:392 (1997)).

[00269] In a further embodiment, shown in Figure 19A, B, and C, an unlabeled ligating NASM such as the lysozyme-dependent LI ligase is shown (see, e.g., Robertson et al, Nucleic Acids Res. 28:1751-1759 (2000)). In the unactivated state, i.e., in the absence of target, no fluorescent emission is detected from the surface-bound NASMs under total internal reflection (TIR)-illumination (see Figure 19A), or epi-illumination (see Figure 19B). Upon recognition of target molecules in the presence of an oligonucleotide substrate with a tag (where the tag is capable of binding to a subsequently added fluorescent label via interactions including, but not limited to, biotm/streptavidin, amine/aldehyde, hydrazide, thiol, or other reactive groups) those oligonucleotide substrates hybridized to NASMs will undergo ligation and become covalently bonded thereto. In order to maximize the probability of hybridization for a given NASM, oligonucleotide substrate can be added in excess relative to NASM, the temperature of the ambient solution in which the reaction takes place can be kept below room temperature (e.g., 4 C), and agitation of the reaction vessel can be employed to overcome the kinetic limitation of diffusion-limited transport of species in solution. Given the above conditions, as well as sufficient time for maximal hybridization and subsequent ligation to occur, fluorescent label with the appropriate reactive group to bind the substrate tag is added to the reaction mixture. Again, the degree of substrate-label binding can be maximized through control of label concentration, solution temperature, and agitation. Once the fluorescent label has bound to all available ligated substrate-NASM target complex, the solution temperature can be raised to drive off all of the hybridized but unligated substrate. With TIR-illumination, the spatial extent of the excitation region above the solid substrate surface to which the ribozymes are bound is only on the order of 100 nm. Therefore, the bulk solution above the substrate surface is not illuminated and the detected fluorescent emission were primarily due to fluorophores which are bound to ligated oligonucleotide substrate-NASM-target molecule complexes tethered to the substrate surface. The fluorescence emission from surface-bound NASM-target molecule complexes in this homogeneous solid phase assay format represents an easily detectable optical signal. In another embodiment, the fluorescence polarization (FP) of the labeled substrate can be monitored, as shown in Figure 19C. Upon ligation, the steady state fluorescence polarization signal from the substrate-NASM complex will increase detectably relative to the FP signal from the free labeled oligonucleotide substrate in solution, due to the difference in the diffusional rotation rates between the free and ligated forms. [00270] In another embodiment, an unlabeled ligating NASM such as the lysozyme- dependent LI ligase (see, e.g., Robertson et al, Nucleic Acids Res. 28:1751-1759 (2000)) is bound to a solid surface. In this embodiment, the oligonucleotide substrate is coupled to an enzyme-linked luminescent moiety, such as horseradish peroxidase (HRP) by a tag (where the tag is capable of binding to a subsequently added label via interactions including, but not limited to, biotin/streptavidin, amine/aldehyde, hydrazide, thiol, or other reactive groups). In the absence of target molecule, no luminescent emission is detected from the surface-bound NASMs. Upon recognition of target molecules in the presence of labeled oligonucleotide substrate, those oligonucleotide substrates hybridized to NASMs will undergo ligation and become covalently bonded to the NASMs. After removal of excess, unbound oligonucleotide substrate, the substrate for activation of the enzyme-linked luminescent label is added to the reaction volume. The resulting luminescent signal (e.g., from HRP, luciferase, etc.) is easily detectable using standard luminometers (e.g., the Fusion multifunction plate reader, Packard Bioscience). In a further embodiment, the activated solution can be precipitated, followed by colorimetric detection. In a particular embodiment, the enzyme linked signal amplification, TSA, (sometimes referred to as CARD-catalyzed reporter deposition) is an ultrasensitive detection method. The technology uses turnover of multiple tyramide substrates per horseradish peroxidase (HRP) enzyme to generate high-density labeling of a target protein or nucleic acid probe in situ. Tyramide signal amplification is a combination of three elementary processes: (1) Ligation (or not) of a biotinylated ligase oligonucleotide substrate oligo, followed by binding (or not) of a streptavidin-HRP to the probe; (2) HRP-mediated conversion of multiple copies of a fluorescent tyramide derivative to a highly reactive radical; and (3) Covalent binding of the reactive, short lived tyramide radicals to nearby nucleophilic residues, greatly reducing diffusion-related signal loss. 5) GENERATING BIOSENSORS

[00271] Optical nucleic acid sensor molecules for the detection of a target molecule of interest are generated by first selecting catalytic nucleic acid molecules with catalytic activity modifiable (e.g., activatable) by a selected target molecule. In one embodiment, at least a portion of the catalytic site of the catalytic NASM is then removed and an optical signal generating unit is either added or inserted. Recognition of the target molecule by the nucleic acid sensor molecule activates a change in the properties of the optical signaling unit.

[00272] The nucleic acid sensor molecules can be, e.g., those which possess either ligating or cleaving activity in the presence of a target molecule.

[00273] One advantage of using nucleic acid sensor molecule arrays as opposed to protein arrays is the relative ease with which nucleic acid sensor molecules can be attached to chip surfaces. Immobilization of nucleic acid sensor molecules on a substrate provides a straightforward mechanism for carrying out multiple arrays in parallel. Initially, the optimal attachment chemistries are determined for use in immobilizing these molecules on a solid substrate. These molecules are further configured such that their activity and allosteric behavior is maintained following immobilization. Generally, the chip is configured such that it may be placed at the bottom of a sample holder and overlaid with sample solution, target and substrate oligonucleotide. Following an incubation to allow target present within the sample to activate catalysis, the sample is washed away and the extent of ribozyme catalysis quantified.

[00274] For example, endonuclease nucleic acid sensor molecules are generated by transcription in the presence of γ-thio-GTP (introducing a unique thiol at their 5 '-end) and subsequently attached to a thiol-reactive surface (e.g. gold-coated polystyrene as described by Seetharaman et al, Nature Biotech 19:336 (2001)). Attachment methodologies are evaluated on the basis of the following criteria: efficiency, e.g., what is the yield of nucleic acid sensor molecule capture; capacity, e.g., what is the maximum concentration of nucleic acid sensor molecules that can be localized in a given spot size; stability, e.g., are ribozymes efficiently retained under a variety of solution conditions and during long-term storage; detection, e.g., do immobilization chemistries interfere with the ability to generate a detectable signal. [00275] To the extent that activity for immobilized nucleic acid sensor molecules is diminished, three different strategies for reconfiguring ribozymes for activity in solid phase applications are available: 1) immobilization chemistries, a variety of different immobilization chemistries are compared on the basis of their ability to maintain allosteric behavior. To the extent that they leave different surfaces available for protein effectors to interact with, that they tether different ends of the nucleic acid sensor molecules, and that they position the NASM either directly at the surface or displaced from the surface (in the case of streptavidin capture), different behaviors are observed depending upon the immobilization method. Protein-target activated NASMs have been shown to function in both direct and indirect attachment scenarios; 2) blocking chemistries, blocking agents (e.g., carrier proteins) are tested to determine whether losses in allosteric responsiveness are due to non-specific interactions between the allosteric activators and the chip surface; 3) tethers, steric effects may cause decreased catalytic activity upon direct end attachment to a solid support. Arbitrary sequence tethers are added as needed to increase the spacing between the attachment end and the core of the ribozyme.

[00276] Immobilized nucleic acid sensor molecules for target are prepared and are assayed for activity by monitoring either retention of end-labeled oligonucleotide substrate (for LI ligase-based ribozymes) or release of end-labeled ribozyme (for endonucleases as originally described by Seetherman et al, Nature Biotech 19:336 (2001)). Radioactive tracers are used for labeling RNAs and substrates.

[00277] In one embodiment, a biosensor is provided which comprises a plurality of optical nucleic acid sensor molecules labeled with first and second signaling moieties specific for a target molecule. In another embodiment, the optical NASMs are labeled with a single signaling moiety. In one embodiment, the labeled nucleic acid sensor molecules are provided in a solution (e.g., a buffer). In another embodiment, the labeled nucleic acid sensor molecules are attached directly or indirectly (e.g., through a linker molecule) to a substrate. In further embodiments, nucleic acid sensor molecules can be synthesized directly onto the substrate. Suitable substrates which are encompassed within the scope include, e.g., glass or quartz, silicon, encapsulated or unencapsulated semiconductor nanocrystal materials (e.g., CdSe), nitrocellulose, nylon, plastic, and other polymers. Substrates may assume a variety of configurations (including, e.g., planar, slide shaped, wafers, chips, tubular, disc-like, beads, containers, or plates, such as microtiter plates, and other shapes). [00278] Different chemistries for attaching nucleic acid sensor molecules to solid supports include: 1) conventional DNA arrays using aldehyde coated slides and 5 '-amino modified oligonucleotides. The attached oligonucleotide serves as a capture tag that specifically hybridizes to a 3 '-end extension on the ribozyme. Nucleic acid sensor molecule RNA treated with periodate to specifically introduce an aldehyde modification at the 3 '-end. Modified RNA can be used either in a subsequent reaction with biotin hydrazide enables RNA capture on commercially-available streptavidin coated slides or in a subsequent reaction with adipic acid dihydrazide enables RNA capture on commercially-available aldehyde coated slides.

[00279] Numerous attachment chemistries, both direct and indirect, can be used to immobilize the sensor molecules on a solid support. These include, e.g., amine/aldehyde, biotin/streptavidin (avidin, neutravidin), ADH/oxidized 3' RNA. In a particular embodiment, the nucleic acid sensor molecules ligate a substrate in the presence of a target molecule. In this embodiment the ribozymes are bound to a solid substrate via the effector oligonucleotide sequence as shown in Figure 20.

[00280] In one embodiment, larger substrates can be generated by combining a plurality of smaller biosensors forming an array of biosensors. In a further embodiment, nucleic acid sensor molecules placed on the substrate are addressed (e.g., by specific linker or effector oligonucleotide sequences on the nucleic acid sensor molecule) and information relating to the location of each nucleic acid sensor molecule and its target molecule specificity is stored within a processor. This technique is known as spatial addressing or spatial multiplexing. Techniques for addressing nucleic acids on substrates are known in the art and are described in, for example, U.S. Patent Number 6,060,252; U.S. Patent Number 6,051,380; U.S. Patent Number 5,763,263; U.S. Patent Number 5,763,175; and U.S. Patent Number 5,741,462.

[00281] In another embodiment, a manual or computer-controlled robotic microarrayer is used to generate arrays of nucleic acid sensor molecules immobilized on a solid substrate. In one embodiment, the arrayer utilizes contact-printing technology (i.e., it utilizes printing pins of metal, glass, etc., with or without quill-slots or other modifications). In a different embodiment, the arrayer utilizes non-contact printing technology (i.e., it utilizes ink jet or capillary-based technologies, or other means of dispensing a solution containing the material to be arrayed). Numerous methods for preparing, processing, and analyzing microarrays are known in the art (see Schena et al, Microarray Biochip Technology, ed. pp. 1-18 (2000); Mace et al, Microarray Biochip Technology, ed. pp. 39- 64 (2000); Heller et al, Academic Press, Inc. pp. 245-256 (1999); Basararsky et al, Microarray Biochip Technology, ed. pp. 265-284 (2000); Schermer, DNA Microarrays a Practical Approach pp. 17-42 (1999)). Robotic and manual arrayers are commercially available including, for example, the SpotArray from Packard Biosciences, Meriden, CT, and the RA-1 from GenomicSolutions, Ann Arbor, MI.

[00282] In another embodiment, different nucleic acid sensor molecules are immobilized on a streptavidin-derivatized substrate via biotin linkers. The individual sensor spots can be manually arrayed. For example, NASM can hybridize to a biotin-linked capture oligo, which in turn will bind to a streptavidin coated surface. [00283] Solution measurements of target molecule concentration can be made by bathing the surface of the biosensor array in a solution containing the targets (analytes) of interest. In practice this is accomplished either by incorporating the array within a microflowcell (with a flow rate of- 25 microliters/min), or by placing a small volume (~ 6- 10 microliters) of the target solution on the array surface and covering it with a cover slip. Detection and quantification of target concentration is accomplished by monitoring changes in the fluorescence polarization (FP) signal emitted from the fluorescein label under illumination by 488 nm laser radiation. The rotational diffusion rate is inversely proportional to the molecular volume; thus the rotational correlation time for the roughly 20-nucleotide unbound sensor (i.e., in the absence of target molecule) were significantly less than that for the target-NASM complex. The fluorescence emission from the target- NASM complex will therefore experience greater residual polarization due to the smaller angle through which the emission dipole axis of the sensor fluorophore can rotate within its radiative lifetime. In another embodiment, different surface attachment chemistries are used to immobilize the NASMs on a solid substrate. As previously noted, these include, e.g., interactions involving biotin/streptavidin, amine/aldehyde, hydrazide, thiol, or other reactive groups.

[00284] One type of array includes immobilized effector oligonucleotides with terminal amine groups attached to a solid substrate derivatized with aldehyde groups. This array can be used to spatially address (i.e., the sequence of nucleotides for each effector oligonucleotide can be synthesized as a cognate to the effector oligonucleotide binding domain of a nucleic acid sensor molecule specific for a particular target molecule) and immobilize the nucleic acid sensor molecules prior to their use in a solid-phase assay (see, e.g., Zammatteo et al, Anal Biochem 280:143 (2000)).

[00285] For example, to attach effector oligonucleotides to aldehyde derivatized substrate, discrete spots of solution containing effector oligonucleotides with amine-reactive terminal groups or linkers with terminal amine groups using microarraying pins, pipette, etc are printed and then allowed to dry for 12 hrs. at room temperature and < 30% relative humidity. The substrate is then rinsed twice with dH₂0 containing 0.2% SDS for 2 min. with vigorous agitation at room temperature. The substrate is then rinsed once in dH₂0 for 2 min. with vigorous agitation at room temperature and transferred to boiling (100 °C) dH₂0 for 3 min. to denature DNA. The denatured substrate is then dried by centrifuging at 500 x g for 1 min. and then treated with 0.1 M NaBKU in phosphate buffered saline (PBS, pH 7) for 5 min. with mild agitation at room temperature. Following NaBH₄ treatment, the substrate is rinsed twice in dH₂0 containing 0.2% SDS for 1 min. with vigorous agitation at room temperature and then washed once with dH₂0 for 2 min. with vigorous agitation at room temperature. The substrate is again boiled in dH₂0 (100 °C) for 10 sec. to denature DNA. The substrate is dried by centrifugation as described above and stored at 4 °C prior to hybridization.

[00286] In the case where it is desirable to immobilize an array of NASMs by direct attachment to a solid surface, the nucleic acid sensor molecules are bound to a solid substrate directly via their 3' termini. The attachment is accomplished by oxidation (using, e.g., N periodate) of the 3' vicinal diol of the nucleic acid sensor molecule to an aldehyde group. This aldehyde group will react with a hydrazide group to form a hydrazone bond. The hydrazone bond is quite stable to hydrolysis, etc., but can be further reduced (for example, by treatment with NaBH₄ or NaCNBH₃). The use of adipic acid dihydrazide (ADH, a bifunctional linker) to derivatize an aldehyde surface results in a hydrazide- derivatized surface which provides a linker of approximately 10 atoms between the substrate surface and point of biomolecular attachment (see Ruhn et al, J. Chromatography A 669:9 (1994); O'Shaughnessy, J. Chromatography 510:13 (1990); Roberston et al, Biochemistry 11(4):533 (1972); Schluep et al, Bioseparation 7:317 (1999); Chan et al, J. Colloid and Interface Sci. 203:197 (1998)).

[00287] A hydrazide-terminated surface can be prepared by ADH treatment of the aldehyde substrate. Briefly, to 50 mL of 0.1 M phosphate buffer (pH 5) 100-fold excess of adipic acid dihydrazide (ADH) relative to concentration of aldehyde groups is added on substrate surface. The substrate is then placed in a 50 mL tube containing the ADH in phosphate buffer and shaken mixture for 2 h. Following incubation, the substrate is washed 4-times with 0.1 M phosphate buffer (pH 7). The free aldehyde groups on the substrate surface are then reduced by treatment with a 25-fold excess of NaBEL; or NaCNBH₃ in 0.1 M phosphate buffer in a 50 ml conical tube with shaking for 90 min. The substrate is then washed 4-times with 0.1 M phosphate buffer (pH 7) and stored 0.1 M phosphate buffer (pH 7) at 4 °C until use.

