WO2007120298A2 - Methods, substrates and probes for assaying n-glycosidase activity - Google Patents

Abstract

Disclosed herein are methods for assaying the RNA N-glycosidase or DNA N-glycosylase activity of protein. Also disclosed are substrates and probes that may be used in the assays.

Description

METHODS, SUBSTRATES AND PROBES FOR ASSAYING N-GLYCOSIDASE ACTIVITY

CROSS-REFERENCE TO RELATED APPLICATIONS

[01] This application claims the benefit of U.S. Provisional Patent Application

Serial Nos. 60/748,231, filed 1 December 2005, and 60/774,797, filed 15 February 2006, both of which are herein incorporated by reference.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

[02] This invention was made by employees of the United States Army Medical

Research and Materiel Command, which is an agency of the United States Government. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. FIELD OF THE INVENTION.

[03] The present invention relates to methods and substrates for assaying N- glycosidase activity. In particular, the present invention relates to methods and substrates for assaying toxins based on their N-glycosidase activity.

2. DESCRIPTION OF THE RELATED ART.

[04] Ribosome -inactivating proteins (RIPs) include adenine-specific N- glycosidases, such as ricin, saporin, and gelonin, which depurinate ribosomal RNA to cause irreversible inhibition of protein synthesis. See Barbieri et al. (1992) Biochem. J. 286:14; Zamboni et al. (1989) Biochem. J. 259:639-643. Referred to as (adenosine) N-glycosidases when acting on RNA, these enzymes also remove adenine from DNA molecules (DNA N-glycosylase activity), including single-stranded or denatured DNA. See Nicolas et al. (2000) J. Biol. Chem. 275:31399-31406; and Barbieri et al. (1997) Nucleic Acids Res. 25:518-522. Likewise, uracil DNA glycosylase (UDG) can remove uracil from deoxyuridine residues in oligodeoxyribonucleotides (ODNs). See Kumar & Varshney (1997) Nucleic Acids Res. 25:2336-2343. The resulting abasic sites have intact phosphodiester backbones when purified N-glycosidases are used, although backbone cleavage (lyase activity) has been reported. See Barbieri et al. (2000) J. Biochem. (Tokyo) 128:883-889; and Nicolas et al. (1998) J. Biol. Chem. 273:17216-17220.

[05] N-glycosidases, when naturally coupled to cell-binding lectins, are also known as potential bioterrorism agents because of their toxic properties. These toxins remove adenine residues from ribosomal RNA, thereby inactivating ribosomes and inhibiting their protein synthesis reactions, which are required for viability of cells. Ricin and abrin are two enzymatic toxins biothreats that contain RNA JV-glycosidase components, or A chains. These A chains catalyze the removal of a specific adenine base from mammalian 28S ribosomal RNA, irreversibly altering its interactions with elongation factors. See Perentesis et al. (1992) Biofactors 3:173-184; and Lord et al. (1994) FASEB J. 8:201-208. Each A chain is linked by a disulfide bond to a B chain, which binds to galactose moieties on cell surfaces and aids in delivering the N- glycosidase to the cytoplasm where it inactivates ribosomes. These heterodimeric, two-chain toxins are referred to as type II RIPs. Variants of the two-chain toxins exist for both ricin and abrin. See Hegde et al. (1992) Eur. J. Biochem. 204:155-164.

[06] Biothreat detection can lead to the misallocation of costly resources if false positive signals cannot be quickly and independently identified. Immunoassay reagents (antibodies) recognize surface features of proteins (epitopes) that may be unrelated to any enzymatic activity or other mechanism of toxicity. Thus, it may be possible for inactive protein toxins to cause positive signals with immunoassays, resulting in an overestimation of the threat.

[07] Activity-based assays previously reported for N-glycosidases involve radiolabeled DNA substrates or the derivatization of released adenine to a fluorescent product using highly toxic chloroacetaldehyde. See Brigotti et al. (1998) Nucleic Acids Res. 26:43064307; Xia & O'Connor (2001) Anal. Biochem. 298:322-326; and Lawrence et al. (1972) J. Pharm. Sci 61 :19-25. In both cases, separation of unreacted DNA substrate was required. The classic approach for assaying RIPs uses inhibition of in vitro translation as a measure of RIP activity. Typically, the complex, unstable reagents necessary for this approach are derived from lysates of rabbit reticulocytes. See Langer et al. (1997) Anal. Biochem. 243:150-153. Since ribosomal inactivation produces a signal that is inversely proportional to the amount of RIP present, inhibitory substances other than RIPs may cause false responses. A fluorescence- based assay has been reported, which directly measures N-glycosidase activity on short ODN substrates having defined sites for enzyme action, but this method is not conducive to signal amplification and thus is limited in sensitivity. See Kreklau et al. (2001) Nucleic Acids Res. 29:2558-2566.

[08] Thus, while prior N-glycosidase assays are known and are generally suitable for their limited purposes, they possess certain inherent deficiencies that detract from their overall utility in field-testing for bioterrorism agents. [09] Therefore, a need exists for N-glycosidase assays and substrates.

SUMMARY OF THE INVENTION

[10] In some embodiments, the present invention provides an assay for an enzyme which comprises hydrolyzing a site on at least one substrate by contacting the substrate with the enzyme, cleaving the substrate at the site hydrolyzed by the enzyme with a cleaving agent to give a cleavage product, and quantifying the amount of the cleavage product. In some embodiments, the enzyme is an N-glycosidase. In some embodiments, the enzyme is ricin toxin A chain (RTA), ricin toxin, saporin, abrin II, or an RNase. In some embodiments, the substrate is an oligonucleotide in the form of a hairpin loop structure. In some embodiments, the oligonucleotide is conjugated to a ligand, a label or both. In some embodiments, the label is an ECL label. In some embodiments, the nucleotide sequence of the loop portion of the hairpin loop structure comprises or consists of GAGA, GAAA, AGAGAC, GUGA, GIGA, or UGCU, and at least one base is an RNA residue or a 2'-O-methyl RNA residue. In some embodiments, the nucleotide sequence of the loop portion of the hairpin loop structure comprises or consists of rGdArGrA, rGrArGrA, rGdArArA, rGdAdGrA, rArGdArGrArC, rGdUrGrA, rGdlrGrA, rUrGrCrU, mGdAmGmA, rGdArGmA, mGdArGmA, or rGdAmGmA, where d is a DNA residue, r is an RNA residue, and m is a 2'-O-methyl RNA residue. In some embodiments, the oligonucleotide sequence is selected from a group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. In some embodiments, the cleavage product is detected by hybridizing with a probe. In some embodiments, the probe is conjugated to a ligand, a label or both. In some embodiments, the probe comprises SEQ ID NO:11. In some embodiments, the label is an ECL label. In some embodiments, the assay further comprises measuring the activity of the enzyme in the presence of an antibody.

[11] In some embodiments, the present invention provides an assay for measuring the activity or amount of an enzyme which comprises hydrolyzing a site on at least one substrate by contacting the substrate with the enzyme, cleaving the substrate at the site hydrolyzed by the enzyme with a cleaving agent to give a cleavage product, quantifying the amount of the cleavage product and correlating the amount of the cleavage product with a standard or control. In some embodiments, the enzyme is an N-glycosidase. In some embodiments, the enzyme is ricin toxin A chain (RTA), ricin toxin, saporin, abrin II, or an RNase. In some embodiments, the substrate is an oligonucleotide in the form of a hairpin loop structure. In some embodiments, the oligonucleotide is conjugated to a ligand, a label or both. In some embodiments, the label is an ECL label. In some embodiments, the nucleotide sequence of the loop portion of the hairpin loop structure comprises or consists of GAGA, GAAA, AGAGAC, GUGA, GIGA, or UGCU, and at least one base is an RNA residue or a 2'- O-methyl RNA residue. In some embodiments, the nucleotide sequence of the loop portion of the hairpin loop structure comprises or consists of rGdArGrA, rGrArGrA, rGdArArA, rGdAdGrA, rArGdArGrArC, rGdUrGrA, rGdlrGrA, rUrGrCrU, mGdAmGmA, rGdArGmA, mGdArGmA, or rGdAmGmA, where d is a DNA residue, r is an RNA residue, and m is a 2'-O-methyl RNA residue. In some embodiments, the oligonucleotide sequence is selected from a group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. In some embodiments, the cleavage product is detected by hybridizing with a probe. In some embodiments, the probe is conjugated to a ligand, a label or both. In some embodiments, the probe comprises SEQ ID NO: 11. In some embodiments, the label is an ECL label. In some embodiments, the assay further comprises measuring the activity of the enzyme in the presence of an antibody. [12] In some embodiments, the present invention provides an assay for characterizing or identifying an unknown enzyme which comprises which comprises hydrolyzing a site on at least one substrate by contacting the substrate with the enzyme, cleaving the substrate at the site hydrolyzed by the enzyme with a cleaving agent to give a cleavage product, quantifying the amount of the cleavage product and correlating the amount of the cleavage product with at least one activity profile for a known enzyme. In some embodiments, the enzyme is an N-glycosidase. In some embodiments, the enzyme is ricin toxin A chain (RTA), ricin toxin, saporin, abrin II, or an RNase. In some embodiments, the substrate is an oligonucleotide in the form of a hairpin loop structure. In some embodiments, the oligonucleotide is conjugated to a ligand, a label or both. In some embodiments, the label is an ECL label. In some embodiments, the nucleotide sequence of the loop portion of the hairpin loop structure comprises or consists of GAGA, GAAA, AGAGAC, GUGA, GIGA, or UGCU, and at least one base is an RNA residue or a 2'-O-methyl RNA residue. In some embodiments, the nucleotide sequence of the loop portion of the hairpin loop structure comprises or consists of rGdArGrA, rGrArGrA, rGdArArA, rGdAdGrA, rArGdArGrArC, rGdUrGrA, rGdlrGrA, rUrGrCrU, mGdAmGmA, rGdArGmA, mGdArGmA, or rGdAmGmA, where d is a DNA residue, r is an RNA residue, and m is a 2'-O-methyl RNA residue. In some embodiments, the oligonucleotide sequence is selected from a group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. In some embodiments, the cleavage product is detected by hybridizing with a probe. In some embodiments, the probe is conjugated to a ligand, a label or both. In some embodiments, the probe comprises SEQ ID NO:11. In some embodiments, the label is an ECL label. In some embodiments, the assay further comprises measuring the activity of the enzyme in the presence of an antibody.