[00288] Nucleic acid molecules for specific coupling to the ADH-terminated surface via their 3' termini are prepared by periodate oxidation of the RNA, see, e.g., Proudnikov et al, Nucleic Acid Res. 24(22):4535 (1996); Wu et al, Nucleic Acids Res. 24(17):3472 (1996). Briefly, up to 20 μg RNA in 5 μl of H₂0 at 20 °C is treated with 1 ml 0.1 M NaI0₄ (~20-fold excess relative to RNA). The RNA is incubated with the NaI0₄ for 30 min. in a light-tight tube prior to the addition of 1 ml 0.2 M Na sulphite (~2-fold excess relative to NaI0₄) to stop the reaction (30 min.; room temperature). The oxidized RNA is then recovered by ethanol precipitation and a spin-separation column.

[00289] The specificity of the biosensors and NASMs according to the invention is determined by the specificity of the target modulation domain of the nucleic acid sensor molecule. In one embodiment, a biosensor is provided in which all of the nucleic acid sensor molecules recognize the same molecule. In another embodiment, a biosensor is provided which can recognize at least two different target molecules allowing for multi- analyte detection. Multiple analytes can be distinguished by using different combinations of first and second signaling molecules. In addition to the wavelength/color and spatial multiplexing techniques previously described, biosensors may be used to detect multiple analytes using intensity multiplexing. This is accomplished by varying the number of fluorescent label molecules on each biosensor in a controlled fashion. Since a single fluorescent label is the smallest integral labeling unit possible, the number of fluorophores (i.e., the intensity from) a given biosensor molecule provides a multiplexing index. Using the combination of 6-wavelength (color) and 10-level intensity multiplexing, implemented in the context of semiconductor nanocrystals derivatized as bioconjugates, would theoretically allow the encoding of million different analyte-specific biosensors (Han et al, Nature Biotechnol. 19:631 (2001)).

[00290] In one embodiment, multiple single target biosensors can be combined to form a multianalyte detection system which is either solution-based or substrate-based according to the needs of the user. In this embodiment, individual biosensors can be later removed from the system, if the user desires to return to a single analyte detection system (e.g., using target molecules bound to supports, or, for example, manually removing a selected biosensor(s) in the case of substrate-based biosensors). In a further embodiment, nucleic acid sensor molecules binding to multiple analytes are distinguished from each other by referring to the address of the nucleic acid sensor molecule on a substrate and correlating its location with the appropriate target molecule to which it binds (previously described as spatial addressing or multiplexing).

[00291] In one embodiment, subsections of a biosensor array can be individually subjected to separate analyte solutions by use of substrate partitions or enclosures that prevent fluid flow between subarrays, and microfluidic pathways and injectors to introduce the different analyte solutions to the appropriate sensor subarray.

NUCLEIC ACID SENSOR MOLECULE AND BIOSENSOR SYSTEMS

[00292] In one embodiment, a nucleic acid sensor molecule or biosensor system is provided comprising a nucleic acid sensor molecule in communication with a detector system. In a further embodiment, a processor is provided to process optical signals detected by the detector system. In still a further embodiment, the processor is connectable to a server which is also connectable to other processors. In this embodiment, optical data obtained at a site where the NASM or biosensor system resides can be transmitted through the server and data is obtained, and a report displayed on the display of the off-site processor within seconds of the transmission of the optical data. In one embodiment, data from patients is stored in a database which can be accessed by a user of the system. [00293] Data obtainable from the biosensors according to the invention include diagnostic data, data relating to lead compound development, and nucleic acid sensor molecule modeling data (e.g., information correlating the sequence of individual sensor molecules with specificity for a particular target molecule). In one embodiment, these data are stored in a computer database. In a further embodiment, the database includes, along with diagnostic data obtained from a sample by the biosensor, information relating to a particular patient, such as medical history and billing information. Although, in one embodiment, the database is part of the nucleic acid sensor molecule system, the database can be used separately with other detection assay methods and drug development methods. [00294] Detectors used with the nucleic acid sensor molecule systems according to the invention, can vary, and include any suitable detectors for detecting optical changes in nucleic acid molecules. These include, e.g, photomultiplier tubes (PMTs), charge coupled devices (CCDs), intensified CCDs, and avalanche photodiodes (APDs). In one embodiment, an optical nucleic acid sensor molecule is excited by a light source in communication with the biosensor. In a further embodiment, when the optical signaling unit comprises first and second signal moieties that are donor/acceptor pairs (i.e., signal generation relies on the fluorescence of a donor molecule when it is removed from the proximity of a quencher acceptor molecule), recognition of a target molecule will cause a large increase in fluorescence emission intensity over a low background signal level. The high signal-to- noise ratio permits small signals to be measured using high-gain detectors, such as PMTs or APDs. Using intensified CCDs, and PMTs, single molecule fluorescence measurements have been made by monitoring the fluorescence emission, and changes in fluorescence lifetime, from donor/acceptor FRET pairs (see, e.g., Sako, et al, Nature Cell Bio. 2:168 (2000); Lakowicz et al, Rev. Sci. Instr. 62(7):1727 (1991)).

[00295] Light sources include, e.g., filtered, wide-spectrum light sources, (e.g., tungsten, or xenon arc), laser light sources, such as gas lasers, solid state crystal lasers, semiconductor diode lasers (including multiple quantum well, distributed feedback, and vertical cavity surface emitting lasers (VCSELs)), dye lasers, metallic vapor lasers, free electron lasers, and lasers using any other substance as a gain medium. Common gas lasers include Argon-ion, Krypton-ion, and mixed gas (e.g., Ar-Kr) ion lasers, emitting at 455, 458, 466, 476, 488, 496, 502, 514, and 528 nm (Ar ion); and 406, 413, 415, 468, 476, 482, 520, 531, 568, 647, and 676 nm (Kr ion). Also included in gas lasers are Helium Neon lasers emitting at 543, 594, 612, and 633 nm. Typical output lines from solid state crystal lasers include 532 nm (doubled Nd:YAG) and 408/816 nm (doubled/primary from Ti:Sapphire). Typical output lines from semiconductor diode lasers are 635, 650, 670, and 780 nm.

[00296] Excitation wavelengths and emission detection wavelengths will vary depending on the signaling moieties used. In one embodiment, where the first and second signaling moieties are fluorescein and DABCYL, the excitation wavelength is 488 nm and the emission wavelength is 514 nm. In the case of semiconductor nanocrystal-based fluorescent labels, a single excitation wavelength or broadband UV source may be used to excite several probes with widely spectrally separated emission wavelengths (see Bruchez et al, Science 281:2013 (1998); Chan et al, J. Colloid and Interface Sci. 203:197 (1998)). [00297] In one embodiment, detection of changes in the optical properties of the nucleic acid sensor molecules is performed using any of a cooled CCD camera, a cooled intensified CCD camera, a single-photon-counting detector (e.g., PMT or APD), or other light sensitive sensor. In one embodiment, the detector is optically coupled to the nucleic acid sensor molecule through a lens system, such as in an optical microscope (e.g., a confocal microscope). In another embodiment, a fiber optic coupler is used, where the input to the optical fiber is placed in close proximity to the substrate surface of a biosensor, either above or below the substrate. In yet another embodiment, the optical fiber provides the substrate for the attachment of nucleic acid sensor molecules and the biosensor is an integral part of the optical fiber.

[00298] In one embodiment, the interior surface of a glass or plastic capillary tube provides the substrate for the attachment of nucleic acid sensor molecules. The capillary can be either circular or rectangular in cross-section, and of any dimension. The capillary section containing the biosensors can be integrated into a microfluidic liquid-handling system which can inject different wash, buffer, and analyte-containing solutions through the sensor tube. Spatial encoding of the sensors can be accomplished by patterning them longitudinally along the axis of the tube, as well as radially, around the circumference of the tube interior. Excitation can be accomplished by coupling a laser source (e.g., using a shaped output beam, such as from a VCSEL) into the glass or plastic layer forming the capillary tube. The coupled excitation light will undergo TIR at the interior surface/solution interface of the tube, thus selectively exciting fluorescently labeled biosensors attached to the tube walls, but not the bulk solution. In one embodiment, detection can be accomplished using a lens-coupled or proximity-coupled large area segmented (pixelated) detector, such as a CCD. In a particular embodiment, a scanning (i.e., longitudinal axial and azimuthal) microscope objective lens/emission filter combination is used to image the biosensor substrate onto a CCD detector. In a different embodiment, a high resolution CCD detector with an emission filter in front of it is placed in extremely close proximity to the capillary to allow direct imaging of the biosensors. In a different embodiment, highly efficient detection is accomplished using a mirrored tubular cavity that is elliptical in cross-section. The sensor tube is placed along one focal axis of the cavity, while a side- window PMT is placed along the other focal axis with an emission filter in front of it. Any light emitted from the biosensor tube in any direction were collected by the cavity and focused onto the window of the PMT.

[00299] In still another embodiment, the optical properties of a nucleic acid sensor molecule are analyzed using a spectrometer (e.g., such as a luminescence spectrometer) which is in communication with the biosensor. The spectrometer can perform wavelength discrimination for excitation and detection using either monochromators (i.e., diffraction gratings), or wavelength bandpass filters. In this embodiment, biosensor molecules are excited at absorption maxima appropriate to the signal labeling moieties being used (e.g., acridine at 450 nm, fluorescein at 495 nm) and fluorescence intensity is measured at emission wavelengths appropriate for the labeling moiety used (e.g., acridine at 495 nm; fluorescein at 515 nm). Achieving sufficient spectral separation (i.e., a large enough Stokes shift) between the excitation wavelength and the emission wavelength is critical to the ultimate limit of detection sensitivity. Given that the intensity of the excitation light is much greater than that of the emitted fluorescence, even a small fraction of the excitation light being detected or amplified by the detection system will obscure a weak biosensor fluorescence emission signal. In one embodiment, the biosensor molecules are in solution and are pipetted (either manually or robotically) into a cuvette or a well in a microtiter plate within the spectrometer. In a further embodiment, the spectrometer is a multifunction plate reader capable of detecting optical changes in fluorescence or luminescence intensity (at one or more wavelengths), time-resolved fluorescence, fluorescence polarization (FP), absorbance (epi and transmitted), etc., such as the Fusion multifunction plate reader system (Packard Biosciences, Meriden, CT). Such a system can be used to detect optical changes in biosensors either in solution, bound to the surface of microwells in plates, or immobilized on the surface of solid substrate (e.g, a biosensor microarray on a glass substrate). This type of multiplate/multisubstrate detection system, coupled with robotic liquid handling and sample manipulation, is particularly amenable to high-throughput, low- volume assay formats.

[00300] In embodiments where nucleic acid sensor molecules are attached to substrates, such as a glass slide or in microarray format, it is desirable to reject any stray or background light in order to permit the detection of very low intensity fluorescence signals. In one embodiment, a small sample volume (~10 nL) is probed to obtain spatial discrimination by using an appropriate optical configuration, such as evanescent excitation or confocal imaging. Furthermore, background light can be minimized by the use of narrow- bandpass wavelength filters between the sample and the detector and by using opaque shielding to remove any ambient light from the measurement system. [00301] In one embodiment, spatial discrimination of nucleic acid sensor molecules attached to a substrate in a direction normal to the interface of the substrate (i.e., excitation of only a small thickness of the solution layer directly above and surrounding the plane of attachment of the biosensor molecules to the substrate surface) is obtained by evanescent wave excitation. Evanescent wave excitation utilizes electromagnetic energy that propagates into the lower-index of refraction medium when an electromagnetic wave is totally internally reflected at the interface between higher and lower-refractive index materials. In this embodiment a collimated laser beam is incident on the substrate/solution interface (at which the biosensors are immobilized) at an angle greater than the critical angle for total internal reflection (TIR). This can be accomplished by directing light into a suitably shaped prism or an optical fiber. In the case of a prism, the substiate is optically coupled (via index-matching fluid) to the upper surface of the prism, such that TIR occurs at the substrate/solution interface on which the biosensors are immobilized. Using this method, excitation can be localized to within a few hundred nanometers of the substrate/solution interface, thus eliminating autofluorescence background from the bulk analyte solution, optics, or substrate. Target recognition is detected by a change in the fluorescent emission of the nucleic acid sensor, whether a change in intensity or polarization. Spatial discrimination in the plane of the interface (i.e., laterally) is achieved by the optical system. [00302] In one embodiment, a large area of the biosensor substrate is uniformly illuminated, either via evanescent wave excitation or epi-illumination from above, and the detected signal is spatially encoded through the use of a pixelated detector, such as CCD camera. An example of this type of uniform illumination/CCD detection system (using epi- illumination) for the case of microarrayed biosensors on solid substrates is the GeneTAC 2000 scanner (GenomicSolutions, Ann Arbor, MI). In a different embodiment, a small area (e.g., 10 x 10 microns to 100 x 100 microns) of the biosensor substrate is illuminated by a micro-collimated beam or focused spot. In one embodiment, the excitation spot is rastered in a 2-dimensional scan across the static biosensor substiate surface and the signal detected (with an integrating detector, such as a PMT) at each point correlated with the spatial location of that point on the biosensor substrate (e.g., by the mechanical positioning system responsible for scanning the excitation spot). Two examples of this type of moving spot detection system for the case of microarrayed biosensors on solid substrates are: the DNAScope scanner (confocal, epi-illumination, GeneFocus, Waterloo, ON, Canada), and the LS IV scanner (non-confocal, epi-illumination, GenomicSolutions, Ann Arbor, MI). In yet another embodiment, a small area (e.g., 10 x 10 microns to 100 x 100 microns) of the biosensor substrate is illuminated by a stationary micro-collimated beam or focused spot, and the biosensor substiate is rastered in a 2-dimensional scan beneath the static excitation spot, with the signal detected (with an integrating detector, such as a PMT) at each point correlated with the spatial location of that point on the biosensor substrate (e.g., by the mechanical positioning system responsible for scanning the substiate). An example of this type of moving substrate detection (using confocal epi-illumination) system for the case of microarrayed biosensors on solid substrates is the ScanArray 5000 scanner (Packard Biochip, Billerica, MA).

[00303] For example, a TIR evanescent wave excitation optical configuration is implemented, with a static substrate and dual-capability detection system. The detection system is built on the frame of a Zeiss universal fluorescence microscope. The system is equipped with 2 PMTs on one optical port, and an intensified CCD camera (Cooke, St. Louis, MO) mounted on the other optical port. The optical path utilizes a moveable mirror which can direct the collimated, polarized laser beam through focusing optics to form a spot, or a beam expander to form a large (> 1cm) beam whose central portion is roughly uniform over the field of view of the objective lens. Another movable mirror can direct the light either to the intensified CCD camera when using large area uniform illumination, or to the PMTs in the scanned spot mode. In spot scanning mode, a polarizing beamsplitter separates the parallel and perpendicular components of the emitted fluorescence and directs each to its designated PMT. An emission filter in the optical column rejects scattered excitation light from either type of detector. In CCD imaging mode, manually adjusted polarizers in the optical column of the microscope must be adjusted to obtain parallel and perpendicular images from which the fluorescence polarization or anisotropy can be calculated. A software program interfaces with data acquisition boards in a computer which acquires the digital output data from both PMTs and CCD. This program also controls the PMT power, electromechanical shutters, and galvanometer mirror scanner, calculates and plots fluorescence polarization in real time, and displays FP and intensity images. [00304] In another embodiment, the detection system is a single photon counter system (see, e.g., U.S. Patent Number 6,016,195 and U.S. Patent Number 5,866,348) requiring rastering of the sensor substrate to image larger areas and survey the different binding regions on the biosensor.