[13] In some embodiments, the present invention provides an assay for identifying substrates for an enzyme which comprises contacting the substrate with the enzyme and determining whether the enzyme hydrolyzes the substrate by detecting the formation of any cleavage products after exposure to a cleaving agent. In some embodiments, the enzyme is an N-glycosidase. In some embodiments, the enzyme is ricin toxin A chain (RTA), ricin toxin, saporin, abrin II, or an RNase. In some embodiments, the substrate is an oligonucleotide in the form of a hairpin loop structure. In some embodiments, the oligonucleotide is conjugated to a ligand, a label or both. In some embodiments, the label is an ECL label. In some embodiments, the nucleotide sequence of the loop portion of the hairpin loop structure comprises or consists of GAGA, GAAA, AGAGAC, GUGA, GIGA, or UGCU, and at least one base is an RNA residue or a 2'-O-methyl RNA residue. In some embodiments, the nucleotide sequence of the loop portion of the hairpin loop structure comprises or consists of rGdArGrA, rGrArGrA, rGdArArA, rGdAdGrA, rArGdArGrArC, rGdUrGrA, rGdlrGrA, rUrGrCrU, mGdAmGmA, rGdArGmA, mGdArGmA, or rGdAmGmA, where d is a DNA residue, r is an RNA residue, and m is a 2'-O-methyl RNA residue. In some embodiments, the oligonucleotide sequence is selected from a group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. In some embodiments, the cleavage product is detected by hybridizing with a probe. In some embodiments, the probe is conjugated to a ligand, a label or both. In some embodiments, the probe comprises SEQ ID NO:11. In some embodiments, the label is an ECL label. In some embodiments, the assay further comprises measuring the activity of the enzyme in the presence of an antibody. [14] In some embodiments, the present invention provides an assay for screening for an agent or a condition which has an effect on the activity of an enzyme on a substrate which comprises conducting an assay as described herein in the presence and absence of the agent or condition and observing any difference between the cleavage products. In some embodiments, the enzyme is an N-glycosidase. In some embodiments, the enzyme is ricin toxin A chain (RTA), ricin toxin, saporin, abrin II, or an RNase. In some embodiments, the substrate is an oligonucleotide in the form of a hairpin loop structure. In some embodiments, the oligonucleotide is conjugated to a ligand, a label or both. In some embodiments, the label is an ECL label. In some embodiments, the nucleotide sequence of the loop portion of the hairpin loop structure comprises or consists of GAGA, GAAA, AGAGAC, GUGA, GIGA, or UGCU, and at least one base is an RNA residue or a 2'-O-methyl RNA residue. In some embodiments, the nucleotide sequence of the loop portion of the hairpin loop structure comprises or consists of rGdArGrA, rGrArGrA, rGdArArA, rGdAdGrA, rArGdArGrArC, rGdUrGrA, rGdlrGrA, rUrGrCrU, mGdAmGmA, rGdArGmA, mGdArGmA, or rGdAmGmA, where d is a DNA residue, r is an RNA residue, and m is a 2'-O-methyl RNA residue. In some embodiments, the oligonucleotide sequence is selected from a group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. In some embodiments, the cleavage product is detected by hybridizing with a probe. In some embodiments, the probe is conjugated to a ligand, a label or both. In some embodiments, the probe comprises SEQ ID NO:11. In some embodiments, the label is an ECL label. In some embodiments, the assay further comprises measuring the activity of the enzyme in the presence of an antibody.

[15] In some embodiments, the present invention provides a substrate for a N- glycosidase selected from the group consisting of an oligonucleotide in the form of a hairpin loop structure wherein the nucleotide sequence of the loop portion of the hairpin loop structure comprises or consists of GAAA, AGAGAC, GUGA, GIGA, or UGCU, and at least one base is an RNA residue or a 2'-O-methyl RNA residue; an oligonucleotide in the form of a hairpin loop structure wherein the nucleotide sequence of the loop portion of the hairpin loop structure comprises or consists of rGdArGrA, rGrArGrA, rGdArArA, rGdAdGrA, rArGdArGrArC, rGdUrGrA, rGdlrGrA, rUrGrCrU, mGdAmGmA, rGdArGmA, mGdArGmA, or rGdAmGmA, where d is a DNA residue, r is an RNA residue, and m is a 2'-O-methyl RNA residue; SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:8; and SEQ ID NO:9.

[16] In some embodiments the present invention provides, a probe for detecting the substrate as described herein. In some embodiments, the probe is Ru-ODN.

[17] Both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed. The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute part of this specification, illustrate several embodiments of the invention, and together with the description serve to explain the principles of the invention.

DESCRIPTION OF THE DRAWINGS

[18] This invention is further understood by reference to the drawings wherein:

[19] Figure IA schematically shows an ECL activity assay using RNA GdAGA

(SEQ ID NO:1) to produce an abasic site which is chemically cleaved to result in a 5' cleavage product which is annealed with a probe having an ECL label. Triangle (arrowhead on substrate) = biotin; cross-shaped dodecagon = streptavidin; Ru = ruthenium label (ECL label); Ru-ODN = ruthenylated oligodeoxynucleotide (probe); circle = paramagnetic support; Me₂ED = N,N'-dimethylethylenediamine; * = abasic site (cleaved by Me₂ED).

[20] Figure IB schematically shows the structure and sequence of the substrate,

RNA GdAGA (SEQ ID NO: 1), in Figure IA which is deadenylated by ricin A chain (RTA) at 2'-deoxyadenosine (dA) to create an abasic site (X) which is chemically cleaved by Me₂ED to give a 5' cleavage product having SEQ ID NO:2). The arrow indicates the depurination site for ricin. All bases of the oligonucleotide are RNA except dA which is a DNA base. Hydrogen bonding interactions are indicated by dots.

[21] Figure 2 is a graph showing ricin holotoxin concentration (in reaction volume of 10 μl) vs. ECL signal. Reaction conditions included 2.5 pmoles RNA GdAGA substrate per reaction, 200 ng MAb 9C3, 37 ⁰C, 3.1 hours, pH 4 (50 mM potassium phthalate buffer). Coefficients of variation were 4.2% for 0.6 ng/ml ricin (lowest concentration) and 4.6% for 30 ng/ml ricin. By linear regression analysis y = 114x + 3387 R² = 0.993 (error bar length = 2 standard deviations).

[22] Figure 3 A is a graph showing the effect of varying incubation time at 37 ⁰C and ricin concentration on signal-to-background ratios from the ricin ECL activity assay (3.5 pmol RNA GdAGA (0.35 μM)) The signal average for a given treatment (n = 4) was divided by the signal average for background samples lacking toxin (n = 4), with propagation of the standard deviations (SD). Error bar length equals 2 X SD. Equations obtained by linear regression analysis are shown in parenthesis as follows: A= O hour incubations (y = 0.022x + 1.003, R² = 0.754); ■ = 1 hour incubations (y = 1.275x + 1.021, R² = 0.998); 4 = 2 hours incubations (y = 3.139x + 1.176, R² = 1.000); 0 = 3 hours incubations (y = 4.366x + 2.068, R² = 0.998); Δ = 4 hours incubations (y = 5.549x + 2.205, R² = 0.999). Ricin concentration is given for the sample volume (5 μl).

[23] Figure 3B is a graph which provides the data of Figure 3 A rep lotted to show that, for a given ricin concentration, the rate of increase in signal-to-background ratio was constant over time up to about 4 hours.

[24] Figure 4 is a graph that compares the responses to varying ricin concentrations for two electrochemiluminescence-based immunoassays and the activity assay using the RNA GdAGA substrate (in 3.2-hour incubations). Calculations are described in Figure 3 A, except n = 6 for background samples. Equations in parentheses were obtained by linear regression analysis with y intercepts fixed at 1.0 as follows: ♦ = activity assay (y = 4.124x + 1.0, R² = 0.995); A = CRP ECL Minitube Immunoassay (y = 5.882x + 1.0, R² = 1.000); ■ = BioVerify™ Ricin Test (y = 10.736x + 1.0, R² = 0.998). MAb 9C3 was included in the activity assay reactions.

[25] Figures 5A-5C are graphs showing toxin differentiation by activity profiling with a set of RNA substrates as provided in Table 1. Reagents were added in 5-μl volumes to a total volume of 10 μl, and provided final concentrations of 0.35 μM oligo substrate with 100 ng MAb 9C3 in 40 mM sodium citrate buffer, pH 4.1, with 2 mM EDTA. Samples (5 μl) of toxins were diluted to the desired concentrations with 0.01% Triton X-100 prior to their addition to the reagents (5 μl).

[26] Figure 5A shows ricin (RT, 5 ng/ml; white bars) vs. Ricinus communis agglutinin (RCA 120, 25 ng/ml; gray bars). Error bar length = 2 X SD.

[27] Figure 5B shows ricin (5 ng/ml; white bars) vs. saporin (1 ng/ml; gray bars).

Error bar length = 2 X SD.

[28] Figure 5C shows ricin (5 ng/ml; white bars) vs. abrin II (26 ng/ml; gray bars).

Error bar length = 2 X SD.

[29] Figure 6 shows the effects of various substitutions of residues of oligo substrates for ricin, and interactions of the substrates with RNase A. The names and sequences of the substrates are described in Table 1. The substrates are stem- loop structures. Most are designed with a variation of the GAGA sequence in the loop in order to serve as a potential substrate for ricin. RNase v2 comprises RNA and DNA residues, with RNA residues in the loop structure that are susceptible to cleavage by RNases, including RNase A; the biotinylated 5 ' stem portion comprises DNA so that it will not be degraded by RNases, but unblocked for annealing to Ru-ODN (Table 1) when the remainder of the substrate is cleaved away. "Activity assay" procedures were as described herein, except that final reaction volumes were 30 μl (20 μl sample plus 10 μl reagents), the substrate concentrations therein were 0.12 μM, and the incubation temperature for the 3 -hour reaction was 43 ⁰C. The samples (20 μl) added to the reactions contained no enzyme (background), 5 ng/ml ricin (light gray bars; Vector Laboratories, Burlingame, CA), or 10 ng/ml RNase A (dark gray bars; Ambion Inc., Austin, TX). Quadruplicate samples gave signal averages with ricin or RNase A, which were divided by the signal average for the background for a given substrate to obtain the signal-to-background ratio. Signal-to-background ratios above 1 indicate transformation of substrate by the toxin/enzyme (to a product that can hybridize to a ruthenylated oligo to result in a signal), ratios near 1 indicate no transformation, and ratios well below 1 indicate undesirable substrate degradation. Error bar length = 2 X standard deviation.

[30] Figure 7 is a graph showing the signal-to-background ratio for UDG (0.16 ng) vs. Ricin (0.2 ng) with a set of substrates as provided in Table 1. Error bar length = 2 X SD, n = 4. Reaction conditions included 2.5 pmoles substrate per reaction (10 μl), 200 ng MAb 9C3, 37 ⁰C, 3.2 hours, pH 4 (50 mM potassium phthalate buffer). Dilutions of UDG were prepared with TE buffer (10 mM Tris, 1 mM EDTA) and reactions with UDG lacked MAb 9C3 and occurred in TE buffer.

[31] Figure 8 is a graph showing the optimum pH for sodium citrate buffer (50 mM) is about 4.1 in a ricin ECL activity assay using RNA GdAGA as the substrate. Reaction conditions included 3.5 pmoles RNA GdAGA substrate per reaction (10 μl), 100 ng MAb 9C3, 37 ⁰C, 3 hours (pH adjusted in substocks using concentrated sodium citrate buffer with KOH or HCl and pH was measured for scaled-up (mock) final reaction mixtures).

[32] Figure 9 graphically shows the optimum amount of RNA GdAGA is about 5 pmol in a ricin ECL activity assay under the following reaction conditions: 10 μl reaction vol.; 50 mM potassium phthalate, pH 4.0; 37 ⁰C; 3.2 hours; 20 pg ricin toxin; 100 ng MAb 9C3.

[33] Figure 10 shows ricin purification with magnetic beads. Error bars = 2 X SD; n = 4.

[34] Figure 11 shows the use of immunomagnetic (IM) prepurification to separate ricin from the representative interfering substance, RNase A. Treatment sets all contained various concentrations of ricin: A= no RNase A or IM -prepurification (y = 4.09x + 1.2, R² = 0.99); X = RNase A (1.25 ng/ml) included without IM- prepurification (y = 0.005x + 0.06, R² = 0.36); ♦ = no RNase A, with IM- prepurification (control; y = 1.98x + 1.4, R² = 0.996); ■ = RNase A (1.25 ng/ml) with IM-prepurification (y = 1.73x + 1.2, R² = 0.99). Error bar length = 1 X standard deviation.

[35] Figure 12 shows the effects of various substitutions of 2' O-methyl groups on

RNA residues of substrates for ricin, and interactions of the substrates with RNase A. The substrates are stem-loop structures. The names and sequences of the substrates are described in Table 1. Reactions contained no enzyme (background), 5 ng/ml ricin (light gray bars), or 10 ng/ml RNase A (dark gray bars). Experimental procedures are described in the legend to Figure 6. Error bar length = 2 X standard deviation.

[36] Figure 13 is a graph showing the effects of MAb 9C3 on the activities of various toxins in the ECL activity assay. Reactions included RNA GdAGA substrate and 100 ng MAb 9C3 (white bars) or no antibody (gray bars). Toxin concentrations were adjusted such that signals with MAb 9C3 were similar to those observed for ricin (5 ng/ml). With toxin reactions lasting about 3 hours, the sensitivity is about 1 ng/ml ricin in an aqueous sample. The total duration of the assay was about 4 hours. Concentrations: RCA 120 (25 ng/ml); saporin (1 ng/ml); abrin II (22 ng/ml).

DETAILED DESCRIPTION OF THE INVENTION

[37] The present invention provides methods, substrates and probes for assaying the hydro lytic N-glycosidase activity of enzymes. These hydrolysis reactions can occur abiotically at low pH and favor DNA residues over RNA. See Amukele & Schramm (2004) Biochemistry 43:4913-4922, which is herein incorporated by reference. These reactions could contribute to a high background signal. However, low concentrations of toxins that hydro lyze bases from RNA or DNA can be detected by the present invention. Indeed, closely related, adenine-specific N-glycosidases can be differentiated. As used herein, "N-glycosidase activity" means the enzyme- catalyzed hydrolysis of the bond between a base and a deoxyribose or ribose residue in a nucleotide residue of an oligonucleotide and includes RNA N-glycosidase and DNA N-glycosylase activities. As used herein, a "substrate" of the present invention comprises an oligonucleotide which is depurinated by a given N-glycosidase, such as ricin A chain (RTA). As used herein, a "probe" of the present invention is a moiety capable of specifically binding to a cleavage product of a substrate of the present invention.

[38] As used herein, a "protein" is used interchangeably with "polypeptide" and

"peptide" and refers to two or more amino acids linked together. As used herein, an "oligonucleotide" is used interchangeably with "oligo" and refers to a nucleic acid molecule which is generally about 10 nucleotides to about 100 nucleotides in length. As used herein, a "nucleic acid molecule" refers to a polymeric compound comprised of covalently linked subunits called nucleotides, such as adenosine, guanosine, uridine, cytidine, deoxyadenosine, deoxyguanosine, deoxythymidine, deoxycytidine, and the like. Nucleic acid molecules include polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which may be single-stranded or double- stranded. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are contemplated. DNA includes cDNA, genomic DNA, synthetic DNA, and semisynthetic DNA. The terms "nucleic acid molecule", "DNA" and "RNA" refer only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms unless explicitly indicated otherwise with respect to a specific nucleic acid molecule described herein. Nucleic acid molecules may be recombinantly or synthetically obtained using methods known in the art.

[39] Nucleic acid molecules can be composed of monomers that are naturally- occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g. α-enantiomeric forms of naturally-occurring nucleotides, including modified nucleotides), or a combination of both. Modified nucleotides can have alterations in sugar moieties and/or in pyrimidine or purine base moieties. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters. Moreover, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs. Examples of modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes, such as 5-propynyl-uracil, 2-thio-5-propynyl-uracil, 5-methylcytosine, pseudoisocytosine, 2-thiouracil and 2-thiothymine, 2-aminopurine, N9-(2-amino-6- chloropurine), N9-(2,6-diaminopurine), hypoxanthine, N9-(7-deaza-guanine), N9-(7- deaza-8-aza-guanine) and N8-(7-deaza-8-aza-adenine).

[40] Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like. The term "nucleic acid molecule" includes "peptide nucleic acids" known in the art. Peptide nucleic acids refers to naturally-occurring or modified nucleic acid bases attached to a polyamide backbone.

[41] An "isolated" nucleic acid molecule or polypeptide refers to a nucleic acid molecule or polypeptide that is in an environment that is different from its native environment in which the nucleic acid molecule or polypeptide naturally occurs. Isolated nucleic acid molecules or polypeptides includes those having nucleotides or amino acids flanking at least one end that is not native to the given nucleic acid molecule or polypeptide. For example, a native nucleic acid sequence Y has sequence X at its 5 ' end and sequence Z at its 3 ' end. When sequence A is inserted between sequence X and sequence Y, sequence Y (as well as sequence X) is considered to be "isolated".

[42] A nucleic acid molecule may be hybridized to itself or another nucleic acid molecule by annealing under the appropriate conditions of temperature and solution ionic strength. The conditions of temperature and ionic strength determine the "stringency" of the hybridization. Low stringency hybridization conditions correspond to a T_m of 55 ⁰C, e.g. 5X SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5X SSC, 0.5% SDS). Moderate stringency hybridization conditions correspond to a higher T_m, 40% formamide, with 5X or 6X SCC. High stringency hybridization conditions correspond to the highest T_m, e.g. 50% formamide, 5X or 6X SCC. Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T_m for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher T_m) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating T_m have been derived. See e.g. Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

[43] The present invention provides substrates which may be used to assay N- glycosidase activity. A substrate of the present invention comprises an oligonucleotide which is hydrolyzed by a given N-glycosidase, such as ricin A chain, at an abasic site. As used herein, an "abasic site" means a site in an oligonucleotide where a base (e.g. adenine, cytosine, guanine, thymine, hypoxanthine, or uracil) has been removed from a nucleotide residue, leaving the corresponding deoxyribose or ribose residue. An abasic site is created by N-glycosidase activity. In some embodiments, the oligonucleotide forms a hairpin loop and the abasic site is located in the loop portion. The abasic site may be chemically cleaved by a chemical, such as Λ/,N'-dimethylethylenediamine (Me₂ED), which specifically cleaves the depurinated nucleotide. See McHugh & Knowland (1995) Nucleic Acids Res. 23:1664-1670, which is herein incorporated by reference. In the methods of the present invention, Me₂ED was found to enhance signals in the ECL activity assays at a pH of about 7.5 to about 8.0, and that signal-to-background ratios were optimal at about 100 mM. Thus, in some embodiments, the concentration OfMe₂ED is about 100 mM, the pH of the solution containing Me₂ED is about 7.5 to about 8.0, or both.

[44] The oligonucleotides may be recombinantly or synthetically made using methods known in the art. Accordingly, the oligonucleotides may contain non- naturally occurring phosphoester analog bonds, such as thioester bonds, and the like. One or more nucleotides of an oligonucleotide of the present invention may be modified nucleotides. The nucleotides of an oligonucleotide may be deoxyribonucleic acid bases, ribonucleic acid bases, or a combination of both. In some embodiments, the abasic site of an oligonucleotide is a deoxyribonucleic acid base. In some embodiments, the abasic site of an oligonucleotide is a deoxyribonucleic acid base and the remainder of the bases in the oligonucleotide are ribonucleic acid bases or modifications thereof. [45] A substrate of the present invention may further comprise a ligand conjugated to the oligonucleotide which is capable of binding with its corresponding receptor. As used herein, "affixed", "attached", "associated", "conjugated", "connected", "immobilized", and "linked" are used interchangeably and encompass direct as well as indirect connection, attachment, linkage, or conjugation unless the context clearly dictates otherwise. As used herein, a "ligand" refers to a molecule that binds with another molecule, which is herein generically referred to as a "receptor", e.g. an antigen binding to an antibody, a hormone or neurotransmitter binding to a cellular receptor, or a substrate or allosteric effector binding to an enzyme and include natural and synthetic biomolecules, such as proteins, polypeptides, peptides, nucleic acid molecules, carbohydrates, sugars, lipids, lipoproteins, small molecules, natural and synthetic organic and inorganic materials, synthetic polymers, and the like. An example of a ligand and receptor pair which specifically bind to each other is biotin and an avidin molecule. As used herein, "avidin" is a meant to include any protein or polypeptide capable of high affinity binding to biotin. Streptavidin is one such example of avidin. As used herein, "specifically binds" refers to the character of a receptor which recognizes and interacts with a ligand but does not substantially recognize and interact with other molecules in a sample under given conditions. In some embodiments, the substrates of the present invention are conjugated to biotin.