[00305] In another embodiment of the invention, the biosensor is used to detect a target molecule through changes in the electrochemical properties of the nucleic acid sensor molecules in close proximity to it which occur upon recognition of the target by the NASM. [00306] In a one embodiment, the biosensor system consists of three major components: 1) optical nucleic acid sensor molecules immobilized on an array of independently addressable gold electrodes. The nucleic acid sensor molecules immobilized on each electrode may be modulated by the same or different target molecules, including proteins, metabolites and other small molecules, etc.; 2) an oligonucleotide substrate which acts as a signaling probe, hybridizing to the oligonucleotide substrate binding domain of the ligase sensor and forming a covalent phosphodiester bond with the nucleic acid sensor molecule nucleotide adjacent to its 3' terminus in the presence of the appropriate target. This oligonucleotide substiate is typically a nucleic acid sequence containing one or more modified nucleotides conjugated to redox active metallic complexes, e.g., ferrocene moieties, which can act as election donors; and 3) an immobilized mixed self-assembled surface monolayer (SAM), comprised of conductive species separated by insulating species, covering the surface of the electrodes, as shown in Figures 15 and 16. Examples of conductive species include thiol-terminated linear molecules, such as oligophenylethyl molecules, while examples of nonconductive thiol-terminated linear molecules, include alkane-thiol molecules terminated with polyethylene glycol (PEG). All immobilized species can be covalently attached to the electrode surface by terminal thiol groups. Upon recognition of the target molecule by the target modulation domain and subsequent ligation of the oligonucleotide substiate, the redox active signaling moieties coupled to the substiate oligo were brought into close proximity to the conductive surface layer, resulting in a detectable increase in electronic surface signal.

[00307] In another preferred embodiment, the biosensor system consists of two major components: (1) Optical nucleic acid sensor molecules immobilized on an array of independent addressable gold electrodes. The nucleic acid sensor molecules immobilized on each electrode may be modulated by the same or different target molecules, including proteins, metabolites and other small molecules, etc. The NASM will contain one or more nucleotides conjugated to redox active metallic complexes, e.g., ferrocene moieties, which can act as electron donors; and (2) an immobilized mixed self-assembled surface monolayer (SAM), comprised of conductive species separated by insulating species, covering the surface of the electrodes. Examples of conductive species include thiol-terminated linear molecules, such as oligophenylethyl molecules, while examples of nonconductive thiol- terminated linear molecules include alkane-thiol molecules terminated with polyethylene glycol (PEG). The SAM-coated molecule can be immobilized via a capture oligonucleotide. In this case, the redox active signaling moieties are coupled to the body of the NASM. Upon recognition of the target molecule by the target modulation domain and subsequent cleavage, the bulk of the NASM, including the nucleotides coupled to the redox active signaling moieties, will dissociate from the surface, resulting in a detectable loss of electronic current signal.

[00308] In another embodiment, the array would be subjected, e.g., by an integrated microfluidic flowcell, to an analyte solution containing the target(s) of interest at some unknown concentration. The range of possible sample analyte solutions may include standard buffers, biological fluids, and cell or tissue extracts. The sample solution will also contain the signaling probe at a saturating concentration relative to the immobilized nucleic acid sensor molecule. This ensures that at any given time during analysis, there is a high probability that each nucleic acid sensor molecule will have a signaling probe hybridized to it. In the presence of the target molecules in the sample solution, the nucleic acid sensor molecule will form a covalent phosphodiester bond, i.e., ligate, with the signaling probe, thus immobilizing it with its redox active electron donor species in electrical contact with the conductive molecules within the mixed self-assembled surface monolayer. After some integration time, during which signal probe ligation occurs, it may be necessary to denature the hybridized but unligated signaling probes. This denaturation step, which effectively removes 'background' signaling probes and their associated redox moieties from the vicinity of the electrode, can be accomplished by a small temperature increase (e.g., from 21 °C to 25 °C), or by a brief negative voltage spike applied to the sensor electrodes followed by the application of a large positive DC voltage to a separate electrode that would collect unligated signaling. For the case of a sufficiently short hybridization region, e.g., 5 base- pairs, on the signaling probe, a separate denaturation step may not be necessary. In either case, following nucleic acid sensor molecule activation by target molecules, a linear electrical potential ramp is applied to the electrodes. The redox species conjugated to the immobilized signaling probe-nucleic acid sensor molecule were electiochemically oxidized, liberating one or more electrons per moiety. The conductive molecules within the surface monolayer will provide an electrical path for the liberated electrons to the electrode surface. [00309] The net electron transfer to or from the electrode were measured as a peak in the faradaic current, centered at the redox potential of the electron donor species (specified for a given reference electrode) and superposed on top of the capacitive current baseline which is observed in the absence of surface-immobilized signaling probes. Quantitative analysis of the sensor signal, and therefore accurate determination of target molecule concentration, is based on the fact that the measured faradaic peak height is directly proportional to number of redox moieties immobilized at the electrode, that is, the number of nucleic acid sensor molecules ligated to signaling probes times the multiplicity of redox moieties per signaling probe molecule. Signal generation by the nucleic acid sensor molecules is thus amplified by virtue of multiple redox species per signaling probe. In addition, if an alternating current (AC) bias voltage is applied (superposed) on top of the DC linear voltage ramp applied to the sensor electrodes, i.e., in the case of AC voltammetry, signal amplification would result from the cyclic repetition of the signal-generating redox reaction.

[00310] The system described above for the case of a surface-immobilized nucleic acid sensor molecule which ligates a signaling probe containing one or more modified nucleotides conjugated to redox active species suggests a general method and instrumentation for the detection and quantitation of an arbitrary target molecule in solution in real time. Detection of a particular target would require development of a nucleic acid sensor molecule that recognizes the target molecule. Additionally, nucleic acid sensor molecules have been developed which are activated only in the presence of two different target molecules. Such dual-effector sensors could be used to detect the simultaneous presence of two or more targets, or could be used in conjunction with single-target molecule sensors to form biological logic (i.e., AND, OR, etc.) circuits. [00311] Multiplexed detection of multiple target molecules simultaneously in a complex sample solution could be accomplished by immobilizing nucleic acid sensor molecules against the target molecules of interest on separate electrodes within a two- dimensional array of electrodes. A complex sample solution containing multiple target molecules and a common signaling probe could then be introduced to the array. All nucleic acid sensor molecules would be exposed simultaneously to all targets, with the target- activated nucleic acid sensor molecule response(s) being observed and recorded only at the spatial location(s) known to contain a nucleic acid sensor molecule specific for the target molecules present in the (unknown) sample. The utility of such a nucleic acid sensor molecule array would be greatly enhanced by the integration of a microfluidic sample and reagent delivery system. Such an integrated microfluidic system would allow the application of reagents and samples to the sensor array to be automated, and would allow the reduction of sample volume required for analysis to < 1 μL.

[00312] The sensor array electrodes may be of any configuration, number, and size.

In a preferred embodiment, the sensor and reference electrodes would be circular gold pads on the order of 100-500 μM in diameter, separated by a center-to center distance equal to twice their diameter. Each electrode would be addressed by separate electrical interconnects. The application of electrical signals to the sensor electiodes can be accomplished using standard commercially available AC and DC voltage sources. Detection of faradaic electrical signals from the sensor electrodes can be accomplished easily using standard commercially available data acquisition boards mounted within and controlled by a microcomputer. Specifically, the raw sensor current signals would need to be amplified, and then converted to a voltage and analyzed via a high resolution (i.e., 16 bit) analog to digital converter (ADC). It is possible to reduce the signal background and to increase the signal to noise ratio (SNR) by using the common technique of phase-sensitive detection. In this detection method, an alternating current (AC) bias voltage (at a frequency between, for example, 100 to 1000 Hz) is superposed on top of the DC linear voltage ramp applied to the sensor electiodes. The frequency of the applied bias voltage is called the fundamental frequency. It can be shown that the sensor response signal contains multiple frequency components, including the fundamental frequency and its harmonics (integral multiples of the fundamental frequency). It can further be shown that the nth harmonic signal is proportional to the nth derivative of the signal. Detecting these derivative signals (by means of a lock-in amplifier) minimizes the effects of constant or sloping backgrounds, and can enhance sensitivity by increasing the signal to noise ratio and allowing the separation of closely spaced signal peaks. It should be noted that digital, computer-controlled AC and DC voltage sources (i.e., digital to analog converters, DACs), current preamplifiers, analog to digital converters (ADCs), and lock-in amplifiers are all available as integrated signal generation/acquisition boards that can be mounted within and controlled by a single microcomputer. [00313] In a preferred embodiment, an integrated nucleic acid sensor molecule system with electrochemical detection would include the following elements: one, an independently addressable multielement electrode array with immobilized surface layer composed of conductive species separated by insulating species and sensors; two, optical nucleic acid sensor molecules immobilized on the electrode array; three, an oligonucleotide substrate/signaling probe which ligates with the nucleic acid sensor molecule in the presence of the appropriate target; four, an automated or semi-automated microfluidic reagent and sample delivery system; and five, a reader instrument/data acquisition system consisting of a microcomputer controlling the appropriate voltage sources, current and lock- in amplifiers, data acquisition boards, and software interface for instrument control and data collection.

[00314] In another embodiment, the change in activity of the nucleic acid sensor molecule can be detected by watching the change in fluorescence of a nucleic acid sensor molecule when it is immobilized on a chip. A ligase can be attached to a chip and its ligase activity monitored. Ligase nucleic acid sensor molecules, labeled with one fluorophore, e.g., Cy3, are attached via an amino modification to an aldehyde chip. The initial Cy3 fluorescence indicates the efficiency of immobilization of the nucleic acid sensor molecules. Next, the chip is exposed to a substrate labeled with a second fluorophore, e.g., Cy5, with or without the target. In the presence of target, the nucleic acid sensor molecule ligates the substrate to itself, and becomes Cy5-labeled. Without target, the ligation does not occur. [00315] The use of a labeled effector oligonucleotide does not change the rate of ligation of the nucleic acid sensor molecule whether target is present or not. When using nucleic acid sensor molecules in the context of a chip based system, in one embodiment, an effector oligonucleotide is used to attach the nucleic acid sensor molecule to the chip. [00316] In another embodiment, a hammerhead nucleic acid sensor molecule could be used to measure the concentration of an analyte through the use of fluorescence. [00317] Any optical method known in the art, in addition to those described above can be used in the detection and/or quantification of all targets of interest in all sensor formats, in both biological and nonbiological media.

[00318] Any other detection method can also be used in the detection and/or quantification of targets. For example, radioactive labels could be used, including ³²P, ³³P, ¹⁴C, ³H, or ¹²⁵I. Also enzymatic labels can be used including horseradish peroxidase or alkaline phosphatase. The detection method could also involve the use of a capture tag for the bound nucleic acid sensor molecule.

Selection of PA-specific peptides

[00319] Bacillus anthracis PA-specific binding peptides of the present invention can be selected for specific binding affinity using methods know to those skilled in the art and used as described for the PA aptamers of the present invention.

[00320] Each of the references cited above, which describe modifications of the basic

SELEX™φrocedure is specifically incorporated by reference herein. [00321] The foregoing being a detailed description of the present invention, persons of skill in the art will understand the following examples to be illustrative of embodiments of aspects of the present invention. Persons of skill in the art will also understand that the foregoing examples are for illustration of the present invention and not limitation thereof. Accordingly, the invention is to be defined not by the preceding illustiative description but instead by the spirit and scope of the following claims.

EXAMPLES

EXAMPLE 1 Methods of Protective Antigen Aptamer Selection

[00322] Aptamers that bind the anthrax protective antigen (PA) were identified using

SELEX™. To expedite the lead optimization process, SELEX™ were carried out using pools containing 2'-fluoropyrimidine nucleotides, eliminating the requirement of identifying which pyrimidine residues in the candidate aptamer molecules are tolerant of 2'-fluoro substitution (Ruckman, 1998).

[00323] IV.A. Pool preparation. Selection for anti-PA aptamers begins with a nucleic acid pool containing 2'- fluoropyrimidines. A DNA template with the sequence (SEQ ID NO: 150)

5'-GCCTGTTGTGAGCCTCCTGTCGAAN₄₀TTGAGCGTTTATTCTTGTCTCCC- TATAGTGAGTCGTATTA-3' (SEQ ID NO: 151) has been synthesized using an ABI EXPEDITE™ DNA synthesizer, deprotected by standard methods and purified using a Poly-Pak™ (Glen Research) purification cartridge (N ₀ denotes a random sequence of 40 nucleotides built uniquely into each aptamer). The pooled templates were amplified with the primers YW.42.30.A (5'TAATACGACTCACTATAGGGAGACAAGAATAAACGCTCAA3') (SEQ ID NO: 152) and YW.42.30.B (5'GCCTGTTGTGAGCCTCCTGTCGAA3') (SEQ ID NO: 160) and then used as a template for in vitro transcription with Y639F T7 RNA polymerase. [00324] rV.A.ii. Selection conditions. Selection was initiated by incubation of 6 x

10¹⁴ RNA pooled molecules with purified recombinant PA (final concentrations 10 μM and 1 μM, respectively) for 30 minutes at room temperature in binding buffer (20 mM Hepes, 1 mM MgCl₂, 1 mM EDTA, pH 6.0). Complexed and free RNA molecules were separated using 0.2 μm nitrocellulose filter disks (Tuerk and Gold, 1990; Conrad et. al., 1996); RNA:PA complexes are expected to be retained on the nitrocellulose membrane, while unbound RNA passed through. RNA was eluted from the nitrocellulose membrane by submerging the membrane in 7 M urea, 100 mM sodium acetate, 3 mM EDTA that had been pre-heated to 90°C, incubating briefly, then collecting the supernatant. The elution process was repeated twice, followed by extraction of the eluate with phenol and ethanol precipitation of the eluted RNA. After annealing to the 3' primer YW.42.30B (5'GCCTGTTGTGAGCCTCCTGTCGAA3') (SEQ ID NO: 160), the RNA was amplified by reverse transcription at 50°C for 30 minutes (Thermoscript™ RT, Invitrogen) followed by PCR under standard conditions (Taq polymerase, Invitrogen) using the primers YW.42.30B and YW.42.30A, yielding the corresponding DNA templates for the round two of selection. Subsequent rounds of selection were conducted using a similar procedure, except that the pooled RNA was passed through a nitrocellulose filter prior to incubation with PA to remove molecules that bind to nitrocellulose. The concentration of PA was decreased to 250 nM at round 5 to increase stringency. After 11 rounds of selection, the pool was significantly enriched for PA binding (62 % at 250 nM) (Figure 21). SELEX enriched the pool for PA binders. The pool RNA from round 10, 11 and round 0 of the selection were incubated with varying concentrations of PA. The mixtures were passed through a nitrocellulose-nylon filter sandwich using a dot blot apparatus. The protein:RNA complexes are captured on the nitrocellulose membrane and the unbound RNA is captured on the nylon filter. After 10 rounds of selection, the extent of binding to PA had significantly increased relative to the starting pool.

[00325] TV.A.iii. Clonal analysis. When the selection has reached the point where further rounds do not increase the fraction of pooled RNA bound to PA, the pooled template DNA was cloned using the TOPO TA cloning system (Invitrogen). 48 individual clones were sequenced (LARK Technologies). 16 individual clones were screened in duplicate for their ability to bind PA using a 96-well nitrocellulose (Protran®, Schleicher and Schuell) - PVDF (Hybond P, Amersham Pharmacia Biotech) sandwich filter binding assay. As both bound and free RNAs were captured, the fraction bound at a fixed concentration of PA can easily be measured, allowing segregation of clones on the basis of binding affinity. Clones that were retained efficiently on nitrocellulose in the absence of PA were discarded. Aptamer clones fell into three distinct sequence groups (Figure 22). Representative clones from each of the three sequence groups were tested for their ability to bind full length PA (PA83), activated PA (PA63) and c-terminal region of PA (PA32) (500 nM protein, 10 mM Hepes pH 6 or 7, 100 mM NaCl, 0.1 mM EDTA, 1 mM MgCl₂). While all three sequences bound PA83 at pH 6, only the Group B sequence bound PA83 at pH 7. Additionally, this sequence bound PA32 at both pH 6 and 7, suggesting that the aptamer binding site is at the c-terminus of protective antigen.

[00326] The ability of truncated PA binding sequences to inhibit PA activity was measured using in vitro cell-based assays. Specifically, we assessed the ability of the aptamers to protect RAW 264.7 macrophage-like cells from challenge with anthrax toxin. The mouse macrophage cell line RAW 264.7 (American Type Culture Collection #TIB-71; Manassas, VA) was maintained in DMEM/F12 with 4 mM L-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose and 10% fetal bovine serum. The viability of RAW 264.7 cells in the presence or absence of PA aptamer - either pre-incubated with toxin (100 ng/ml PA; 50 nf/ml LF) for 30 min or added 10, or 20 min after toxin challenge - was measured using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). MTT was diluted in PBS and then added to the cells at 1 mg/ml three hours after the addition of anthrax toxin. Following a 1 hour incubation in MTT, the cells were solubilized with acidic isopropanol (90%) isopropanol containing 40 mM HCl and 0.5% sodium dodecyl sulfate) and the absorbance were measured at ^₅₉₅. The percentage of cells surviving toxin challenge in the presence of PA aptamer, as compared to controls, was determined.