[46] A substrate of the present invention may further comprise a label conjugated to the oligonucleotide. As used herein, a "label" refers to a moiety that can be attached to molecular entity such as an oligonucleotide which then renders the entity detectable by means and methods known in the art.

[47] In some embodiments, the substrate of the invention comprises an oligonucleotide having a quencher molecule conjugated to one end which quenches the detection of a label conjugated to its other end. In these embodiments, the label becomes detectable when the distance between the quencher molecule and the label is increased by cleaving the oligonucleotide. Quencher molecules and their corresponding labels are known in the art and contemplated herein. See e.g. Tyagi, S. and Kramer, F. R. (1996) Nat. Biotechnol. 14:303-308; Tyagi, S., et al. (1998) Nat. Biotechnol. 16:49-53; Kostrikis, L. G., et al. (1998) Science 279:1228-1229; Sokol, D. L., et al. (1998) PNAS 96:11538-11543; Knemeyer, J. P., et al. (2000) Anal. Chem. 72:3717-3724; and US 20040158051, which are herein incorporated by reference. [48] In some embodiments, the substrates are conjugated to a support. As used herein, a "support" may comprise a wide range of material such as biological material, nonbiological material, organic material, inorganic material, and the like, or a combination of any of these, including polymers, plastics, resins, silica or silica- based materials, carbon, metals, inorganic glasses and the like. In some embodiments, the support is a magnetic or paramagnetic material.

[49] The supports may be in any shape such as plates, beads, pellets, disks, fibers, or the like. In preferred embodiments, the support is in the form of a superparamagnetic bead ranging in size from about 1 μm to about 3 μm in diameter.

[50] The surface of the support can be etched using well known techniques to provide for desired surface features such as trenches, v-grooves, mesa structures, and the like. The surfaces on the support may comprise a material different from the support which may be affixed thereto by chemical or physical methods known in the art. In some embodiments, the surface of the support is coated with a receptor, such as avidin molecules.

[51] The present invention provides probes which may be used to assay N- glycosidase activity. A probe of the present invention is a moiety capable of specifically binding or hybridizing to a cleavage product of a substrate of the present invention. In some embodiments, the probe comprises or is a nucleic acid molecule which is complementary to the 5' cleavage product of a substrate. In some embodiments the probe comprises or is a nucleic acid molecule which is complementary to the 3' cleavage product. As used herein, a "5' cleavage product" of a given substrate is the nucleic acid molecule sequence upstream of the abasic site that remains after hydrolysis by an N-glycosidase and cleavage. Similarly, a "3' cleavage product" of a given substrate is the nucleic acid molecule sequence downstream of the abasic site that remains after hydrolysis by an N-glycosidase and cleavage. As used herein, "cleavage products" refers to 5' cleavage products, 3' cleavage products or both.

[52] In some embodiments, the probe is conjugated to a label. In some embodiments, the label is an ECL label. As used herein, an "ECL label" refers to a chemical substance that, when electrochemically oxidized or reduced under appropriate conditions, emits light. The term "ECL label" includes the substance itself, a chemical derivative that has been modified to allow attachment to substrate or other reagent, a chemical derivative that is attached to a substrate or other reagent, and the various products or intermediates formed from the label during electrochemiluminescent reactions. Various ECL labels known in the art are contemplated herein. See Knight et al.(1994) Analyst 119:879, US Patent Publication Nos. 20060035248, and the like, which are herein incorporated by reference. Some ECL labels emit electromagnetic radiation in the visible spectrum while others might emit other types of electromagnetic radiation, such as infrared or ultraviolet light, X- rays, microwaves, and the like. Use of the terms "electrochemiluminescence", "electrochemiluminescent", "electrochemiluminesce", "luminescence", "luminescent" and "luminesce" in connection with the present invention does not require that the emission be light, but admits of the emission being such other forms of electromagnetic radiation.

[53] Although ruthenium based ECL labels are exemplified herein, other ECL labels known in the art may be used in accordance with the present invention. In some embodiments, the substrate may be conjugated to a support, either with or without a ligand, prior to its interaction with the N-glycosidase, and the cleaved, single-stranded product of this reaction would specifically anneal to a probe conjugated to a label or (another) ligand. In addition, although biotin is conjugated to the substrates and the ECL labels are conjugated to the probes as exemplified in the ECL activity assays herein, in some embodiments, the ECL labels may be conjugated to the substrates and biotin may be conjugated to the probes. In some embodiments, the substrate, comprising a stem-and-loop hairpin structure, may not be conjugated to a ligand or a label, but instead may also contain a single-stranded oligo segment appended to a stem segment; this single-stranded segment may anneal specifically to one or more probe molecules, each of which may be conjugated to a label or a ligand (not both). In this embodiment, the enzymatic and chemical cleavage of the unconjugated substrate would unblock a stem segment immediately adjacent to the single-stranded segment such that this product would crosslink two oligos, one linked to a label and the other linked to a ligand or directly to a support.

[54] The present invention provides methods for assaying the N-glycosidase activity of protein of interest. Generally, such an assay involves mixing a sample containing an unknown quantity of the protein with a predetermined quantity of one or more substrates and determining the N-glycosidase activity by quantifying any cleavage products. The amount of the protein in the sample can be correlated to the N-glycosidase activity using methods described herein and known in the art. Thus, the present invention also provides methods of assaying the amount of a protein which exhibits N-glycosidase activity. The present invention also provides methods for characterizing or determining an unknown protein by its N-glycosidase activity profile.

[55] The present invention can also be used to assay conditions or factors, such as temperature, pH, enzyme inhibitors, denaturing compounds, enzyme activators, enzyme deactivators and the like, that may influence the N-glycosidase activity of a protein. In these embodiments, the N-glycosidase activity assayed under conditions or factors of interest is compared with a standard or a control using methods known in the art. In some embodiments, the influence of a candidate compound on N- glycosidase activity of a given protein on a substrate may be determined by assaying the N-glycosidase activity of the protein in the presence of the candidate compound and comparing with a standard or a control using methods known in the art. For example, the activity assays of present invention may be used to screen compounds and biomolecules, such as antibodies, for those which inhibit the activity of an N- glycosidase, such as ricin, and identify candidates for treating or inhibiting intoxication. Similarly, the methods of the present invention may be used to examine various sample matrices, e.g. blood and plasma constituents, that may have an effect on intoxication by a given toxin, such as ricin.

[56] The present invention provides assays for screening candidate substrates for a given N-glycosidase. Generally, such an assay involves mixing a sample containing a candidate substrate with the N-glycosidase and measuring the formation of any cleavage products.

[57] The activity assays of the present invention may be used to detect N- glycosidases in or on a sample such as food, air, water, liquids, biological fluids, clothing, equipment, and the like. The activity assays of the present invention may be used to determine the efficacy of a procedure for decontamination of an N- glycosidase, such as ricin. For example, after a substance or area exposed to ricin is cleaned or neutralized by a given procedure, one may determine the efficacy of the procedure by detecting any remaining N-glycosidase activity using the activity assay of the present invention.

[58] With modifications, detect specific endonucleases of various types. For example, in order to provide a substrate for a pyrimidine-specific RNase, the residues in the loop of a hairpin substrate could be pyrimidine RNA bases while the stem residues are DNA bases. As another example, in order to detect a particular restriction endonuclease that recognizes double-stranded DNA and cuts both strands, a hairpin DNA substrate would be comprised entirely of DNA, and the recognition site would be positioned near the loop but with several base pairs occurring between the recognition site and the loop as required by the endonuclease. In both cases, cleavage by the enzyme of interest would generate a biotinylated, single-stranded product capable of annealing to a probe.

[59] In some embodiments, the present invention provides reagents and kits for carrying out the methods of the invention. A kit in accordance with the present invention contain, packaged together, at least two of the following components: enzyme, substrate, supports, probes, buffers appropriate for carrying out the enzymatic reaction (e.g. mixtures of pH buffering substances, detergents, salts, metal ions, cofactors, proteins, sugars, excipients, and the like), solutions appropriate for carrying out a measurement, solutions appropriate for cleaning and/or conditioning a measuring device, calibration solutions containing known concentrations of a protein, calibration solutions containing a known concentration of an enzyme inhibitor, calibration solutions for calibrating the response of a measuring instrument, assay controls and standards. In some embodiments, the kits of the present invention further contain instructions for use.

[60] The N-glycosidase activity assays of the present invention are advantageous over prior art methods as they are compatible for use in ECL based detection assays, separation of unreacted substrate molecules is unnecessary, the detected signal (substrate cleavage) is directly proportional to the concentration of the N-glycosidase, its specificity may be modified (e.g. by using different anti-toxin capture antibody, a different monoclonal rate-enhancing antibody, or changing the structure/sequence of RNA/DNA biotinylated substrate) for given N-glycosidases in order to avoid false positive results, all steps of the activity assay may be conducted in one reaction container, allows simple sample prepurification (e.g. in a separate container) to remove contaminants and interfering agents, no radioisotopes or hazardous chemicals are required, and small substrate molecules allow defined site(s) of action for given N-glycosidases. ECL ACTIVITY ASSAYS

[61] As provided herein, the assays of the present invention may be adapted for electrochemiluminescence (ECL) detection. Thus, the present invention provides ECL assays for N-glycosidase activity. ECL assays involve signal amplification through the redox cycling of an ECL label. As provided herein, the ECL activity assays of the present invention comprise three simple steps without the need for separate reaction tubes or separation steps. The three steps comprise (1) combining the reagents including reaction buffer, substrate, and sample to be assayed in one reaction tube and incubating for a suitable time at a suitable temperature, e.g. 3 hours at 37 ⁰C, (2) adding detection reagents and stop solution and incubating for a suitable time at a suitable temperature, e.g. 15 minutes at 37 ⁰C, and (3) measuring the ECL signals.

[62] The premise of the ECL activity assays of the present invention is that N- glycosidase-dependent cleavage of a biotinylated, hairpin substrate leads to the unblocking of the 5' half of its stem, the 5' cleavage product, once the 3' cleavage product diffuses away, which allows an ECL labeled probe to anneal to the 5 ' cleavage product. Upon addition of the avidin-labeled supports, Dynabeads® M-270 Streptavidin (Invitrogen Corp., Carlsbad, CA), the biotinylated hybridization product noncovalently cross-links the ECL labeled probe to the supports.

[63] Figure IA schematically shows an embodiment of an ECL activity assay according to the present invention. All steps occurred in one reaction tube and included a toxin reaction at about 37 ⁰C (for about 3 hours), addition of stop/detection reagents and further incubation at about 37 ⁰C (for about 15 minutes), then quantification of ECL (about 1.5 hours per 96-well plate). In the first step of the Ricin ECL activity assay ricin holotoxin cleaves an adenine base from the RNA substrate at 37 ⁰C to produce an intermediate containing an abasic site. In the second step, the addition of stop/detection reagents brings the pH up to about 8.0 to stop the reaction, cleaves the sugar-phosphate backbone of the intermediate, and immobilized substrate molecules and cleavage products conjugated to ECL-labels onto the supports. The tubes are then incubated at 37 ⁰C for 15 minutes. In the third step, an M-SERIES® MlR Analyzer (BioVeris, Corp., Gaithersburg, MD) is used to process the samples and read the ECL signals which includes magnetically separating the paramagnetic supports from the solution and injecting a solution comprising tripropylamine (which serves as an electron source in cyclic, light-generating, redox reactions of the ECL-label). Because the reactions are electrochemically initiated at an electrode surface, only ECL-labels immobilized on the supports that are magnetically appressed to the electrode contribute to luminescence. Repeated light emission by each ECL-label provide signal amplification and confer high sensitivity.

[64] As shown in Figure IB, a substrate having biotin conjugated to one end of its oligonucleotide is depurinated by an N-glycosidase and then chemically cleaved at its abasic site indicated by the arrow. After the cleavage product which does not have biotin conjugated thereto (in this case the 3 ' cleavage product) is separated or diffuses away from the cleavage product conjugated to biotin (the 5' cleavage product), a probe comprising an ECL label is hybridized to the 5 ' cleavage product. The resulting biotin conjugated complexes are bound to avidin coated magnetic supports which are magnetically appressed to an electrode where the ECL labels undergo repeated, cyclic reactions, resulting in detectable electrochemiluminescence. Thus, the deposition of ECL labels onto magnetic supports and their subsequent detection is dependent on and indicative of the N-glycosidase activity of ricin.

[65] Test samples of various concentrations of ricin holotoxin were assayed as schematically shown in Figures IA and IB. Reaction conditions included 2.5 pmoles RNA GdAGA substrate per reaction, 200 ng MAb 9C3, 37 ⁰C, 3.1 hours, pH 4 (50 mM potassium phthalate buffer). Figure 2 is a graph showing that the ECL signal is directly proportional to the concentration of ricin.

[66] As provided herein, the N-glycosidase activities of Ricinus communis agglutinin (RCA 120), saporin, and abrin II were assayed. In the experiments disclosed herein, ricin holotoxin (RCA II, RCA₆₀) and RCA 120 (RCA I, RCAi₂₀) were obtained from Vector Laboratories (Burlingame, CA). Saporin (Advanced Targeting Systems, San Diego, CA) and uracil DNA glycosylase (UDG; uracil N- glycosidase; Epicentre, Madison, WI) were also obtained commercially. Abrin II, which was purified as described previously, was provided by Dr. Eric Garber (Food and Drug Administration, College Park, MD). See Hegde et al. (1991) Anal. Biochem. 194: 101-109, which is herein incorporated by reference. It is noted that those skilled in the art may assay the activities of other N-glycosidases in accordance with the present invention.

[67] For the ECL activity assays disclosed herein, unless otherwise indicated, reagents were obtained from Sigma-Aldrich (St. Louis, MO). Table 1 shows the substrates used. Table 1 Oligonucleotides used in this study

Name Sequence ^a' ^b

RNA 5' -/BiOtJnZrArGrCrGrGrGrArGrArGdArGrArLJrCrLJrCrCrC -3' GdAGA^c (SEQ ID N0:l)

5 ' -/B±ot±nITArGrCrGrGrGrArGrArGrArGrArUrCrUrCrCrC -3'

RNA GAGA (SEQ ID NO: 1)

5 ' -/B±ot±n/TArGrCrGrGrGrArGrArGdArArArUrCrUrCrCrC -3'

RNA GdAAA (SEQ ID N0:3)

RNA 5' -/B±ot±n/TATGrCrGrGrGrArGrArGdAdGrArUrCrUrCrCrC -3'

GdAdGA (SEQ ID NO:1)

RNA 5 ' -/B±ot±n /TArGrCrGrGrGrArGrArGdArGrArCrCrUrCrCrCrG -3 '

AGdAGAC ( SEQ I D NO : 4 )

5 ' -/BiOtJnZrArGrCrGrGrGrArGrArGdUrGrArLJrCrLJrCrCrC -3 '

RNA GdUGA ( SEQ ID N0 : 5 )

5 ' -/B±ot±n/ TArGrCrGrGrGrArGrArGdIrGrArUrCrUrCrCrC -3 '

RNA GdIGA ( SEQ ID NO : 6 )

DNA 5 ' -/Biotin/dGdAdGdCdGdGdGdAdGdAdGdGdAdGdAdCdLJdCrdCdCdC -

GAGA(CU) 3 ' ( SEQ ID NO : 7 )

CNA GdAGA 5 ' -/B±ot±n/ UGdAdGdCdGdGdGrArGrArGrGdArGrAdCTdCTdCdCdC -

_V1 3 ' ( SEQ ID N0 : 8 )

CNA GdAGA 5 ' - /B±ot±n/ dGdAdGdCdGdGdGrArGrArGrGdArGrAdCTdCTdCdCdCdG

_V2 -3 ' ( SEQ I D N0 : 9 )

This substrate is similar to CNA GdAGA v2 substrate except that the 2' hydoxyl moieties of the RNA residues mDNA are methylated to form 2'-0-methyl RNA residues, which mGdAmGmA should render the mDNA substrate resistant to various RNases .

This substrate is similar to mDNA mGdAmGmA substrate mDNA above, except that the first and third loop positions rGdArGmA are RNA residues rather than 2'-0-methyl RNA residues. This substrate is similar to mDNA mGdAmGmA substrate mDNA above, except that the third loop position is an RNA mGdArGmA residue rather than a 2'-0-methyl RNA residue. This substrate is similar to mDNA mGdAmGmA substrate mDNA above, except that the first loop position is an RNA rGdAmGmA residue rather than a 2'-0-methyl RNA residue.

This substrate is similar to mDNA mGdAmGmA substrate mDNA above, except that the first, third, and fourth loop rGdArGrA positions are RNA residues rather than 2'-0-methyl RNA residues .

/BiOtJnZdGdAdGdCdGdGdGdAdGdArGrLJrUrGrCrUrArCrLJrCrLJrCrCrC RNase v2 -3' (SEQ ID NO:10)

Designed to be a substrate for RNase A, RNase Tl, and

RNase Vl (Ambion, Inc., Austin, TX). RNA product 5' -/Biotin/rArGrCrGrGrGrArGrArG (SEQ ID NO: 11)

5' -/Ruthenium/TTTTTdAdCdCTdCTdC (T) dCdGdCTdC (SEQ ID

Ru-ODN NO : 12 ) d = 2-deoxyribosyl moiety (DNA residues; T = dT); r = ribosyl moiety (RNA residue); m = 2-0-methylribosyl moiety (2'-0-methyl RNA residues) ^b Oligonucleotide segments: bold, hairpin loops; italics, stem segments; underline, segments that hybridize to Ru-ODN. The T in parentheses in the Ru-ODN sequence participates in a G-T mismatch (upon annealing to a product oligo) that disfavors the annealing of Ru-ODN to unreacted substrate molecules, wherein the hairpin configuration is maintained without mismatches. ^c The names of substrates that begin with "CNA" or "RNA" include the loop sequence denoted by capital letters; those capital letters not preceded by lower case letters in the name are understood to represent RNA residues. (CNA is an abbreviation for chimeric nucleic acid.) Likewise, for the substrate with the "DNA" prefix, capital letters in the name are DNA residues.

[68] Substrate oligonucleotides were obtained from Integrated DNA Technologies with purification by RNase-free high performance liquid chromatography (HPLC) (Coralville, IA). Mouse monoclonal antibody 9C3 (MAb 9C3, ATCC Accession Number PTA-6106 (American Type Culture Collection, Manassas, VA)) was provided by Dr. Mark Dertzbaugh (U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD). See Dertzbaugh et al. (2005) Hybridoma (Larchmt) 24:236-243, which is herein incorporated by reference. The antibody was affinity purified using an UltraLink® Immobilized Protein A/G column according to the manufacturer's instructions (Pierce Biotechnology, Inc., Rockford, IL). The eluate was dialyzed against distilled water, then frozen and lyophilized. The purified protein residue was weighed, then dissolved in nuclease-free water (Ambion, Inc., Austin, TX) to a concentration of 35 μg/ml. Aliquots (1 ml) in 1.5-ml microcentrifuge tubes were vortexed 5 seconds after additions of diethylpyrocarbonate (0.3 μl of DEPC per tube; Sigma- Aldrich, St. Louis, MO); afterward, tubes were rotated at 20 rpm at room temperature for 1 hour, then stored at 4 ⁰C.

[69] Reactions were performed in quadruplicate samples in 0.2-ml PCR tubes in eight-tube strips (USA Scientific Inc., Ocala, FL). Total reaction volumes were 10 μl, with 5 μl of Reagent Solution plus 5 μl of sample in diluent (nuclease-free water (Ambion, Inc., Austin, TX) with 0.01% v/v Triton® X-IOO (Triton)). Reagent Solution for 100 reactions, freshly combined before use, included: 200 μl of 200 mM sodium citrate with 10 mM EDTA (pH 4.1 after dilution to the final reaction concentrations of 40 mM Na citrate, 2 mM EDTA), 293 μl of 35 μg/ml MAb 9C3, and 7 μl of 50 pmol/μl substrate in TE buffer (10 mM Tris, ImM EDTA, pH 8). Tubes were incubated in a thermocycler at a constant 37 ⁰C with a lid temperature of 105 ⁰C.