[00327] To verify the mechanism of action and further characterize our PA aptamers, the ability of the aptamers to inhibit binding of Lethal Factor (LF) to PA are determined using CHO cells as described in the literature (Mourez, 2001). CHO cells (American Type Culture Collection #CCL-61) are grown in Ham's F12K medium with 2mM L glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 10% fetal bovines serum. Confluent cells are incubated on ice for lh in DMEM/F12 medium buffered with 20 mM HEPES, pH 7.4, in the presence of 2 x 10^"18 M PA cleaved by trypsin as described previously (Miller et al, 1999). 255 residues of the N terminus of Lethal Factor (LFN) are labeled with [³⁵S]methionine in vitro coupled transcription and translation (Wesche et al, 1998). After one wash with ice cold PBS, cells pretieated with PA are incubated for lh with radiolabeled LF_N on ice in the presence of various amounts of the PA aptamer. The cells are then washed and lysed, and the radioactivity in the lysate measured. The level of LF_N bound to cells in absence of PA is measured and subtracted as background.

[00328] IV.A.iv. Minimization. The in vitro screens for PA binding and functional activity are designed to identify those aptamers that inhibit the action of PA. These aptamers were next refined through minimization. Depending on the number of different sequence families represented by the candidate aptamers, the most potent representative of each family (1-3) were used as a basis sequence for the preparation of a doped pool. Approximately 80% of the residues in each aptamer sequence was mutated at a frequency of 15%, such that at each mutated position there is an 85%> chance of having the wild type nucleotide and a 5% chance of having each of the other three nucleotides. SELEX™ was performed on the doped pools to enrich for sequences with high affinity for PA. Enriched pool templates were cloned and sequenced, and minimal binding elements were deduced based on sequence conservation and covariation.

[00329] Limited hydrolysis may also be used to identify the minimal binding element required for high affinity binding of the aptamers (Jellinek et. al., 1994). Candidate aptamers were radiolabeled at the 5 '-end using γ-³²P ATP and T4 polynucleotide kinase or at the 3'- end using ³²pCp and T4 RNA ligase. The labeled aptamers were partially hydrolyzed by incubation at 90°C for 15 minutes in 50 mM bicarbonate, pH 9. Next, the RNA fragments were incubated with a range of concentrations of PA, and free from bound fragments were separated using nitrocellulose filter disks. After elution from the filter, the bound RNA fragments were resolved on a denaturing polyacrylamide gel. The length of the smallest fragment of RNA bound at the lowest PA concentration defined the boundary of the end opposite the labeled one. Results from 5'- and 3'- labeled material combined define the boundary of the minimal binding element.

[00330] On the basis of the doped reselection and alkaline hydrolysis experiments, minimized forms of each aptamer class were synthesized and characterized. These minimized forms serve as the starting point for engineering directed at optimization of pharmacokinetics (as they relate to biological efficacy). [00331] TV.B. Lead optimization for pharmacokinetics. The pharmacokinetic properties of the minimized aptamer leads identified in Section IV.A.i. were characterized and used to guide further efforts to engineer the aptamers. Efforts were focused in two areas: (1) optimizing resistance to nucleases while maintaining high affinity PA-binding, and (2) adjusting clearance/distribution as needed to optimize biological efficacy. Nuclease resistance can be easily assessed ex vivo using human plasma. Clearance and distribution were initially assessed through standard pharmacokinetic analyses (Tucker, 1999; Bendele, 1997) in mice prior to initiating animal intoxication and infection models. [00332] Whereas initial generation and characterization of the aptamer can be accomplished largely using material generated by in vitro transcription, subsequent optimization requires the ability to engineer specific chemical variants. Modified aptamers are chemically synthesized using commercially available phosphoramidites, an AKTA oligopilot 10 DNA/RNA synthesizer, and standard coupling chemistries optimized for 5- 100 μmol-scale synthesis. Following cleavage from a solid support and deprotection, aptamers are preparatively purified using ion pair reverse phase chromatography on the Transgenomic WAVE HPLC system. Following desalting and concentration via lyophilization, aptamers were analyzed by LC/MS (using a Fi negan LC-Q ) to confirm the synthesis.

[00333] TV.B.i. Nuclease resistance. Given the composition of the starting pools used for selection, all pyrimidines in our candidate aptamers were 2'-fluoro-substituted, affording the aptamers enhanced stability in vivo through nuclease resistance. Introduction of 2'-0-methylpurine residues will increase their stability further. It is anticipated that not all of the purine sites will tolerate a 2'-0-methyl substitution, since the ligand: aptamer complexes may contain contacts between key 2' -OH groups and PA. We will therefore test the tolerance of 2'-0-methyl substitution at the purine residues using 2'-0-methyl purine nucleotide interference analysis (Green et. al., 1995).

[00334] In brief, partially 2'-0-methyl substituted aptamer pools that contain all 2'-F- pyrimidine nucleoside phosphoramidites and a 2:1 (mokmol) mixture of 2'-0-Me:2'-OH purine phosphoramidites at specified positions was chemically synthesized. Each pool had three to four 2'-0-methylpurine substitutions, therefore the number of pools synthesized was determined by the number of purine residues in the candidate aptamer molecules. Each pool was end labeled using γ-³²P ATP and T4 polynucleotide inase and purified by denaturing gel electiophoresis. Pools were then incubated with various concentrations of PA in selection buffer and the PA: aptamer complexes were partitioned with nitrocellulose filters. After elution and precipitation, the bound RNA molecules were subject to limited alkaline hydrolysis under the conditions described for aptamer minimization. Non-affinity selected pools were also subjected to hydrolysis to provide reference information. The hydrolytic fragments were analyzed on high resolution denaturing gels. Since the 3'- phosphodiester linkage of a 2'-0-methyl containing nucleotide is not susceptible to alkaline hydrolysis, purines that tolerate substitution by 2'-0-methyl groups are expected to show less cleavage when a 2'-0-methyl is present than when a 2'-OH is present relative to the non-affinity selected pools. Aptamers containing 2'-fluoropyrimidines and 2^,-0- methylpurines at all tolerated positions were chemically synthesized and assayed for PA binding and inhibition of PA function in vitro.

[00335] It has been demonstrated that addition of cap structures to the 5'- and 3'-ends of an aptamer can be used to effectively block the activity of exonucleases. For example, a 3 '-3' thymidine cap can dramatically increase the half-life of aptamers in vivo (Floege, 1999; Tucker, 1999; Ruckman, 1998), as can a 3'-biotin tag (Dougan, 2000). andidate 2'- fluoropyrimidine- and 2'-0-methylpurine-containing aptamers were synthesized with these modifications and tested them for PA binding and inhibition of PA function in vitro using the assays described in Section III.B. The 3'-3' thymidine and 3'-biotin caps can be introduced into the aptamer using a commercially available inverted dT phosphoramidite CPG and biotin TEG CPG (both available from Glen Research) respectively. [00336] rV.B.ii. Clearance. As described in section IILA.iv.b., elimination of aptamers via the kidneys may be virtually eliminated by the addition of high molecular weight PEG. To the extent that maintaining a high concentration of aptamer in the intravascular space is likely to be important for detoxification (and as suggested by comparison of scAb and scFv anti-PA antibody constructs (Maynard, 2002)), PEGs were tested for their ability to alter aptamer half-life, effective dose concentiation, and duration of effect. PEGylated aptamer were synthesized as described previously using an N- hydroxysuccinimide-activated 2 x 20 KD PEG from Shearwater Corporation, a supplier of GMP-grade PEGylation reagents for pharmaceutical applications (Janjic, 2001). NHS- activation allows site specific attachment to an aptamer whose 5 '-end has been previously derivatized by attachment with an 5'-amino modifier phosphoramidite. [00337] rV.B.iii. Assays for evaluating aptamer pharmacokinetics. Methods for plasma pharmacokinetic analysis of aptamers in rodent and primate models include assays based on probe hybridization, HPLC analysis, and mass spectioscopy. [00338] Probe hybridization. In a dual capture assay, two hybridization probes are used; a capture probe attached to a solid support such as magnetic beads or a 96-well plate bottom, and a biotinylated detection probe. The capture probe forms a hybrid with the 5' end of the oligo (aptamer) to be detected and the detect probe forms a hybrid with the 3' end of the aptamer to be detected. These assays are highly sensitive to full length oligo and yield a positive signal. The limit of quantitation for these assays is approximately 10 fmoles in 5 μl plasma (Bendele, 1997). Concentrations of positive control aptamer are determined by an approximate extinction coefficient for the oligonucleotide of 37 microgram/ mL/A₂₆₀ unit. Plasma or tissues samples from which aptamer levels are being determined are diluted into buffer and the test aptamer is allowed to hybridize with both the capture and detect probes. In the 96-well assay format, the capture probe is attached via an amino linker to the wells of the plate by reacting the plate surface and capture probe in 100 microliter buffer (1M Hepes, pH 7.5, lmM EDTA, lOOpmol/mL capture probe) overnight 4 °C. Plates are then blocked with 100 mM Hepes, pH 7.5, 0.1 mM EDTA, 1% w/v bovine serum albumin. Duplicate test samples are diluted 10-fold into 4X SSC, with 0.5% sarcosyl and 40 pmol mL detect probe, and then heated for 10 minutes at 95 °C. 100 microliters of the test samples are transferred to the captured plates and incubated for 2 hours at 45 °C. Plates are washed three times (350 uL) with buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 0.1% v/v Tween 20). Alkaline phosphatase conjugated streptavidin is added and incubated at room temperature for 30 minutes. Wells are washed (five times) as before, followed by the addition of a 100 uL alkaline phosphatase substrate (0.1 M diethanolamine, pH 10, 10% v/v Sapphire solution [Tropix, Inc], 17 uL/mL CSPD [Tropix, Inc.], 1 mM MgCl₂, and 0.02% NaN₃). After 20 minutes, chemiluminescence is measured in a luminometer. [00339] HPLC. Serum levels of therapeutic aptamers can also be determined using standard HPLC procedures. In a typical example for a PEGylated and nucelease stabilized aptamer (Tucker, 1999), plasma stability and serum levels were determined by HPLC, in order to support human clinical development of NXl 838. Separations can be performed on Dionex Nucleopac TM PA- 100 column, or other suitable matrix useful for separation of PEGylated oligonucleotides. Aptamers can be eluted in standard mobile phases of acetonitiile-water mixtures buffered atpH 8.0 using Tris(hydroxymethyl) aminomethane or other suitable buffer system.

[00340] Mass Spectrometry. PEGylated oligonucletide aptamers can also be characterized by using electiospray mass spectrometry (Tarasow, 1997). The process of chemically altering aptamers (and proteins) by PEG modification results in multiple reaction products especially when polymers are involved. Detailed characterization of these molecules is crucial to gaining knowledge of their mode of action, their stability and chemical state in vivo. Hence, the relationship between product characterization and in vivo properties is essential for development of an aptamer that were used in humans to treat anthrax infection. The mass spectrometry technique which offers the appropriate level of resolution, accuracy and sensitivity is electiospray mass spectiometry. ESMS generates and detects multiple charge states of the sample, which upon reconstruction yields the correct molecular weights of all species present in a complex mixture. ESMS is particularly useful for large biomolecules including proteins and oligonucleotides (Fen, 1989). ESMS when coupled to HPLC by standard instrumentation (e.g., Finigan LC-Q) allows for the identification of individual aptamer-PEG conjugates differing in molecular weight a single ethylene glycol unit with an accuracy of 0.02% of the molecular weight. [00341] IV.B.iv. Pharmacokinetic analysis of anti-PA aptamers in mice is to be carried out with the initial aim of defining basic pharmacokinetic parameters to understand aspects of the aptamers' distribution, elimination, and metabolism as a function of both modifications to the aptamers (outlined above) and as a result of different routes of administration (IV versus SC). Studies utilize standard ICR mice grouped in 4-8 mice per time point. Animals are to be sacrificed according to IAACUC-approved procedures and blood samples removed by coronary puncture. Parenteral administration of anti-PA aptamers is to be evaluated by subcutaneous and intravenous injection. Blood samples were taken before administration and time intervals typically spaced at 1, 3, 5, 10, 20, 30, 60, 120, 180, 300, 600 and 1440 minutes. For pharmacokinetic measurements to support studies in rat models of anthrax infection, Fischer 344 rats (250-300g), 4-6 rats per group, were dosed with aptamer as described (Maynard, 2002). In rat disease models, serum levels of PA and lethal factor (LF) which produce disease symptoms can be as high as 0.5 0.1 nanomoles injected/rat (Mourez, 2001). Therefore, pharmacokinetic analyses were completed at multiple aptamer dosing levels to determine the relationship between dose level and apparent plasma C_max, and AUG. Clearance rates and volumes of distribution were determined by nonlinear regression fitting to a biphasic exponential function. [00342] Distribution of the anti-PA aptamer outside the vascular compartment and into discrete tissues were assessed by extraction of the tissue followed by dual capture assay (described above). In addition, methods for assessing tissue distribution using technicium- 99 labeled aptamers have been previously described and were tested as needed (Hicke, 2000). Animals dosed with ⁹⁹Tc labeled aptamer were sacrificed and tissue distribution of the radiolabeled aptamer determined by radiometric analyses.

[00343] IV.C. In vivo proof of concept (rat purified toxin model). The in vivo efficacy of our 1-3 most potent PA aptamers identified using cell-based models were determined using a rat purified toxin model developed previously and described in detail below (Maynard, 2002). The aptamers were tested under three experimental conditions: (1) co-administration of aptamer, PA and LF; (2) PA added first, followed by LF plus aptamer; and (3) PA and LF added initially followed by aptamer at various timepoints subsequent to toxin addition. The protective ability of the aptamers were measured as delayed time to death. Each of the subsequent treatment conditions is increasingly stringent. An aptamer that is successful in all three tests should be well suited to proceed to inhalation models. Each of the aptamers selected for optimization in their various modified forms (e.g. PEGylated, unPEGylated) were tested for efficacy. Aptamers that demonstrate the most protection in the rat toxin model were tested further in a rabbit anthrax inhalation model. Efficacy was correlated with pharmacokinetics and be used to direct further engineering to alter half-life and distribution.

[00344] The general experimental design is as follows: Fischer 344 rats (~250 g) were anesthetized by intiaperitoneal (IP) injection of ketamine (80 mg per kg) and xylazine (10 mg per kg). PA Aptamer (or vehicle control) and anthrax toxin (40 μg PA, 8 μg LF) were administered at varying times with respect to each other as outlined above. Aptamer were administered at various times as noted above, with dosing ranging from 0 -> 1 mg/kg. Five animals were used for each test condition, and the rats were monitored for symptoms of intoxication. Rats were maintained under anesthesia for five hours or until death to minimize discomfort. Surviving rats were killed by overdose of sodium phenobarbitol given by IP injection. The protective ability of the aptamers was measured as a delayed time to death. All animal experiments were performed under the Animal Welfare Regulations 9CFR, Chapter 1. [00345] IV.D. Manufacturing process development. Summarized below are the basic steps in aptamer manufacturing.

[00346] Solid-phase synthesis. Monomers (phosphoramidites) are sequentially coupled to an immobilized support to grow out the oligonucleotide.

[00347] Cleavage/deprotection. Oligonucleotide is cleaved from the solid support, protecting groups on the bases on the bases are chemically removed.

[00348] Purification. Ion exchange and/or ion paired reverse phase chromatography is used to isolate full-length oligonucloetide.

[00349] PEGylation. An activated PEG molecule is covalently coupled to the deprotected aptamer.

[00350] Ultrafltration. concentration, and desalting. Salts and solvents introduced during chromatographic purification are removed and the sample is prepae for final packaging.

[00351] Lyophilization. Purified oligonucleotide is dried down into powder for storage prior to use.

[00352] IV.E. In vivo dose ranging. PA aptamers shown to be effective in rats are subsequently tested in a rabbit and rhesus macaque monkey anthrax inhalation model described in detail below. To effectively model the desired indication (post-symptomatic inhalational anthrax), aptamer and ciprofloxacm administration are initiated simultaneously after the first symptoms appear.

[00353] As a general method, animals are initially challenged with aerosol anthrax spores, followed by a co-administration of PA aptamer and ciprofloxacm. Respiratory rates and volumes will be measured in New Zealand white rabbits (Charles River) and Rhesus macaques (USAMRIID) prior to anthrax administration. Rabbits and Rhesus monkeys are exposed to anthrax spores with a spore aerosol nebulizer. The spore concentiation in the inhaled dose (LD₅₀) will be calculated on all animals after plating diluted samples onto

Tripticase Soy Agar (TSA) plates. Rabbits and monkeys are observed regularly in regards to appetite, activity and respiratory distress. Bacteremia studies are performed by drawing blood 1 ml of blood daily for 1 week post-challenge in the monkeys and on day 2 after the challenge in rabbits. 100 μl aliquots of blood serial dilutions are plated onto TSA plates, and the plates are incubated at 37°C for 18 h and colonies counted. The protective ability of the aptamer in rabbits and monkeys are measured as delayed time to death. [00354] rV.F.i. Genotoxicity. Aptamer genotoxicity is determined using the Ames test carried out in both S. typhimurium and E. coli. Both tests are based on the reversion of amino acid auxotiophy.

[00355] Salmonella typhimurium. Seven mutant tester strains (TA97, TA98, TA100,

TA102, TA1535, TA1537, TA1538) deficient inhistidine synthesis are used. Aptamer (at concentrations ranging from 0 - 10 mg/ml) and bacteria are mixed in combinations both with and without rat liver microsome enzyme homogenate (S9). Each combination is mixed with molten agar and poured on the surface of a minimal glucose agar plate. Visible colonies (corresponding to His+ revertants) are counted after 2 days growth. Mutagenicity is established by comparing reversion rates in the presence and absence of the aptamer. [00356] Escherichia coli. Escherichia coli WP2 uvrA (ATCC 49979) or WP2 uvrA pKMlOl strains deficient in tryptophan synthesis are used in experiments exactly paralleling those with S. typhimurium. Mammalian genotoxicity is assessed in cell culture to define the likelihood that the aptamer induces chromosomal aberrations or gene mutations.

[00357] Chromosomal aberrations. Primary cultures of human lymphocytes are exposed to varying concentrations of aptamer both with and without metabolic activation with S9 homogenate. At 4, 24, and 48 hours after initial exposure, cell cultures are treated with colchicine to induce metaphase arrest. Following harvesting and staining, cells are analyzed microscopically for chromosomal aberrations (scoring 200 metaphases per culture).

[00358] Gene mutations. The mutagenic potential of the aptamer are measured by its ability to induce TK+/ — > TK-/- mutations in cultured mouse lymphoma cells. Assays are performed by exposing duplicate cultures of L5178Y/TK -/- cells to a range of aptamer concentrations for 4-24 hours in the presence and absence of an S9 activation system. Following a two day expression period, cultures demonstrating 0% to 90% growth inhibition are cloned, in triplicate, in restrictive medium containing soft agar to select for the mutant phenotype. After a 14 day selection, mutant colonies are counted and scored on the basis of size.

[00359] IV.F.U. Acute toxicity. Toxicity of the anti-PA aptamers and its component monomers is assessed in intravenous dose escalation studies (single dose per group) to determine the apparent LD50 level and the maximum tolerated dose (MTD). Rats and monkeys are dosed by intravenous infusion (doses up to 100 mg/kg) or subcutaneous injection (single bolus, 100 mg/kg). Animals are monitored closely for any signs of acute toxicity for up to 3-7 days. End point measurements of blood pressure, electrocardiogram (ECG), clinical chemistry, hematology, complement factors, coagulation parameters, and plasma concentrations are made as previously described (Sandberg, 2000). [00360] IV.F.iii. One-month and Three-month Chronic Toxicity Studies. The effects of the anti-PA aptamer in 1 -month and 3 -month chronic toxicity studies are assessed in rats and monkeys. Animals are dosed over a range of levels to be defined on the basis on the animal infection studies. Rats and monkeys are dosed by intravenous injection twice daily for 28 and 90 days, respectively. Animals are monitored continuously for adverse effects and monitored for changes in clinical signs, appetite, and physical condition. Electrocardiograms are obtained prior to the first dose and prior to necropsy. At weekly (1- mo) and monthly (3 -mo) intervals blood and urine samples are collected and evaluated for standard hematology, serum chemistry, urinalysis, and coagulation parameters. At the end of each study, animals are sacrificed and their organs weighed. Microscopic histopathology analysis will be focused on the liver and the kidney. Serum collected at the end of each trial is analyzed for the presence of anti-aptamer antibodies using an ELISA-based assay as described previously for the anti-VEGF aptamer.

[00361] EXAMPLE 2 Optimized PA aptamer ligands

[00362] Clone sequences isolated from Protective Antigen Binding Pool from

Example 1.

[00363] Sequence Group A

[00364] >SEQ ID NO 1: PAcloneDll

GGGAGACAAGAAUAAACGCUCAAGAGGU1UUCAACUGCUGUGAUGAGUAACAGGCACGAAU CCUUCGACAGGAGGCUCACAACAGGC

[00365] SEQ ID NO 2 PAcloneB7

GGGAGACAAGAAUAAACGCUCAAGAGGUUUUCAACUGCUGUGAUGAGUAACAGGCACGAAU CCUUCGACAGGAGGCUCACAACAGGC

[00366] SEQ ID NO 3 PAcloneCl

GGGAGACAAGAAUAAACGCUCAAGAGGUUUUCAACUGCUGUGAUGAGUAACAGGCACGAAU CCUUCGACAGGAGGCUCACAACAGGC [00367] SEQ ID NO 3 PAcloneC12

[00368] SEQ ID NO 4 PAcloneDIO

[00369] SEQ ID NO 5 PacloneAll

[00370] SEQ ID NO 6 PAcloneD8

[00371] SEQ ID NO 7 PAcloneC9

[00372] SEQ ID NO 8 PAcloneDδ

[00373] SEQ ID NO 9 PAcloneA2

[00374] SEQ ID NO 10 PAcloneCδ

[00375] SEQ ID NO 11 PAcloneB8

[00376] SEQ ID NO 12 PAcloneD3

[00377] SEQ ID NO 13 PAcloneB12

[00378] SEQ ID NO 14 PAcloneB5

GGGAGACAAGAAUAAACGCUCAAGAGGUUUUCAACUGCUGUGAUGAGUAAAGGCACGAAUC CUUCGACAGGAGGCUCACAACAGGC [00379] SEQ ID NO 15 PAcloneC5

[00380] SEQ ID NO 16 PAcloneA4

GGGCCCCUNAAAANAAACGCUCAAGAGGUUUUCAACUGCUGUGAUGAGUAACAGGCACGAA UCCUUCGACAGGAGGCUCACAACAGGC

[00381] SEQ ID NO 17 PAcloneAl

[00382] SEQ ID NO 18 PAcloneC4

[00383] SEQ ID NO 19 PAcloneD4

GGGAGACAAGAAUAAACGCUCAAGAGGUUUUNAACUGCUGUGAUGAGUAACAGGCACGAAU CCUUCGACAGGAGGCUCACAACAGGC

[00384] SEQ ID NO 20 PAcloneD12

GGGAGACAAGAAUAAACGCUCAANAGGUUUUCAACUGCUGNGAUGAGUAACAGGCACNAAU CCUUCNACAGGAGGCUCACAACAGGC

[00385] SEQ ID NO 21 PAcloneA6

GGGAGACAAGAAUAAACGCUCAAGAGGNUUUCAACUGNUGNGAUGANUAACAGGNACGAAU CCUUCNACAGGAGGNUCACAACAGGN

[00386] SEQ ID NO 22 PAcloneD2

GGGAGACAAGAAUAAACGCUCAAGAGGUCUUCAACUGCUGUGAUGAGUAACAGGCACGAAU CCUUCGACAGGAGGCUCACAACAGGC

[00387] SEQ ID NO 23 PAcloneAlO

[00388] Sequence Group B

[00389] SEQ ID NO 24 PAcloneD9

GGGAGACAAGAAUAAACGCUCAAGGGGUAAUCGACAACAUUAUGGGAAUUCACGCAGGUAG CUUUCGACAGGAGGCUCACAACAGGC

[00390] SEQ ID NO 25 PAcloneB4

GGGAGACAAGAAUAAACGCUCAACUGGGUGACCGACAAUUAUGGGAGUCGAAUUGUUGUGA GUUCGACAGGAGGCUCACAACAGGC [00391] SEQ ID NO 26 PAcloneB9

GGGAGACAAGAAUAAACGCUCAAUUGGGUGACCGACAAUUAUGGGAGUCGAAUUGUUGUGA GUUCGACAGAGGCUCACAACAGGC

[00392] SEQ ID NO 27 PAcloneC2

GGGAGACAAGAAUAAACGCUCAAUUGGGUGACCGACAAUUAUGGGAGUCGAAUUGUUGUGA GUUCGACAGGAGGCUCACAACAGGC

[00393] SEQ ID NO 28 PAcloneB2

[00394] SEQ ID NO 29 PAcloneD5

[00395] SEQ ID NO 30 PAcloneCδ

[00396] Sequence Group C

[00397] SEQ ID NO 31 PAcloneDl

GGGAGACAAGAAUAAACGCUCAACGUCCUGUAGCUUGGGUAAGAUAAAGAGUGAUCCUUCG ACAGGAGGCUCACAACAGGC

[00398] SEQ ID NO 32 PAcloneC3

[00399] SEQ ID NO 33 PAcloneCll

GGGAGACAAGAAUAAACGCUCAACGUCCUGUAGCUUGGGUAAGAUAAAGAGUGAUCCUUGG ACAGGAGGCUCACAACAGGC

[00400] SEQ ID NO 34 PAcloneBβ

[00401] SEQ ID NO 35 PAcloneB3

GGGAGACAAGAAUAAACGCUCAACGUCUGUAGCUUGGGUAAGAUAAAGAGUGAUCCUUCGA CAGGAGGCUCACAACAGGC

[00402] SEQ ID NO 36 PAcloneA5

GGGAGACAAGAAUAAACGCUCAACGUCUGUAGCUUGGGUAAGAUAAAGAGUGAUCCUUCGA CAGGAGGCUCACAACAGGC [00403] Single, non-homologous sequences

[00404] SEQ ID NO 37 PAcloneBll

GGGAGCACAAGAAUAAACGCUCAAGCCACCUACUUUACUUUUCUCACUCACACGGCUCGUU UAUUCGACAGGAGGCUCACAACAGGC

[00405] SEQ ID NO 38 PAcloneBl

GGGAGACAAGAAUAAACGCUCAAUUCUUCAUCUUUCUAAAGUUCUUGAUCCCGCUGUGUGA CGUUCGACAGGAGGCUCACAACAGGC

[00406] SEQ ID NO 39 PAcloneCIO

GGGAGACAAGAAUAAACGCUCAACCUGUGAGCCGAAAGGACAUACUUUAGUGAAGGAUUAG CCUUCGACAGGAGGCUCACAACAGGC

[00407] Optimization of PA binding sequences. The dominant group B (represented by PAcloneB2) sequence was expanded via doped reselection. A synthetic template was prepared such that each nucleotide originating from the random region of the original pool had a 70% probability of being the wild type nucleotide, and a 30% probability of being one of the other 3 nucleotides (in the sequences below, the italicized residues are mutagenized).

[00408] SEQ ID NO 40 SCK. 92 .42 .b (Group B)

5 ' GGGAGACAAGAAUAAACGCUCAA ^GGG ^TGACCGACAaOT7A ^TGGGAG!7CGAaiJL^TGt7L^TG ^T

GAGUUCGACAGGAGGCUCACAACAGGC3'

[00409] The template was amplified and transcribed to generate approximately 3 x

10¹³ different pool RNA sequences. Selection with the doped pool SCK.92.42.b was carried out using a modified version of the de novo selection procedure. In brief, PA (10 - 100 pmoles) was incubated in one well of a NUNC Maxisorp UA-well plate in selection buffer

(20 mM Hepes, 1 mM MgCl₂, 0.1 mM EDTA, 100 mM NaCl , pH 7; 100 μL) for 2 hours at

37°C to immobilize the protein to the plate surface. The supernatant was removed and the well was washed three times with 120 μL selection buffer. The well was then blocked with binding buffer plus 1 mg/niL casein and 0.1 % Tween-20 for 1 hour at room temperature followed by four washes with 20 μl selection buffer.

[00410] The pool RNA was incubated in 100 μL selection buffer in an empty well for

30 minutes at room temperature to remove plate binding sequences then the supernatant was transferred to a fresh well to repeat the process. Finally, the unbound RNA contained in the supernatant was transferred into the well coated with PA and incubated for one hour at

37°C. The supernatant was then removed and discarded and the well was washed 6 times with 120 μL selection buffer. [00411] The RNA that had bound to the PA coated plate was amplified. The reverse transcription was carried out directly in the well by adding the reaction components and heating the plate at 65°C for 30 minutes. The cDNA was then amplified by PCR under standard conditions and the resulting template was transcribed to yield the pool RNA for the next round of selection. After four rounds of selection the pool was enriched with PA binding sequences (~ 25% bound; 500 nM PA).

EXAMPLE 3 Aptamers emerging from SCK.92.42.b (PAcloneB2)

[00412] The enriched SCK.92.42.b was cloned after 4 rounds of selection and clone sequences were obtained. The following are the individual sequences:

[00413] SEQ ID NO 43 SCK.92.88 F2

GGGAGACAAGAATAAACGCTCAATTGGGTGACCGACAATTATGGGAGTCGAATTGTTGTGAGTTCGACAGGAGG CTCACAACAGGC

[00414] SEQ ID NO 44 SCK.92.88 D7

[00415] SEQ ID NO 45 SCK.92.88 C6

[00416] SEQ ID NO 46 SCK.92.88 D3

[00417] SEQ ID NO 47 SCK.92.88 A12

GGGAGACAAGAATAAACGCTCAATAGGGTGACCGACAAATATGGGAGTCGAATGGTTGTGAGTTCGACAGGAGG CTCACAACAGGC

[00418] SEQ ID NO 48 SCK.92.88 G8

GGGAGACAAGAATAAACGCTCAATTGGGTGACCGACAATTATGGGAGTCAAATTGTTGTGTGTTCGACAGGAGG CTCACAACAGGC

[00419] SEQ ID NO 49 SCK.92.88 C9

GGGAGACAAGAATAAACGCTCAATGGGGTGACCGACAATAATGGGAGTCCAATTGTTGTGTGTTCGACAGGAGG CTCACAACAGGC

[00420] SEQ ID NO 50 SCK.92.88 B7

GGGAGACAAGAATAAACGCTCAATGGGGTGACCGACAATTATGGGAGTCGATTTGTTGTGAATTCGACAGGAGG CTCACAACAGGC

[004213 SEQ ID NO 51 SCK.92.88 E6

GGGAGACAAGAATAAACGCTCAATGGGGTGACCGACAATAATGGGAGTCAATTTGTTGTGAGTTCGACAGGAGG CTCACAACAGGC [00422] SEQ ID NO 52 SCK.92.88 A5