[70] Reactions were typically stopped by adding 240 μl of combined Stop Solution and Detection Solution to each tube after a 3 -hour incubation at 37 ⁰C. Stop Solution (about pH 8) for 100 tubes consisted of reagents added in the following order: 20.2 ml TE buffer containing 1.09 M NaCl, 1.54 ml 2 N HCl, 260 μl N,N'- dimethylethylenediamine, and 48 μl of Triton X-IOO, filtered through a 0.2-μm-pore- size filter. Detection Solution for 100 tubes consisted of: 1.94 ml TE buffer with 1.09 M NaCl, 55 μl of 10 mg/ml Dynabeads® M-270 Streptavidin (Invitrogen Corp., Carlsbad, CA), and 10.5 μl of 50 pmol/μl Ru-ODN in TE buffer (Biosource Intl., Camarillo, CA; ruthenium in BV-TAG™ label (BioVeris, Corp., Gaithersburg, MD). For the data shown in Figure 3, reactions were stopped by adding 220 μl of Stop Solution to each tube; stopped tubes were recapped, then mixed by repeated inversions and kept at room temperature. After all reactions were stopped, detection reagents were added (20 μl per tube), followed by mixing. Tubes were then incubated 15 minutes at 37 ⁰C (lid temperature = 45 ⁰C). For data in other figures and tables, the reactions were stopped by adding 240 μl of combined Stop/Detection Solution. [71] In order to maintain a one-tube-per-test format, quadruplicate tubes were cut from the eight-tube strips, uncapped, and transferred to a modified Costar® round- bottom, polypropylene, 96-well microtiter plate (Corning Inc., Corning, NY; product no. 3365). The modification involved drilling a 3.5-mm diameter hole at the bottom (center) of each well such that the PCR tubes could be inserted as four-tube strips held upright across four wells. The plate was then analyzed on an M-SERIES® MlR instrument (BioVeris, Corp., Gaithersburg, MD) using the following parameters: plate type, standard round l; plate layout, standard BioVeris; volume in well, 250 μl; volume sampled, 200 μl; bead type, 2.8; clean type, level 2; bead wash, level 5; detection sequence, standard.

RiciN ECL ACTIVITY ASSAY

[72] As provided herein, the present invention provides methods for assaying the

N-glycosidase activity of proteins, such as ricin. Prior art assay methods include immunoassays, ECL immunoassays, and activity assays without ECL detection. Prior art ECL immunoassays include Bio Verify™ Ricin Test ("Bio Verify immunoassay", BioVeris, Corp., Gaithersburg, MD) and Ricin Toxin ECL Minitube Immunoassay (CRP) ("CRP immunoassay" Critical Reagents Program, Aberdeen Proving Ground, MD). The kits were used according to manufacturer instructions, and ECL measurements were made on the an M-SERIES® MlR instrument (BioVeris, Corp., Gaithersburg, MD) using the recommended parameters. M-SERIES® BV- DILUENT™ solution (BioVeris, Corp., Gaithersburg, MD) was used in place of the "ECL Buffer" in the CRP protocol. None of the prior art methods assay the N- glycosidase activity of ricin using ECL detection. Thus, the ricin ECL activity assays provided herein, may be used as a complement to ricin ECL immunoassays as a confirmatory assay or as a substitute to ricin ECL immunoassays.

[73] As provided in Figure 3 A, the ricin ECL activity assay of the present invention exhibited sensitivity towards ricin which is comparable to prior art ECL immunoassays. Reactions included 3.5 pmoles RNA GdAGA substrate per tube (0.35 μM). Figure 3 A shows that, for various toxin incubation times of 1 to 4 hours, the activity-dependent ECL response was proportional to the toxin concentration.

[74] Moreover, data in Figure 3A can be replotted as provided in Figure 3B, to show that, for a given ricin concentration, the rate of increase in signal-to-background ratio was constant over time up to about 4 hours. Background signals typically ranged from about 2,000 to about 4,000 ECL units in different experiments. In one experiment, factors that contributed to an average background signal of 2,600 ± 100 included baseline signal (4 ± 0% of signal; no beads or Ru-ODN, n = 4), interaction of Ru-ODN with beads (supports) in the absence of RNA GdAGA substrate (3 ± 0% of signal), said interaction in the presence of unreacted substrate or perhaps a trace contaminant similar to RNA product (i.e. '5 cleavage product) (70 ± 4%; see Table 1 footnotes), and abiotic hydrolysis that occurred at pH 4.1 at 37 ⁰C over 3 hours (23 ± 5%; TE buffer, pH 8.0, provided the control for no hydrolysis). Since dA bases undergo abiotic hydrolysis (deadenylation) much faster than A bases at pH 4.0, in some embodiments, deadenylation occurs at a pH of about 4.0. Longer incubations (6 hours; not shown) did not improve the sensitivity due to a higher background signal, which was attributed primarily to abiotic hydrolysis of RNA GdAGA.

[75] Figure 4 is a graph that shows that a 3.2-hour incubation provided activity data complementary to two prior art ECL immunoassays. Specifically, in the range of 0.1 to 10 ng/ml ricin, the ricin ECL activity assay and the prior art ECL immunoassays exhibited signal-to-background ratios that were comparable and directly (linearly) proportional to the ricin concentration. Table 2 shows that two methods of determining the limit of detection (LOD) gave similar values for each assay; thus, signal-to-background ratios (Figure 4) greater than 1.2 represented positive signals. Table 2 Limits of Detection and Coefficients of Variation*

Ricin Coefficients of

Limits of Detection⁸ (0.1 ng/ml) Variation

Bkg avg Bkg avg Signal avg 0.1 ng/ml 10 ng/ml

Assay + (3 X SD) X 1.2 - SD ricin ricin

Activity (inc. MAb 9C3)^ϋ 3653 3664 3915 8% 8%

Activity (no MAb 9C3) 2952 3082 3043 6% 8%

CRP Immunoassay 138 140 173 7% 6%

BioVerify 274 275 403 9% 17% Immunoassay

^* from data used to calculate signal-to-background ratios in Figure 4. ^aAverage ECL signal values for background samples ("Bkg avg"; n=6) were used to calculate limits of detection by two simple formulas given in the subheadings (SD, standard deviation). ^b Activity assay reactions were performed with and without Mab 9C3 (100 ng per reaction) as described above. Figure 4 does not include activity data without the MAb.

[76] For the ECL activity assay including MAb 9C3 and the two prior art ECL immunoassays, 0.1 ng/ml ricin gave signals above the LODs. See Table 2. Only the BioVerify immunoassay gave signals significantly above the LOD when samples with 0.05 ng/ml ricin were tested (not shown). This result is attributed to the fact that the BioVerify immunoassay uses 100 μl of sample per test, whereas the CRP immunoassay uses 50 μl. Coefficients of variation in Table 2 indicate that individual tests performed with all of the assays were highly reproducible. Thus, Figure 3 A and Table 2 shows that 3 -hour incubations were sufficient to detect, using the ECL activity assay of the present invention, ricin concentrations as low as those detectable with the prior art ECL immunoassays (Table 2).

[77] Data points for the three assays beyond 10 ng/ml of ricin (up to 1,000 ng/ml) are not shown in Figure 4, but asymptotically approached maximum signals. Because nonspecific cleavage of adenine residues near the 5' (biotinylated) end of the substrate RNA GdAGA would yield a product incapable of cross-linking biotin to Ru-ODN, it may be inferred from the asymptotic response that non-target adenine residues were not significantly cleaved by ricin. If the opposite were true, false negative signals would be possible with high concentrations of ricin in test samples. The high apparent specificity of ricin toward the dA target residue is consistent with previous findings. As shown in Figures 5A-5C, these rate differences may account for the undetectable activity with ricin and RNA GAGA.

SUBSTRATES

[78] Previous research showed that RNA substrates for ricin A chain containing a

GdAGA loop exhibited large rate enhancements relative to substrates with a GAGA loop. See Amukele & Schramm (2004) Biochemistry 43:4913-4922; and Orita et al. (1996) Nucleic Acids Res. 24:611-618, which are herein incorporated by reference. Therefore, RNA GdAGA, a biotinylated RNA substrate, which has a 2'- deoxyadenosine (dA) residue within the four-residue loop, GdAGA, in a hairpin structure was designed and used. See Table 1 and Figure IB.

[79] To define the site of action of ricin, the dA residue was replaced with adenosine (GAGA loop), 2'-deoxyinosine (GdIGA loop), or 2'-deoxyuridine (GdUGA loop; Table 1); no activity was detected when these variations were exposed to ricin (5 ng/ml in 5-μl sample), saporin (1 ng/ml), abrin II (26 ng/ml), or RCA 120 (25 ng/ml). Thus, dA is preferred in the second position of the loop of the hairpin substrates for these adenine-specific depurinating enzymes. The results are shown in Figure 6. In Figure 6, the bar height at signal-to-background ratio of 1 indicates the substrate is not modified by the enzyme (ricin, light gray bars; RNase A, dark gray bars). Height well below 1 indicates substrate is degraded (by RNase A); this is apparent for RNA GdAGA. Height above 1 indicates the oligonucleotide is a substrate for the given enzyme. Thus, RNA GdAGA and CNA GdAGA v2 are suitable substrates for ricin activity assays in accordance with the present invention.

[80] To further verify that enzymatic hydrolysis at the second loop residue would cause signal generation, three RNA substrates (RNA GdAGA, RNA GAGA, and RNA GdUGA) and the DNA substrate (DNA GAGA(CU) as provided in Table 1) were exposed to uracil DNA Glycosylase (UDG, Epicentre, Madison, WI) in TE buffer, which cleaves uracil from DNA; only RNA GdUGA and the DNA substrate yielded signals above background as provided in Figure 7. As shown in Figure 7, the uracil at the dU residue in RNA GdUGA was cleaved by UDG. Observations of ECL signals with the dU-containing DNA substrate suggest that the U residues in the stems of the RNA substrates (GAGA, GdAGA) were also available to UDG but not cleaved by it, so the 5' halves of their stems remained blocked to Ru-ODN. Thus, base cleavage at sites other than the second loop residue can lead to signal generation.

[81] In some embodiments, the substrates of the present invention can be used to define the substrate specificity and site(s) of action of a protein such as a toxin and to enable the rapid differentiation of adenine-specific N-glycosidases. For example, replacing the second G of the GAGA loop (third loop position) with riboinosine has been shown to dramatically reduce the cleavage rate by ricin, thereby suggesting that the amino group of that G is important. Thus, in some embodiments, a substrate having an A residue in the third position of the GAGA loop will present a GdAAA loop, which also lacks the amino group as presented on the third residue of the GAGA loop. In some embodiments, the second G residue may be replaced with dG to give the substrate, RNA GdAdGA. In some embodiments, the loop structures of the substrates contain more than four residues, e.g. RNA AGdAGAC, a hexaloop hairpin structure. See Gluck et al. (1992) J. MoI. Biol. 226:411-424, which is herein incorporated by reference.