GGGAGACAAGAATAAACGCTCAATGGGGTGACCGACAATAATGGGAGTCGAAATGTTGTGAGTTCGACAGGAGG CTCACAACAGGC

[00423] SEQ ID NO 53 SCK.92.88 C5

GGGAGACAAGAATAAACGCTCAATAGGGTGACCGACAAAAATGGGAGTCCAATCGTTGTGAGTTCGACAGGAGG CTCACAACAGGC

[00424] SEQ ID NO 54 SCK.92.88 A9

GGGAGACAAGAATAAACGCTCAATGGGGTAACCGACAATTATGGGAGTTCAATTGTTCTGAGTTCGACAGGAGG CTCACAACAGGC

[00425] SEQ ID NO 55 SCK.92.88 F5

GGGAGACAAGAATAAACGCTCAATTGGGTGACCGACAAGAATGGGAGTCTAATTGTTGTGAGTTCGACAGGAGG CTCACAACAGGC

[00426] SEQ ID NO 56 SCK.92.88 H8

GGGAGACAAGAATAAACGCTCAATAGGGTGACCGACAAGAATGGGAGTCCAATTGTTGTGAGTTCGACAGGAGG CTCACAACAGGC

[00427] SEQ ID NO 57 SCK.92.88 E8

GGGAGACAAGAATAAACGCTCAATTGGGTGACCGACAATAATGGGAGTCAAATCGTTGTGAGTTCGACAGGAGG CTCACAACAGGC

[00428] SEQ ID NO 58 SCK.92.88 B8

GGGAGACAAGAATAAACGCTCAATAGGGTGACCGACAAGTATGGGAGTCCAATTGTTTTGAGTTCGACAGGAGG CTCACAACAGGC

[00429] SEQ ID NO 59 SCK.92.88 Gil

GGGAGACAAGAATAAACGCTCAATGGGGTGACCGACAATTATGGGAGTCTAAATGTTGTGATTTCGACAGGAGG CTCACAACAGGC

[00430] SEQ ID NO 60 SCK.92.88 D6

GGGAGACAAGAATAAACGCTCAATAGGGTGACCGACAAAGATGGGAGTCCAATTGTTTTGAGTTCGACAGGAGG CTCACAACAGGC

[00431] SEQ ID NO 61 SCK.92.88 C8

GGGAGACAAGAATAAACGCTCAATAGGGTGACCGACAATTATGGGAGTCGAAATGTTATGAGTTCGACAGGAGG CTCACAACAGGC

[00432] SEQ ID NO 62 SCK.92.88 D12

GGGAGACAAGAATAAACGCTCAATTGGGTGACCGACAATTATGGGAGTCCGGTTGTTGTGTGTTCGACAGGAGG CTCACAACAGGC

[00433] SEQ ID NO 63 SCK.92.88 B10

GGGAGACAAGAATAAACGCTCAATAGGGTGACCGACAGTTATGGGAGTCGAATGGTTGTGAGTTCGACAGGAGG CTCACAACAGGC

[00434] SEQ ID NO 64 SCK.92.88 H7

GGGAGACAAGAATAAACGCTCAATTGGGTGACCGACAATTATGGGAGTCCGCTTGTTGTTAGTTCGACAGGAGG CTCACAACAGGC

[00435] SEQ ID NO 65 SCK.92.88 A8

GGGAGACAAGAATAAACGCTCAATTGGGTGACCGACAANTGTGGGAGTCTA TTGTTGTGAGTTCGACAGGAGG CTCACAACAGGC [00436] SEQ ID NO 66 SCK.92.88 F8

GGGAGACAAGAATAAACGCTCAATTGGGTGACCGACAATTATGGGAGTCCGCTTGATTTGAGTTCGACAGGAGG CTCACAACAGGC

[00437] SEQ ID NO 67 SCK.92.88 G12

GGGAGACAAGAATAAACGCTCAATAGGGTGACCGACAATAATGGGAGTCGAATGTTTGTGAGTTCGACAGGAGG CTCACAACAGGC

[00438] SEQ ID NO 68 SCK.92.88 E5

GGGAGACAAGAATAAACGCTCAATTGGGTGACCGACAATAATGGGAGTCTAAATGTTTTGAGTTCGACAGGAGG CTCACAACAGGC

[00439] SEQ ID NO 69 SCK.92.88 Cll

GGGAGACAAGAATAAACGCTCAATAGGGTGACCGACAATTTTGGGAGTCGACTTGTTCTGAGTTCGACAGGAGG CTCACAACAGGC

[00440] SEQ ID NO 70 SCK.92.88 H5

GGGAGACAAGAATAAACGCTCAATTGGGTGACCGACAGCTATGGGAGTCNATTTGTTGTGAGTTCGACAGGAGG CTCACAACAGGC

[00441] SEQ ID NO 71 SCK.92.88 D8

GGGAGACAAGAATAAACGCTCAATTGGGTGACCGACAATGATGGGAGTCAACTTGTTATGAGTTCGACAGGAGG CTCACAACAGGC

[00442] SEQ ID NO 72 SCK.92.88 Cl

GGGAGACAAGAATAAACGCTCAATGGGGGTATCCGACAAACATTATGGGAGATGAGTTGTAGCTTCGACAGGAG GCTCACAACAGGC

[00443] SEQ ID NO 73 SCK.92.88 B4

[00444] SEQ ID NO 74 SCK.92.88 D5

GGGAGACAAGAATAAACGCTCAATTGGGTGACCGACAAATGTGGGAGTCAAATCGTTGTGAGTTCGACAGGAGG CTCACAACAGGC

[00445] SEQ ID NO 75 SCK.92.88 D2

GGGAGACAAGAATAAACGCTCAATTGGGTGACCGACAANTATGGGAGTCGAANTGTTGTGAATTCGACAGGAGG CTCACAACAGGC

[00446] SEQ ID NO 76 SCK.92.88 Al

GGGAGACAAGAATAAACGCTCAATGGGGTAACCGACAACATTTTATGGGAGTTAGGGCCTGTTCGACAGGAGGC TCACAACAGGC

[00447] S SEQ ID NO 77 CK.92.88 B5

GGGAGACAAGAATAAACGCTCAATGGGGTGACCGACAATTATGGGAGTCGAANTGNTGTGA TTCGACAGGAGG CTCACAACAGGC

[00448] SEQ ID NO 78 SCK.92.88 G7

GGGAGACAAGAATAAACGCTCAATTGGGTGACCGACAANTGTGGGAGTCNAATTGTTGTGA TTCGACAGGAGG CTCACAACAGGC

[00449] SEQ ID NO 79 SCK.92.88 F6

GGGAGACAAGAATAAACGCTCAATTGGGTGACCGACAGTTATGGGAGTCTGTTCTTTGTGAGTTCGACAGGAGG CTCACAACAGGC [00450] SEQ ID NO 80 SCK.92.88 H9

GGGAGACAAGAATAAACGCTCAATGGGGTGACCGACAAATTTGGGAGTCTGCCTTTATGAGTTCGACAGGAGGC TCACAACAGGC

[00451] SEQ ID NO 81 SCK.92.88 A3

GGGAGACAAGAATAAACGCTCAATAGGGTNACCGACAATTGTGGGAGTATGATTGTTGTGATTTCGACAGGAGG CTCACAACAGGC

[00452] SEQ ID NO 82 SCK.92.88 Dll

GGGAGACAAGAATAAACGCTCAATAGGGTGACCGACAATAATGGGAGTCAAACTGTTGTGTGTTCGACAGGAGG CTCACAACAGGC

[00453] SEQ ID NO 83 SCK.92.88 C7

GGGAGACAAGAATAAACGCTCAATGGGGTGACCGACAATTATGGGAGTCTGCTTGTTCTGATTTCGACAGGAGG CTCACAACAGGC

[00454] SEQ ID NO 84 SCK.92.88 B3

GGGAGACAAGAATAAACGCTCAATAGGGTGACCGACATCAATGGGAGTCTAATTGCTGTGAGTTCGACAGGAGG CTCACAACAGGC

[00455] SEQ ID NO 85 SCK.92.88 E4

GGGAGACAAGAATAAACGCTCAATTGGGTGACCGACAAATTTGGGAGTCCGCTTGTTTTGCATTCGACAGGAGG CTCACAACAGGC

[00456] SEQ ID NO 86 SCK.92.88 D9

GGGAGACAAGAATAAACGCTCAATAGGGTGACCGACAGTTTTGGGAGTCCAAATGTAGTGAGTTCGACAGGAGG CTCACAACAGGC

[00457] SEQ ID NO 87 SCK.92.88 D4

GGGAGACAAGAATAAACGCTCAATAGGGTGACCGACAAAACTGGGAGTCCAATTGTGGTGAGTTCGACAGGAGG CTCACAACAGGC

[00458] SEQ ID NO 88 SCK.92.88 F12

GGGAGACAAGAATAAACGCTCAATGGGGTNACCGACATTATGGGAGT N NTTAGTTGTGATTTCGACAGGAG GCTCACAACAGGC

[00459] SEQ ID NO 89 SCK.92.88 A4

[00460] SEQ ID NO 90 SCK.92.88 G10

GGGAGACAAGAATAAACGCTCAATGGGGTGACCGACAATTATGGGAGTCCNNNTGNNNTCTGTTCGACAGGAGG CTCACAACAGGC

[00461] SEQ ID NO 91 SCK.92.88 C2

GGGAGACAAGAATAAACGCTCAATTGGGTGACCGACANNTATGGGAGTC NCTTGTTTTGAGTTCGACAGGAGG CTCACAACAGGC

[00462] SEQ ID NO 92 SCK.92.88 E10

GGGAGACAAGAATAAACGCTCAATCGGGTGACCGACAATTTTGGGAGTCCGCTTGTTGTCGTTTCGACAGGAGG CTCACAACAGGC

[00463] SEQ ID NO 93 SCK.92.88 HI

GGGAGACAAGAATAAACGCTCAATGGGGGTATCCGACAAACATTATGGGAGATGAGTTGTAGCTTCGACAGGAG GCTCACAACAGGC [00464] SEQ ID NO 94 SCK.92.88 All

GGGAGACAAGAATAAACGCTCAATTGGGTGACCGACAGATGTGGGAGTCTGCTTGATGTGATTTCGACAGGAGG CTCACAACAGGC

[00465] SEQ ID NO 95 SCK.92.88 H10

GGGAGACAAGAATAAACGCTCAATAGGGTGACCGACATTTCTGGGAGTCAATAGTTTGTGAGTTCGACAGGAGG CTCACAACAGGC

[00466] SEQ ID NO 96 SCK.92.88 E2

[00467] SEQ ID NO 97 SCK.92.88 G9

GGGAGACAAGAATAAACGCTCAAATGGGTGACCGACAGTTATGGGAGTCGAATTGCTGTGAGTTCGACAGGAGG CTCACAACAGGC

[00468] SEQ ID NO 98 SCK.92.88 C3

GGGAGACAAGAATAAACGCTCAAANGGGTGACCNACAATTATGGGANTCTGTCTGGTGCGAGTTCGACAGGAGG CTCACAACAGGC

[00469] SEQ ID NO 99 SCK.92.88 D10

GGGAGACAAGAATAAACGCTCAAATGGGTGACCGACAATTATGGGAGTCGTATTGTTGTGAGTTCGACAGGAGG CTCACAACAGGC

[00470] SEQ ID NO 100 SCK.92.88 B9

GGGAGACAAGAATAAACGCTCAAATGGGTGACCGACAATTATGGGAGTCCNATNGNTGTGAGTTCGACAGGAGG CTCACAACAGGC

[00471] SEQ ID NO 101 SCK.92.88 H6

GGGAGACAAGAATAAACGCTCAAATGGGTGACCGACAATCATGGGAGTCGAATTGTTGTGAGTTCGACAGGAGG CTCACAACAGGC

[00472] SEQ ID NO 102 SCK.92.88 F7

GGGAGACAAGAATAAACGCTCAAATGGGTGACCGACAATAATGGGAGTCGAATATTTGTGCNTTCGACAGGAGG CTCACAACAGGC

[00473] SEQ ID NO 103 SCK.92.88 Gl

GGGAGACAAGAATAAACGCTCAATGGGTGACCNANAATTATGGGANTCNAATTGTTGTGAGTTCGACAGGAGGC TCACAACAGGC

[00474] SEQ ID NO 104 SCK.92.88 A6

GGGAGACAAGAATAAACGCTCAAATGGGTGACCGACGATAGTGGGAGTCAACTTGTTGTGATTTCGACAGGAGG CTCACAACAGGC

[00475] SEQ ID NO 105 SCK.92.88 H4

GGGAGACAAGAATAAACGCTCAAATGGGTGACCGACATATGTGGGAGTCAAATTGTNGTGGGTTCGACAGGAGG CTCACAACAGGC

[00476] SEQ ID NO 106 SCK.92.88 F10

GGGAGACAAGAATAAACGCTCAAAGGGGTGACCGACAAACATGGGAGTCCGATTGTTCTGAGTTCGACAGGAGG CTCACAACAGGC

[00477] SEQ ID NO 107 SCK.92.88 Hll

GGGAGACAAGAATAAACGCTCAAAGGGGTGACCGACAATTATGGGAGTCGAATTGTTGTGAGTTCGACAGGAGG CTCACAACAGGC [00478] SEQ ID NO 108 SCK.92.88 E12

GGGAGACAAGAATAAACGCTCAAAGGGGTGACCGACAATCATGGGAGTCTGATCGTTGTGAGTTCGACAGGAGG CTCACAACAGGC

[00479] SEQ ID NO 109 SCK.92.88 Bll

GGGAGACAAGAATAAACGCTCAAAGGGGTAACCGACAATCATGGGAGTTCGATTGTTCTGAGTTCGACAGGAGG CTCACAACAGGC

[00480] SEQ ID NO 110 SCK.92.88 CIO

GGGAGACAAGAATAAACGCTCAAAGGGGTGACCGACAAAGATGGGAGTCGGCTTGTTTTGAGTTCGACAGGAGG CTCACAACAGGC

[00481] SEQ ID NO 111 SCK.92.88 C12

GGGAGACAAGAATAAACGCTCAAGGGGTGACCGACAATNATGGGAGTCTNNTTGTTGTGAGTTCGACAGGAGG CTCACAACAGGC

[00482] SEQ ID NO 112 SCK.92.88 B12

GGGAGACAAGAATAAACGCTCAANNGGGTGACCGACAATTNTGGGAGTCCAATTGTTGTGTGTTCGACAGGAGG CTCACAACAGGC

[00483] SEQ ID NO 113 SCK.92.88 E7

GGGAGACAAGAATAAACGCTCAANGGGTGACCGACAATTNTGGGAGTC NANTGTTNTGAGTTCGACAGGAGG CTCACAACAGGC

[00484] SEQ ID NO 114 SCK.92.88 E9

GGGAGACAAGAATAAACGCTCAANNGGGTAACCGACAAGTGTGGGAGTTCANTTGTAGTGAGTTCGACAGGAGG CTCACAACAGGC

[00485] SEQ ID NO 115 SCK.92.88 Fll

GGGAGACAAGAATAAACGCTCAANGGGGTGACCGACAANTNTGGGAGTCNAGANNG NTGAGTTCGACAGGAGG CTCACAACAGGC

[00486] SEQ ID NO 116 SCK.92.88 H12

GGGAGACAAGAATAAACGCTCAANTGGGGTGACCGACAATTATGGGAGTCTACTTGTTGTGAGTTCGACAGGAG GCTCACAACAGGC

[00487] SEQ ID NO 117 SCK.92.88 C4

GGGAGACAAGAATAAACGCTCAAGGGGTAATCGACAACATTATGGGAATTCGCAGGTAGCTTTCGACAGGAGGC TCACAACAGGC

[00488] SEQ ID NO 118 SCK.92.88 F4

GGGAGACAAGAATAAACGCTCAAGGGGTAATCGACAACATTATGGGAATTCGCGCAGGTAGCTTTCGACAGGAG GCTCACAACAGGC

[00489] SEQ ID NO 119 SCK.92.88 H3

GGGAGACAAGAATAAACGCTCAAGGGGTAATCGACAACATTATGGGAATTCGCANGTNNCTTTCGACAGGAGGC TCACAACAGGC

[00490] SEQ ID NO 120 SCK.92.88 G3

GGGAGACAAGAATAAACGCTCAACGGGGTGATCGACAGAATGAAAATTGGGAATCAATGCCTGTTCGACAGGAG GCTCACAACAGGC

[00491] SEQ ID NO 121 SCK.114.5 G5

GGGAGACAAGAATAAACGCTCAATTGGGTGATCCGACAATTATGGGAGTCGGTTTGTTGTGTGTTCGACAGGAG GCTCACATATCAGGC [00492] SEQ ID NO 122 SCK.114.5 A6

GGGAGACAAGAATAAACGCTCAATTGGGTGCCGACAATTTTGGGAGTCGACTTGTGGTGAGTTCGACAGGAGGC TCACAACAGGC

[00493] SEQ ID NO 123 SCK.114.5 F7

GGGAGACAAGAATAAACGCTCAATTGGGTGACCGACAATTATGGGAGTCGCATTGTTGTGTGTTCGACAGGAGG CTCACAACAGGC

[00494] SEQ ID NO 124 SCK.114.5 D5

GGGAGACAAGAATAAACGCTCAATTGGGTGACCGACAANTNTGGGAGTCAAATTGTTGTGNATTCGACAGGAGG CTCACAACAGGC

[00495] SEQ ID NO 125 SCK.114.5 A8

GGGAGACAAGAATAAACGCCCAATTGGGTGACCGACAATTATGGGAGTCGAATTGTTGTGAGTTCGACAGGAGG CTCACAACAGGC

[00496] SEQ ID NO 126 SCK.114.5 A7

GGGAGACAAGAATAAACGCTCAATTGGGTGACCGACAATTATGGGAGTCNAATTGTTGTGAGTTCGACAGGAGG CTCACAACAGGC

[00497] SEQ ID NO 127 SCK.114.5 D7

GGGAGAAAGAATAAACGCTCAANTGGGTGACCGACAATTATGGGAGTCNGACTTGTTNTAGAGTTCGACAGGAG GCTCACAACAGGC

[00498] SEQ ID NO 128 SCK.114.5 F8

GGGNGNCAAGAATAA CGCTCAANTGGGTGACNGACACTTATGGGAGTCAATTTGNTT NATTCGACAGGAGG CTCACAACAGGC

[00499] SEQ ID NO 129 SCK.114.5 H8

GGGAGACAAGAATAAACGCTCAATGGGGTGACCGACAATTATGGGAGTCCACTTGTTGTTAGTTCGACAGGAGG CTCACAACAGGC

[00500] SEQ ID NO 130 SCK.114.5 E8

GGGAGACAAGAATAAACGCTCAATGGGGTGACCGACAATAATGGGAGTCTAATTGTTGTGAGTTCGACAGGAGG CTCACAACAGGC

[00501] SEQ ID NO 131 SCK.114.5 G8

GGGAGACAAGAATAAACGCTCAATGGGGTGACCGACAATTATGGGAGTCCAATTGTTTTGAGTTCGACAGGAGG CTCACAACAGGC

[00502] SEQ ID NO 132 SCK.114.5 F5

GGGAGACAAGAATAAACGCTCAATGGGGTGACCGACAATATGGGAGTCCAAATGTTTTGAGTTCGACAGGAGGC TCACAACAGGC

[00503] SEQ ID NO 133 SCK.114.5 C5

GGGAGACAAGATAAACGCTCAATGGGGTGACCGACAAATATGGGAGTCCAATCGTTGTGAGTTCGACAGGAGGC TCACAACAGGC

[00504] SEQ ID NO 134 SCK.114.5 C7

GGGAGAAAGAATAAACGCTCAATGGGGTGACCGACAATTATGGGAGTCNAACTGTTGTGTGTTCGACAGGAGGC TCACAACAGGC

[00505] SEQ ID NO 135 SCK.114.5 A5

GGGAGACAAGAATAAACGCTCAATGGGGTGACCGACAATAATGGGAGTCAACTTTTGTGAGTTCGACAGGAGGC TCACAACAGGC [00506] SEQ ID NO 136 SCK.114.5 C8

GGGAGAAAGAATAAACGCTCAATAGGGTGACCGACAATTATGGGAGTCCAATTTTTGTGAGTTCGACAGGAGGC TCACAACAGGC

[00507] SEQ ID NO 137 SCK.114.5 E6

GGGAGACAAGAATAAACGCTCAATAGGGTGACCGACAATTATGGGAGTCCGCTTGTTGTGANTTCGACAGGAGG CTCACAACAGGC

[00508] SEQ ID NO 138 SCK.114.5 E5

GGGAGACAAGAATAAACGCTCAATAGGGTGACCGACAGTAATGGGAGTCGAATTGTTGTGACTTCGACAGGAGG CTCACAACAGGC

[00509] SEQ ID NO 139 SCK.114.5 C6

GGGAGACAAGAATAAACGCTCAATAGGGTGACCGACATTTATGGGAGTCCAATTGTTGTAAGTTCGACAGGAGG CTCACAACAGGC

[00510] SEQ ID NO 140 SCK.114.5 B7

GGGAGACAAGAATAAACGCTCAATAGGGTGACCGACATTTTTGGGAGTCCACTTATTGTGAGTTCGACAGGAGG CTCACAACAGGC

[00511] SEQ ID NO 141 SCK.114.5 D8

GGGAGACAAGAATAAACGCTCAATAGGGTGACCGACAATCATGGGAGTCTGAATGTTTTGAGTTCGACAGGAGG CTCACAACAGGC

[00512] SEQ ID NO 142 SCK.114.5 H7

GGGAGACAAGATAAACGCTCAATAGGGTGACCGACAATTATGGGAGTCCAATTGTTGTAACTTCGACAGGAGGC TCACAACAGGC

[00513] SEQ ID NO 143 SCK.114.5 H5

GGGAGACAAGAATAAACGCTCAAAAGGGTGACCGACAATAATGGGAGTCCTATTGTTGGGAGTTCGACAGGAGG CTCACAACAGGC

[00514] SEQ ID NO 144 SCK.114.5 D6

GGGAGACAAGAATAAACGCTCAACTGGGTGACCGACAATTATGGGAGTCCTAATGTTGTGAGTTCGACAGGAGG CTCACAACAGGC

[00515] SEQ ID NO 145 SCK.114.5 B6

GGGAGACAAGAATAAACGCTCAAGGGGGTGACCGACAATTATGGGAGTCCAATAGTTCTGAGTTCGACAGGAGG CTCACAACAGGC

[00516] SEQ ID NO 146 SCK.114.5 F6

GGGAGACAAGAATAAACGCTCAAGAAGCTTTCCCCTTCTCTAGTGTCTGACTTGCTCGATTCCTTCGACAGGAG GCTCACAACAGGC

[00517] SEQ ID NO 147 SCK.114.5 E7

GGGAGAACAAGAATAAACGCTCAATGTTGTGAATGGAAAAACCGGACACTGAGCAAAACCTCCTCTTCGACAGG AGGCTCACAACAGGC

[00518] SEQ ID NO 148 SCK.114.5 G6

GGGAGACAAGAATAAACGCTCAATTATTGTAGCTTGGTTAAGATAATGAGACATCAAGTTCGACAGGAGGCTCA CAACAGGC

[00519] SEQ ID NO 149 SCK.114.5 B8

GGGAGACAAGAATAAACGCTCAACATCCTAGCGNTTGTGAGAGNTAAAGAGTGATNCAATTCGACAGGAGGCTC ACAACAGGC

[00520] Analysis of the sequences revealed three conserved domains, which are highlighted in the following consensus sequence (primer sequences are underlined): [00521] SEQ ID NO : 167

GGGAGACAAGAATAAACGCTCAAynsrGGGTMCCGACA NlTOTGGGAN.^ TCACAACAGGC

EXAMPLE 4 Characterization of PA binding sequences

[00522] Templates for several of the clone sequences were made synthetically and used to prepare 2'-F pyr, 2'-OH pur containing transcripts. The transcripts were assayed for their ability to bind PA83 (20 mM Hepes pH 7, 100 mM NaCl, 1 mM MgCl₂, 0.1 mM EDTA, 25°C). None of the PAcloneB2 derivatives bound more tightly to PA83 than PAcloneB2 (Kd ~ 500 nM).

[00523] The majority of the calculated (M-Fold) secondary structures of PAcloneB2 and its derivatives contain a long 3 '-terminal stem that we reasoned was not part of the core PA binding element. Constructs lacking that domain (SEQ IDs 168, 169, 170) retained PA binding activity.

[00524] SEQ ID NO 168 STC.104.35.A GGGAGACAAGAATAAACGCTCAATTGGGTGACCGACAATTATGGGAGTCGAA

[00525] SEQ ID NO 169 STC.104.35.B GGGAGACAAGAATAAACGCTCAATAGGGTGACCGACAATCATGGGAGTCTGAA

[00526] SEQ ID NO 170 STC.104.35.C GGGAGACAAGAATAAACGCTCAATGGGGTGACCGACAATAATGGGAGTCAAC

[00527] Clones STC.104.35.A, STC.104.35.B, and STC.104.35.C, were tested in a cell-based assay that measures inhibition of PA activity. In brief, cultured mouse macrophages were challenged by addition of protective antigen and lethal factor (references are in the provisional draft). Cytotoxicity is assessed using MTT, a chromogenic substrate that monitors mitochondrial activity. Active aptamers are expected to rescue macrophages from PA/LF mediated cell killing. PA₃₂ is used as a positive control in the reaction, as it competes with PA₈₃ for binding to the cell surface receptor ATR. All of the aptamers demonstrated inhibtion at 10 μM in this assay (Figure 10).

[00528] Figure 10 shows Anti-PA aptamers protect RAW 264.7 cells from PA/LF- induced cell death. RAW 264.7 cells were plated at 30,000 cells per well in a 96-well plate. Aptamers at the indicated concentrations or PA₃₂ at 20μg/ml were pre-incubated at the indicated concentrations with lOOng/ml PA₈₃ for 15 minutes. Aptamer and PA₈₃ were added to the cells followed 10 minutes later by the addition of 50 ng/ml of LF. The cells were incubated for 10 hours with or without aptamer, PA₈₃ or LF at 37°C. Cell viability was assessed with MTT (Promega). Control scrambled sequence aptamers to unrelated targets were included to demonstrate specificity (SEQ ID No 171 and 172).

[00529] SEQ ID NO 171 Control Aptamer 1 GGGGUUAUUACAGAGUCUGUAUAGCUGUACCCT

[00530] SEQ ID NO 172 Control Aptamer 2

40KPEG-CAGCGUACG-PEG-CGTACCGATUCA-PEG-TGAAGCUGT

EXAMPLE 5 PA Nucleic Acid Sensor Molecules

[00531] Figure 11 illustrates an RNA ribozyme library derived from a hammerhead sequence pool consisting of up to 10¹ variants of randomized sequences appended to the hammerhead ribozyme motif. The starting pool of nucleic acids comprising a target modulation domain (TMD), linker domain (LD) and catalytic domain (CD) is prepared on a DNA synthesizer. Random nucleotides are incorporated during the synthesis to generate pools of roughly 10¹⁶ molecules. Randomized stem region scanning library is designed to identify cis-hammerhead NASMs that are modulated by PA. The linker library is generated by appending a PA target modulation domain to the randomized linker domain to create a library of potential PA-modulated cis-hammerhead NASMs. The linker library of PA- modulated cis-hammerhead NASMs consists of up to 65,000 variants. Most molecules in the randomized NASM pools are non-functional NASMs. In some libraries, the catalytic site is a known sequence (a ligase site or a hammerhead catalytic core) and is at least a portion of either the 5' and/or 3' fixed region (the other portion being supplied by the random sequence), or is a complete catalytic site. However, the catalytic site may be selected along with the target molecule binding activity of oligonucleotides within the oligonucleotide pool.

[00532] Sorting among the PA sensors candidates to find the desired molecules starts from the complex sequence pool, whereby desired PA-modulated sensors are isolated through an iterative in vitro selection process: in addition to the target-activated NASMs that one desires, the starting pool is usually dominated by either constitutively active or completely inactive ribozymes. The selection process removes both types of contaminants. In a following amplification stage, thousands of copies of the surviving sequences are generated to enable the next round of selection. During amplification, random mutations can be introduced into the copied molecules — this 'genetic noise' allows functional NASMs to continuously evolve and become even better adapted as target-activated enzymes. The entire experiment reduces the pool complexity from 10¹⁷ molecules down to around 100 PA sensor candidates that require detailed characterization. [00533] The nucleic acid sensor molecules identified through in vitro selection comprise a catalytic domain (i.e., a signal generating moiety), coupled to a target modulation domain, (i.e., a domain which recognizes PA and which transduces that molecular recognition event into the generation of a detectable signal). In general, the target modulation domain is defined by the minimum number of nucleotides sufficient to create a three-dimensional structure which recognizes PA. In addition, the nucleic acid sensor molecules of the present invention use the energy of molecular recognition to modulate the catalytic or conformational properties of the nucleic acid sensor molecule. The selection process as described in detail in the present invention identifies novel nucleic acid sensor molecules through target modulation of the catalytic core of a ribozyme. [00534] The NASM selection procedures place selective pressure on catalytic effectiveness of potential NASMS by modulating both PA concentration and reaction time- dependence. Either parameter, when optimized throughout the selection, can lead to nucleic acid molecular sensor molecules which have custom-designed catalytic properties, e.g., NASMs that have high switch factors, and or NASMs that have high specificity. [00535] PA sensor candidates which are derived from in vitro selection are tested as target modulated biosensors. The pool of PA sensor candidates is cloned into various plasmids transformed into E. coli. Individual PA sensor encoded DNA clones are isolated, PCR amplified and the PA sensor candidate is transcribed in vitro to generate PA sensor RNA. The PA sensor RNAs are then tested in target modulation assays which determine the rate or extent of ribozyme modulation. For hammerhead PA sensor RNAs, the extent of target dependent and independent reaction is determined by quantifying the extent of self cleavage of an oligonucleotide substrate in the absence or presence of PA. The extent of reaction can be followed by electrophoretic separation of the reaction products on a denaturing PAGE gel, and subsequently analyzed by standard radiometric methods. [00536] Individual PA sensor clones which display high target dependent switch factor values, or high k_aC rate values are subsequently chosen for further modification and evaluation.

[00537] Hammerhead derived NASM clones are then further modified to render them suitable for the optical detection applications that are described herein. [00538] The NASMs of the present invention are added to a sample to be tested for the presence of Bacillus anthracis and/or its toxins. The sample can be an environmental swab or other sample in solution mixed with the NASMs of the present invention. The sample can also be a bodily fluid of a subject or patient suspected to have been exposed to Bacillus anthracis and/or its toxins. The NASM provides a signal that can be interpreted versus a control for a positive result when there is Bacillus anthracis and/or its toxins present in the sample.

EXAMPLE 6 Peptides with specific binding affinity to PA

[00539] PA32 and PA18 are peptides with specific binding affinity to Bacillus anthracis protective antigen and can be used to inhibit formation of functional protective antigen thus hindering anthrax pathogenesis.

[00540] PA32 is a peptide having 283 amino acids, MW : 32214,33 Molecular extinction coefficient at 280 nm (1cm) : 14080 Absorption 0,1% (lg/1) a 280 nm (1cm) : 0.437 Theoretical isoelectric point: - classic : 5,60. [00541] Method to isolate PA-specific binding peptides

[00542] The full length cDNA of protective antigen from B. anthracis was obtained from USAMRIID (PA1086Δ5 or pET22PA). The 3'-region of cDNA coding the domain 3 and 4 of protective antigen was amplified by PCR using primers PA32f (AAGGATCCGAAACAACTGCACGTATCATT) (SEQ ID NO: 161) and PA32r (ATCTCGAGTTATCCTATCTCATAGCCTTTTTT) (SEQ ID NO: 162). The amplified approximately 770 bp DNA fragment was cloned into pRESTB vector (Invitrogen) using Xhol and BamHI sites. The resulted plasmid, pRSETPA32, was transformed into BL21(DE3) pLys stiain of E. coli for expression. Expression of his-PA32 was induced with ImM IPTG (isopropyl-beta-D-thiogalactopyranoside), at 37 degree for 3 hours. The soluble fraction was obtained using commercial lysis buffer containing detergent (e.g. BPER, Pierce biotechnology) and directly applied onto Ni-NTA superflow (Qiagen) column. The resin was washed with 20mM HEPES pH 7.5 with 20mM NaCl, 20mM Imidazol, and 10% Glycerol. His-PA32 was eluted from the column using 50~200mM Imidazol with -90% purity. The elute was then applied onto High Q column (Bio Rad) directly. After washing with 20mM HEPES pH7.5 with 20mM NaCl and 10% Glycerol, his-PA32 was eluted with 200mM NaCl with >95% purity. [00543] Method to generate his-PA32mut

[00544] The full length cDNA of protective antigen from B. anthracis was obtained from USAMRIID (PA1086Δ5 or pET22PA). The 3'-region of cDNA coding the domain 3 and 4 of protective antigen was amplified by PCR using primers PA32f (AAGGATCCGAAACAACTGCACGTATCATT) (SEQ ID NO: 161) and PA32r (ATCTCGAGTTATCCTATCTCATAGCCTTTTTT) (SEQ ID NO: 162). The amplified approximately 770 bp DNA fragment was cloned into pRESTB vector (Invitrogen) using Xhol and BamHI sites. The mutation at 232 from lysine to arginine was generated by spontaneous mutation during PCR reaction. The resulted plasmid, pRESTPA32mut, was transformed into BL21(DE3) pLys strain of E. coli for expression. Expression of his- PA32mut was induced with ImM IPTG (isopropyl-beta-D-thiogalactopyranoside), at 37 degree for 3 hours. The soluble fraction was obtained using lysis buffer containing detergent and directly applied onto Ni-NTA superflow (Qiagen) column. The resin was washed with 20mM HEPES pH 7.5 with 20mM NaCl, 20mM Imidazol, and 10% Glycerol. His-PA32mut was eluted from the column using 50~200mM Imidazol with -90% purity. The elute was then applied onto High Q column (Bio Rad) directly. After washing with 20mM HEPES pH7.5 with 20mM NaCl and 10% Glycerol, his-PA32mut was eluted with 200mM NaCl with >95% purity. [00545] Method to generate his-PA18

[00546] The full length cDNA of protective antigen from B. Anthrax was obtained from USAMRIID (PA1086Δ5 or pET22PA). The 3 '-region of cDNA coding the domain 4 of protective antigen was amplified by PCR using primers PA18f (GCCGGATCCGAGAATAACATAGCAGTT) (SEQ ID NO: 163) and PA32r (ATCTCGAGTTATCCTATCTCATAGCCTTTTTT) (SEQ ID NO: 162). The amplified approximately 430 bp DNA fragment was cloned into pRESTB vector (Invitrogen) using Xhol and BamHI sites. The resulted plasmid was transformed into BL21(DE3)pLys strain of E. coli for expression. The cells were incubated at 37 degree until OD600 reached 0.3. Then, expression of PA18 was induced with 0.5mM IPTG at 30 degree for overnight. The soluble fraction was obtained using lysis buffer containing detergent and directly applied onto Ni-NTA superflow (Qiagen) column. The resin was washed with two column volumes of 20mM HEPES pH 7.5 with 20mM NaCl, 50mM Imidazol, and 10% Glycerol. PA18 was eluted from the column using 200mM Imidazol with -90%) purity. The eluted PA18 fraction was further purified using either High S or High Q column (BioRad).