[82] In some embodiments, other substrates, such as DNA GAGA(CU) instead of

RNA GdAGA, may be used to avoid or reduce potential RNase interference in the ricin ECL activity assays. Even though the DNA GAGA(CU) substrate may be less affected by RNases, the lower limit of detection for the DNA substrate is about two orders of magnitude higher than that of the RNA GdAGA substrate (data not shown). Similarly, the DNA substrate "dA12" was reported to have a much lower turnover number (£_cat) for ricin A chain than an RNA substrate with a GdAGA loop. Nevertheless, those skilled in the art may readily select the substrates and assay conditions that are optimum sensitivities and limits of detection for a given Ricin ECL activity assay.

[83] For example, nuclease-resistant substrates suitable for the N-glycosidase ECL activity assays of the present invention include the substrates CNA GdAGA vl, CNA GdAGA v2, mDNA mGdArGmA, mDNA rGdArGmA, and mDNA rGdArGrA, as provided in Table 1 ; these substrates were transformed to signal-yielding products by ricin but were resistant to RNase A. In contrast, the oligos mDNA mGdAmGmA and mDNA rGdAmGmA were not significantly transformed by ricin or RNase A. For each substrate tested, the effect of 200 pg of RNase A (20 μl of 10 ng/ml), was evaluated and compared with appropriate controls (a blank solution with no RNase A added). The results are shown in Figure 6 and Figure 12. [84] As shown in Figure 6, RNA GdAGA was highly susceptible to RNase A,

CNA GdAGA v2 was completely resistant to RNase A and gave a higher signal-to- background ratio with 5 ng/ml of ricin, mDNA mGdAmGmA was resistant to RNase A, but acted as a poor substrate for ricin, and RNase v2 was not a substrate for ricin, but was an excellent substrate for RNase A. mDNA rGdArGmA and mDNA rGdArGrA yielded very high signals with ricin and no apparent degradation by RNase A. The background averages were similar and acceptable for RNA GdAGA substrate and CNA GdAGA substrate (ca. 8400), and was a little better for the RNase v2 substrate (ca. 6000). Thus, the present invention provides nuclease-resistant substrates suitable for use in the ECL activity assays of the present invention which includes RNA GdAGA, CNA GdAGA VZ, mDNA mGdArGmA, mDNA rGdArGmA, and mDNA rGdArGrA. Those skilled in the art may readily determine the RNA bases of a substrate that are required by incrementally replacing the RNA residues of a the substrate with 2' O-methyl RNA residues and testing the modified substrate in accordance with the present invention.

[85] In some embodiments, the background signal in the ECL activity assays may be lowered by purifying the substrates using methods known in the art, such as RNase-free HPLC purification methods, and the like. In some embodiments, variants of the substrates may be pretreated with Me₂ED and then the signal-generating impurities may be separated out by affinity chromatography. The variants have poly- T segments at their 3 ' ends (about 20 residues in length) which anneal to a poly-dA oligonucleotide immobilized on a solid support in the presence of 50 mM NaCl at room temperature. Rinsing this support with 100 mM Me₂ED in TE buffer (pH 8) with 50 mM NaCl will cleave at any (abiotically-generated) abasic sites and wash out any biotinylated oligonucleotides with structures similar to RNA product (that contribute to background signals). Finally, elution with water without salt will cause melting of the intact, poly-T containing substrate from the poly-dA support, thereby providing a purified substrate.

SCREENING FOR FACTORS WHICH MODULATE N-GLYCOSIDASE ACTIVITY [86] The ECL activity assays of the present invention may be used to identify or determine what factors have an effect on N-glycosidase activity and to identify means of mitigating the effects of the factors. Such factors include solvent effects, e.g. pH, salinity, reagent degradation, e.g. by nucleases, direct interactions with the toxin, e.g. dithiothreitol, and the like. For example, Figure 8 shows that the ricin-catalyzed deadenylation of RNA GdAGA was optimal at pH 4.1 in sodium citrate buffer, which is consistent with previous work. See Chen et al. (1998) Biochemistry 37:11605- 11613, which is herein incorporated by reference. This observation is reflected in Table 3, which shows that about 75% of ricin activity remained when 10 mM acid (HCl) or base (NaOH) was present in the unbuffered 5-μl sample.

Table 3

Residual activity of ricin with RNA GdAGA substrate in the presence of various chemical inhibitors

Residual ricin activity at selected agent concentrations (mM) '

Agent 200 ^b 100 20 10 4

NaCI 7% ± 1^C 30% ± 2 90% ± 6 94% ± 10 n.d.^d

KCI 5% ± 1 25% ± 2 79% ± 8 90% ± 7 n.d.

NaOH n.d. n.d. 41% ± 2 77% ± 7 99% ± 4

HCI n.d. n.d. 38% ± 5 74% ± 8 93% ± 10

DTT^e n.d. n.d. 74% ± 7 81 % ± 6 92% ± 7

MgCI₂ n.d. n.d. n.d. 65% ± 9 91% ± 7

Ricin incubations at 37 ⁰C (3 hours) and assay procedures are described above. ^b Concentrations of agents are given for a 5-μl sample volume. ^c Values (± propagated SD) were determined by subtracting the background signal average from the signal averages for ricin (5 ng/ml) with the agent (n=4), then dividing these differences by the difference for ricin only (no agent or DEPC); values are expressed as a percentage (residual activity). ^d not determined. ^e dithiothreitol.

[87] Less inhibition was observed when representative salts were added (10 mM

KCl or NaCl). Still, clinical samples containing 0.9% saline (154 mM NaCl; e.g. nasal swabs) may require desalting or dilution. Dithiothreitol, which can reductively cleave the disulfide bond linking the A and B chains of ricin, moderately inhibited ricin activity. See Table 3. In contrast to the need to reductively separate the chains of ricin for optimal activity on ribosomes, reduction is not needed when artificial substrates are transformed by ricin and related type II RIPs. See Barbieri et al. (1997) Nucleic Acids Res. 25:518-522, which is herein incorporated by reference. Thus, the present invention is advantageous over prior art methods in that the substrates are stable to long-term storage and the ricin ECL activity assay does not require reductive activation of the toxin. Figure 9 shows that optimum concentration of RNA GdAGA is about 5 pmol in under the following reaction conditions: 10 μl reaction vol.; 50 mM K phthalate, pH 4.0; 37 ⁰C; 3.2 hours; 20 pg ricin toxin. Higher concentrations of substrate lead to lower signals because unreacted (biotinylated) substrate can compete with RNA product for binding to the magnetic Streptavidin M- 270 Dynabeads®.

[88] DNases reportedly contaminate many preparations of RIPs. See Day et al.

(1998) Eur. J. Biochem. 258:540-545, which is herein incorporated by reference. However, as shown in Table 4, DNase I had no effect on the ricin ECL activity assay.

Table 4 Residual activity of ricin with RNA GdAGA substrate in the presence of nucleases

Residual activity at selected nuclease concentrations

(P9/μl)^a

Agent 5000 ^b 50 10 2 0.4

DNase I 101 % ± 8^C n.d.^d n. d. n. .d. n.d.

DNase I + 4 mM MgCI₂ 98% ± 5 n.d. n. d. n. .d. n.d.

RNase A n.d. -4% ± 0 -4% + 0 3% ± 1 87% ± 7

RNase A + DEPC ^e n.d. -4% ± 0 -4% ± 0 12% ± 1 60% ± 5 ^a Ricin incubations at 37 ⁰C (3 hours) and assay procedures are described above. Ricin was present at 5 ng/ml. ^b Concentrations of agents are given for a 5-μl sample volume. ^c Values were determined as described in the Table 3 footnotes. Negative percentages reflect substrate degradation. ^d not determined ^e Mixtures of ricin and RNase A in 1-ml aliquots were treated with diethylpyrocarbonate

(DEPC) as described for MAb 9C3 in above.

[89] The chelating agent EDTA was included in the toxin reaction (2 mM) and also in the stop/detection reagents (0.7 mM) to avoid degradation of oligonucleotides due to divalent cations (e.g., Mg²⁺; Table 3) or to magnesium-dependent DNase activity. See Wiame et al. (2000) Biotechniques 29:52-256, which is herein incorporated by reference. Although a reaction buffer pH of 4.1 should be suboptimal for most nucleases, RNase A caused degradation of the substrate, RNA GdAGA. Although ricin A chain has been reported to be unaffected by diethylpyrocarbonate (DEPC), inhibition of ricin (not shown) that limited the usable DEPC concentration such that treatment with this chemical rendered only a slight mitigation of the interference by RNase A. See Table 4. [90] Therefore, the activity assays of the present invention may be optimized accordingly. For example, the N-glycosidase activity of ricin occurs in aqueous buffer at a pH of about 4.0 to about 4.1 and the N-glycosidase activity of ricin may be enhanced by the addition of the anti-ricin antibody, MAb 9C3. Thus, in some embodiments, about 40 mM sodium citrate buffer, at pH 4.1, and anti-ricin antibodies are used for ricin activity assays. Those skilled in the art may readily select other buffers such as potassium citrate, potassium phthalate, and sodium/potassium acetate and antibodies for desired N-glycosidase and optimizations. All other reagents used in the assay may be similarly optimized.

SAMPLE PREPROCESSING

[91] Since RNases are known to be present in castor bean extracts, and perhaps also in illicit weapons made therefrom, the following experiment was conducted to determine whether a polyclonal anti-ricin antibody linked to supports might be useful in removing ricin from potential interferences, such as RNase, in a sample. See Winchcombe & Bewley (1992) Phytochemistry 31 :2591-2597, which is herein incorporated by reference. As provided herein, the supports ("beads") were Dynabead® M270 Streptavidin (Invitrogen) beads with polyclonal goat anti-ricin antibody. See PoIi et al. (1994) Toxicon 32(11): 1371-1377, which is herein incorporated by reference.