[00547] Protection Assay with RAW264.7 Cells

[00548] RAW 264.7 cells were plated at 30,000 cells per well in a 96-well plate with lOOul of DMEM (Dulbecco's Modification of Eagle's Media) containing 10% FBS supplemented with penicillin and streptomycin. The cells were incubated at 37°C overnight in a C0 incubator. PA18 or PA32 at various concentrations and PA83 (100 ng/ml final concentration) were added to the cells followed 10 minutes later by the addition of 50 ng/ml (final concentration) of LF. The cells were incubated for approximately 16 hours with or without testing reagents, PA and/or LF at 37°C. Cell viability was assessed with MTT according to the manufacture's instruction (Promega).

[00549] PA32 Amino Acid Sequence SEQ ID NO. 153. :

MRGSHHHHHH GMASMTGGQQ MGRDLYDDDD KDPETTARI I FNGKDLNLVE RRIAAVNPSD PLETTKPDMT LKEALKIAFG FNEPNGNLQY QGKDITEFDF NFDQQTSQNI KNQLAELNAT NIYTVLDKIK LNAKMNILIR DKRFHYDRNN IAVGADESW KEAHREVINS STEGLLLNID KDIRKILSGY IVEIEDTEGL KEVINDRYDM LNISSLRQDG KTFIDFKKYN DKLPLYISNP NYKVNVYAVT KENTI INPSE NGDTSTNGIK KILIFSKKGY EIG

[00550] PA18 is a peptide with a 136 amino acid sequence having an isolectric point at 5.1, 3.7%> Arg content, and 15%> positive amino acid content, and a molecular weight of 15357,39.

[00551] PA18 Amino Acid Sequence SEQ ID No. 154 :

MRGSHHHHHH GMASMTGGQQ MGRDLYDDDD KDPRNNIAVG ADESWKEAH REVINSSTEG LLLNIDKDIR KILSGYIVEI EDTEGLKEVI NDRYDMLNIS SLRQDGKTFI DFKKYNDKLP LYISNPNYKV NVYAVTKENT IINPSENGDT STNGIKKILI FSKKGYEIG

[00552] PA32 Nucleotide sequence SEQ ID No. 155 :

ATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATGACTGGTGGACAGCAAA TGGGTCGGGATCTGTACGACGATGACGATAAGGATCCGGAAACAACTGCACGTATCATTTT TAATGGAAAAGATTTAAATCTGGTAGAAAGGCGGATAGCGGCGGTTAATCCTAGTGATCCA TTAGAAACGACTAAACCGGATATGACATTAAAAGAAGCCCTTAAAATAGCATTTGGATTTA ACGAACCGAATGGAAACTTACAATATCAAGGGAAAGACATAACCGAATTTGATTTTAATTT CGATCAACAAACATCTCAAAATATCAAGAATCAGTTAGCGGAATTAAACGCAACTAACATA TATACTGTATTAGATAAAATCAAATTAAATGCAAAAATGAATATTTTAATAAGAGATAAAC GTTTTCATTATGATAGAAATAACATAGCAGTTGGGGCGGATGAGTCAGTAGTTAAGGAGGC TCATAGAGAAGTAATTAATTCGTCAACAGAGGGATTATTGTTAAATATTGATAAGGATATA AGAAAAATATTATCAGGTTATATTGTAGAAATTGAAGATACTGAAGGGCTTAAAGAAGTTA TAAATGACAGATATGATATGTTGAATATTTCTAGTTTACGGCAAGATGGAAAAACATTTAT AGATTTTAAAAAATATAATGATAAATTACCGTTATATATAAGTAATCCCAATTATAAGGTA AATGTATATGCTGTTACTAAAGAAAACACTATTATTAATCCTAGTGAGAATGGGGATACTA GTACCAACGGGATCAAGAAAATTTTAATCTTTTCTAAAAAAGGCTATGAGATAGGATAA

[00553] His-PA32mut Amino Acid Sequence SEQ ID No. 156 (his to arg mutation underlined) :

MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPETTARIIFNGKDLNLVERRIAAVNPSDP LETTKPDMTLKEALKIAFGFNEPNGNLQYQGKDITEFDFNFDQQTSQNIKNQLAELNATNI YTVLDKIKLNAKMNILIRDKRFHYDRNNIAVGADESWKEAHREVINSSTEGLLLNIDKDI RKILSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDFKKYNDRLPLYISNPNYKV NVYAVTKENTIINPSENGDTSTNGIKKILIFSKKGYEIG

[00554] His-PA32mut Nucleotide Sequence SEQ ID No. 157 (His to Arg Mutation) :

ATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATGACTGGTGGACAGCAAA TGGGTCGGGATCTGTACGACGATGACGATAAGGATCCGGAAACAACTGCACGTATCATTTT TAATGGAAAAGATTTAAATCTGGTAGAAAGGCGGATAGCGGCGGTTAATCCTAGTGATCCA TTAGAAACGACTAAACCGGATATGACATTAAAAGAAGCCCTTAAAATAGCATTTGGATTTA ACGAACCGAATGGAAACTTACAATATCAAGGGAAAGACATAACCGAATTTGATTTTAATTT CGATCAACAAACATCTCAAAATATCAAGAATCAGTTAGCGGAATTAAACGCAACTAACATA TATACTGTATTAGATAAAATCAAATTAAATGCAAAAATGAATATTTTAATAAGAGATAAAC GTTTTCATTATGATAGAAATAACATAGCAGTTGGGGCGGATGAGTCAGTAGTTAAGGAGGC TCATAGAGAAGTAATTAATTCGTCAACAGAGGGATTATTGTTAAATATTGATAAGGATATA AGAAAAATATTATCAGGTTATATTGTAGAAATTGAAGATACTGAAGGGCTTAAAGAAGTTA TAAATGACAGATACGATATGTTGAATATTTCTAGTTTACGGCAAGATGGAAAAACATTTAT AGATTTTAAAAAATATAATGATAGATTACCGTTATATATAAGTAATCCCAATTATAAGGTA AATGTATATGCTGTTACTAAAGAAAACACTATTATTAATCCTAGTGAGAATGGGGATACTA GTACCAACGGGATCAAGAAAATTTTAATCTTTTCTAAAAAAGGCTATGAGATAGGATAA

[00555] His-PAl8 Amino Acid Sequence SEQ ID No.158 :

MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPRNNIAVGADESWKEAHREVINSSTEGL LLNIDKDIRKILSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDFKKYNDKLPLY ISNPNYKVNVYAVTKENTIINPSENGDTSTNGIKKILIFSKKGYEIG

[00556] His-PAl 8 Nucleotide Sequence SEQ ID No. 159 :

ATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATGACTGGTGGACAGCAAA TGGGTCGGGATCTGTACGACGATGACGATAAGGATCCGAGAAATAACATAGCAGTTGGGGC GGATGAGTCAGTAGTTAAGGAGGCTCATAGAGAAGTAATTAATTCGTCAACAGAGGGATTA TTGTTAAATATTGATAAGGATATAAGAAAAATATTATCAGGTTATATTGTAGAAATTGAAG ATACTGAAGGGCTTAAAGAAGTTATAAATGACAGATACGATATGTTGAATATTTCTAGTTT ACGGCAAGATGGAAAAACATTTATAGATTTTAAAAAATATAATGATAAATTACCGTTATAT ATAAGTAATCCCAATTATAAGGTAAATGTATATGCTGTTACTAAAGAAAACACTATTATTA ATCCTAGTGAGAATGGGGATACTAGTACCAACGGGATCAAGAAAATTTTAATCTTTTCTAA AAAAGGCTATGAGATAGGATAA

[00557] The present invention having been described by detailed description and the non-limiting examples, is now defined by the spirit and scope of the following claims.

Claims

We claim:

1. A nucleic acid aptamer having binding affinity to one or more pathogen targets, whereby binding of said aptamer to said target reduces virulence of said target, wherein said pathogen is Bacillus anthracis.

2. The nucleic acid aptamer of claim 1 wherein said target is protective antigen.

3. The nucleic acid aptamer of claim 2 wherein said binding of said aptamer prevents the formation of protective antigen multimers required for virulence of said pathogen.

4. The nucleic acid aptamer of claim 3 selected from the group consisting of SEQ ID No: 1, SEQ ID No: 2 through SEQ ID No: 40, SEQ ID No: 43 through SEQ ID No: 148, SEQ ID No: 149 and SEQ ID No: 167.

5. A nucleic acid sensor molecule comprising:

(a) a target modulation domain, wherein said target modulation domain recognizes a Bacillus anthracis protective antigen; .

(b) a linker domain; and

(c) a catalytic domain.

6. The nucleic acid sensor molecule of claim 5 wherein the catalytic domain comprises an optical signal generating unit.

7. The nucleic acid sensor molecule of claim 6, wherein said optical signal generating unit comprises at least one optical signaling moiety.

8. The nucleic acid sensor molecule of claim 6, wherein said optical signal generating unit comprises at least a first optical signaling moiety and a second optical signaling moiety.

9. The nucleic acid sensor molecule of claim 8, wherein said first and second signaling moieties change proximity to each other upon recognition of a target by the target modulation domain.

10. The nucleic acid sensor molecule of claim 9, wherein said first and second signaling moieties comprise a fluorescent donor and a fluorescent quencher, and recognition of a target by the target modulation domain results in an increase in detectable fluorescence of said fluorescent donor.

11. The nucleic acid sensor molecule of claim 9, wherein said first signaling moiety and said second signaling moiety comprise fluorescent energy transfer (FRET) donor and acceptor groups, and recognition of a target by the target modulation domain results in a change in distance between said donor and acceptor groups, thereby changing optical properties of said molecule.

12. The nucleic acid sensor molecule of claim 1, wherein said optical signaling moiety changes conformation upon recognition of a target by the target modulation domain, thereby resulting in a detectable optical signal.

13. The nucleic acid sensor molecule of claim 5, further comprising a detectable label.

14. The nucleic acid sensor molecule of claim 13 wherein the detectable label comprises at least one radioactive moiety.

15. The nucleic acid sensor of claim 13, wherein the detectable label comprises a fluorescent label.

16. The nucleic acid sensor of claim 5, wherein said nucleic acid sensor further comprises an affinity capture tag label.

17. The nucleic acid sensor molecule of claim 5, wherein said nucleic acid sensor molecule comprises RNA, DNA, or both RNA and DNA.

18. The nucleic acid sensor molecule of claim 5, wherein said nucleic acid sensor molecule comprises at least one modified nucleotide.

19. A composition comprising at least one nucleic acid sensor molecule according to claim 5, affixed to a substrate.

20. The composition of claim 19, wherein the nucleic acid sensor molecule is immobilized to the substiate via hybridization of a terminal portion of the nucleic acid sensor molecule to an oligonucleotide that is bound to the surface of the substrate.

21. The substiate of claim 20, wherein said substrate comprises at least 50 nucleic acid sensor molecules.

22. The substiate of claim 20, wherein said substrate comprises at least 250 nucleic acid sensor molecules.

23. A system for detecting a Bacillus anthracis protective antigen, comprising a composition according to claim 1 , and a detector in communication with said composition, wherein said detector is capable of detecting a signal generated upon recognition of a Bacillus anthracis protective antigen by the nucleic acid sensor molecule of claim 5.

24. The system of claim 23, further comprising a light source in optical communication with said composition.

25. The system of claim 23, further comprising a processor for processing optical signals detected by the detector.

26. A method of identifying or detecting a Bacillus anthracis protective antigen in a sample, the method comprising: contacting a sample suspected of containing Bacillus anthracis protective antigen with a nucleic acid sensor molecule according to claim 6 or claim 13, wherein a change in the signal generated by the optical signal generating unit or detectable label indicates the presence of Bacillus anthracis protective antigen in said sample.

27. The method of claim 26 wherein the sample is selected from the group consisting of: environmental samples, biohazard materials, organic samples, drugs and toxins, flavors and fragrances, and biological samples.

28. The method of claim 26 wherein the sample is a biological sample selected from the group consisting of cells, cell extracts, cell lysates, tissues, tissue extracts, bodily fluids, serum, blood, and blood products.

29. The method of claim 26 wherein said detectable comprises a fluorescent label.

30. The method of claim 26, wherein said optical signal generating unit comprises at least a first optical signaling moiety and a second optical signaling moiety.

31. The method of claim 30, wherein said first and second signaling moieties change proximity to each other upon recognition of a target by the specific binding agent.

32. The method of claim 31 , wherein said first and second signaling moieties comprise a fluorescent donor and a fluorescent quencher, and recognition of a target by the specific binding agent results in an increase in detectable fluorescence of said fluorescent donor.

33. The method of claim 32, wherein said first signaling moiety and said second signaling moiety comprise fluorescent energy tiansfer (FRET) donor and acceptor groups, and recognition of a target by the specific binding agent results in a change in distance between said donor and acceptor groups, thereby changing optical properties of said molecule.

34. The method of claim 26, wherein said optical signaling moiety changes conformation upon recognition of a target by the target modulation domain, thereby resulting in a detectable optical signal.

35. The method of claim 26, further comprising quantifying the change in signal generated by the detectable label or optical signal generating unit to quantify the amount of Bacillus anthracis protective antigen in the sample.

36. A peptide therapeutic having binding affinity to Bacillus anthracis protective antigen.

37. The peptide therapeutic of claim 36 selected from SEQ ID No. 153, SEQ ID No. 154, SEQ ID No. 156, and SEQ ID No. 158.

38. The peptide therapeutic of claim 37 having 75 % sequence identity to the peptide selected from SEQ ID No. 153, and SEQ ID No. 154.

39. A method of treating Bacillus anthracis disease in subjects exposed to Bacillus anthracis spores comprising the steps of administering to said subjects a therapeutically effective amount of a therapeutic selected from an aptamer, a NASM, and a peptide, said therapeutic having binding affinity to Bacillus anthracis protective antigen.

40 The method of claim 39 wherein said aptamer is selected from the group consisting of SEQ ID No: 1, SEQ ID No: 2 through SEQ ID No: 40, SEQ ID No: 43 through SEQ ID No: 148, SEQ ID No: 149 and SEQ ID No: 167.

41. The method of claim 39 wherein said peptide is selected from the group consisting of SEQ ID No. 153, SEQ ID No. 154, SEQ ID No. 156 and SEQ ID. 158.

42. The method of claim 41 wherein said peptide has greater than 75 percent amino acid sequence identity to SEQ ID No. 153 or SEQ ID No. 154.