[92] Figure 10 shows the results of a ricin ECL activity assay wherein the ricin was immunomagnetically (IM) purified with beads prior to assaying. The reaction conditions were: RNA GdAGA substrate (2.5 pmoles) and 200 ng MAb 9C3 in 10 μl final volume; 1.5 hours, 37 ⁰C; phthalate buffer at pH 4.0 (treatments 1, 2) or at 4.5 (treatments 3, 4). A pH of 4.5 was used so that its combination with glycine buffer at pH 3.1 would yield pH 4.0. Tubes containing 1 ng ricin in 1 ml were combined with 100 μg beads in 8 μl additions (treatments 2-4 only); tubes were inverted repeatedly at room temperature to mix. Beads were pulled to tube sides by a magnet and supernatants were discarded. To these beads, 10-μl aliquots of 12.5 mM glycine buffer (pH 3.1) were added for treatments 3 and 4, whereas 10 μl water was added for treatment 2. Beads were suspended briefly and beads were again drawn to the magnet. Five μl aliquots were transferred to fresh reaction tubes. These aliquots were combined with 5 μl aliquots of reagents including RNA GdAGA (2.5 pmoles) with phthalate buffer at pH 4.0 (or 4.5 for treatments 3, 4). [93] As shown in Figure 10, Treatment 3 is above background (#4), and is equivalent to about 20 pg RT. Treatment 2 shows the same signal as background and suggests that water may be used for further rinsing, if necessary. Treatments 1 and 3 show that this approach can be used to concentrate ricin. Note that 5 pg ricin was delivered by adding 5 μl of 1 ng/ml (1 pg/μl); therefore, treatment 1 shows the signal from testing ricin at the original concentration (1 ng/ml) prior to concentrating the ricin on the beads, releasing it with a small volume of glycine buffer, and testing that eluate (treatment 3). Thus, the assays of the present invention may be effectively combined with concentrating and purifying the proteins of interest.

[94] The N-glycosidase activities of (1) ricin without RNase, (2) ricin with RNase

A (Ambion, Inc., Austin, TX), (3) ricin without RNase + purification with beads (4) ricin with RNase A + purification with beads, and (5) positive and negative controls were assayed in accordance with the ricin ECL activity assays disclosed herein. Specifically, four sets of one -ml samples contained various concentrations of ricin with and without RNase A prepared in diluent of 0.01% Triton X-100 in nuclease-free water. Treatment sets all contained various concentrations of ricin. Each set had one tube for each ricin concentration. Magnetic bead suspension (20 μl of 10 mg/ml) was added to tubes of two sets. Dynabead® M270 Streptavidin beads from Invitrogen were bound to biotinylated polyclonal goat anti-ricin antibody as described in PoIi et al. (1994) Toxicon 32:1371-1377, which is herein incorporated by reference. Tubes were rotated at 20 rpm for 1 hour at room temperature. The steps for rinsing and elution of beads were: pull beads to magnet and discard supernatants; add 1.5 ml diluent (above) per tube, resuspend beads, rotate 5 minutes; pull beads to magnet and discard supernatants; add 100 μl of 10 mM glycine buffer, pH 3.2, with 0.01% Triton X-100 per tube as the eluent, vortex gently; draw beads to magnet and transfer supernatants with eluted proteins to new tubes. For each of these transferred supernatants, and for each of the aforementioned tubes not exposed to beads, quadruplicate activity assays were performed as described in Figure 6. Note: The glycine buffer in the eluate did not significantly affect the pH in the final reaction mixture due to the overriding effects of the citrate buffer.

[95] As shown in Figure 11 , immunomagnetic (IM) prepurification effectively removed RNase A from the ricin samples, given that the lines through the diamond and square symbols are essentially the same. Without IM prepurification, there is no signal in the presence of RNase A (X symbols) which degrades the RNA GdAGA substrate.

[96] The N-glycosidase activities of ricin samples without RNase A and without purification provided activity ratios which were consistent with prior experiments. The N-glycosidase activities of ricin samples with RNase A and without purification provided ratios that are less than one as the RNase reduced ECL signals to less than the background, thereby demonstrating that RNase interferes with ECL activity assays. The N-glycosidase activities of ricin samples without RNase A and with purification provided ratios that were lower than the ratios where beads are not present. However, the ricin could have been eluted from the beads using a much smaller volume (e.g., 25 μl) than the 100 μl required for quadruplicate testing, which would deliver a more concentrated sample of ricin (20 μl) to a 10 μl aliquot of Reagent Solution. Thus, the purification step could increase the sensitivity of the ECL activity assays. The N-glycosidase activities of ricin samples with RNase A with purification provided ratios that were slightly lower than those not containing any RNase, but the difference is not significant, thereby indicating that purification prevents the RNase from interfering with the ECL activity assay.

[97] These results indicate that purification of a toxin or an N-glycosidase prior to conducting the ECL activity assays of the present invention reduces the interference of RNase. Therefore, in some embodiments, a sample to be assayed is purified using methods known in the art prior to assaying in order to prevent or inhibit the interference by contaminants or other constituents in the sample.

N-GLYCOSIDASE ACTIVITY PROFILING

[98] The ECL activity assays of the present invention may be used to distinguish one N-glycosidase from another. For example, although saporin cleaves RNA GdAGA efficiently, Figure 12 shows that slight modifications of the tetraloop sequence leads to differing cleavage efficiencies for ricin and saporin. Thus, one may select a substrate for a desired N-glycosidase which provides a cleavage efficiency that is distinguishable from that of other N-glycosidases. For example, RNA GdAAA would preferred over RNA GdAGA for distinguishing between ricin and saporin.

[99] Further a set of substrates may be used to provide an activity profile of an N- glycosidase which may be used to identify or characterize proteins exhibiting N- glycosidase activity. For example, Figures 5A-5C also show the activity profiles for ricin (was tested as a standard), RCA 120, saporin, and abrin II for a set of substrates. The concentrations of RCA 120, saporin, and abrin II were adjusted so that the signals with RNA GdAGA substrate were comparable to that obtained with 5 ng/ml ricin. As shown in Figures 5A-5C, these four substrates: RNA GdAGA; RNA GAGA; RNA GdAAA; and RNA AGdAGAC, yielded a reproducible pattern with ricin, i.e. ricin activity profile. Figure 5 A shows that the activity profiles for ricin and RCA 120 are very similar, which might be expected given that the A chains of the two toxins share 93% homology. See Chen et al. (2005) J. Agric. Food Chem. 53:2358-2361, which is herein incorporated by reference. Figure 5B and Figure 5C show the activity profiles for saporin and abrin II, which are similar to each other, but different from the activity profiles of ricin and RCA 120.

[100] Thus, in some embodiments, the present invention provides activity profiles obtained by ECL activity assays which may be used to identify or characterize unknown proteins which exhibit N-glycosidase activity. Once an activity profile for a given protein is determined for a desired set of different substrates, the activity profile may be used to characterize or identify an unknown. For example, the N-glycosidase activity of ricin is determined for the four substrates as provided in Figure 5 A. Then the activity profile of the unknown sample of interest is determined for the same substrates under similar conditions. If the activity profile of the unknown sample is similar to that of ricin as provided in Figure 5 A, the unknown is characterized as ricin, RCA 120 or similar to ricin or RCA 120. Further testing may be conducted to definitively identify the sample. However, one could determine with a high degree of confidence that the unknown sample is not saporin or abrin II based on the initial activity profiling. Therefore, the present invention may be used as a preliminary assay to determine whether additional expensive and timely assays are necessary.

[101] As provided herein, the addition of an antibody, MAb 9C3, against N- glycosidases assayed in accordance with the present invention enhanced the N- glycosidase activities toward substrates. For example, as shown in Figure 13, MAb 9C3 was found to enhance the signal-to-background ratios of various toxins, ricin (5 ng/ml); RCA 120 (25 ng/ml); saporin (1 ng/ml); abrin II (22 ng/ml), in an ECL activity assay according to the present invention. Reactions included RNA GdAGA as the substrate, Ru-ODN as the probe (Biosource Intl., Camarillo, CA; ruthenium in BV-TAG™ label from BioVeris, Corp. (Gaithersburg, MD), and 100 ng MAb 9C3 (white bars) or no antibody (gray bars). Toxin concentrations were adjusted such that signals with MAb 9C3 were similar to those observed for ricin (5 ng/ml). The addition of MAb 9C3 enhanced signals by about 20 to about 25% for ricin and saporin, but caused more dramatic enhancements with RCA 120 and abrin II. It is hypothesized that antibodies may act as a specific, non-inhibitory solubilizing agent for the N-glycosidases, which thereby increases the signal in the ECL activity assays of the present invention. Therefore, in some embodiments, the ECL activity assays of the present invention further include using antibodies against a desired N-glycosidase. [102] When the activities measured for an unknown with and without antibody is determined, they may be combined with the activity profiles for a set of substrates, such as in Figures 5A-5C, in order to fully differentiated and characterize the unknown. Therefore, the present invention provides methods for identifying unknown toxins and N-glycosidases in a sample by determining the activity profiles, determining any change in activity by the addition of an antibody in an ECL activity assay and comparing the results with a control or standard.

OTHER ACTIVITY ASSAYS

[103] Although assays for the activities of a few proteins and their substrates are exemplified herein, other ribosome-inactivating proteins may likewise be assayed using the substrates of the present invention or those known in the art such as those provided in Table 5.

04] To the extent necessary to understand or complete the disclosure of the present invention, all publications, patents, and patent applications mentioned herein are expressly incorporated by reference therein to the same extent as though each were individually so incorporated. Having thus described exemplary embodiments of the present invention, it should be noted by those skilled in the art that the within disclosures are exemplary only and that various other alternatives, adaptations, and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments as illustrated herein, but is only limited by the following claims.

Claims

We claim:

1. An assay for an enzyme which comprises hydrolyzing a site on at least one substrate by contacting the substrate with the enzyme; cleaving the substrate at the site hydrolyzed by the enzyme with a cleaving agent to give a cleavage product; and quantifying the amount of the cleavage product.

2. The assay of claim 1, wherein the enzyme is an N-glycosidase.

3. The assay of claim 1, wherein the enzyme is ricin toxin A chain (RTA), ricin toxin, saporin, abrin II, or an RNase.

4. The assay of claim 1, wherein the substrate is an oligonucleotide in the form of a hairpin loop structure.

5. The assay of claim 4, wherein the oligonucleotide is conjugated to a ligand, a label or both.

6. The assay of claim 5, wherein the label is an ECL label.

7. The assay of claim 4, wherein the nucleotide sequence of the loop portion of the hairpin loop structure comprises GAGA, GAAA, AGAGAC, GUGA, GIGA, or UGCU, and at least one base is an RNA residue or a 2'-O-methyl RNA residue.

8. The assay of claim 7, wherein the nucleotide sequence of the loop portion of the hairpin loop structure comprises rGdArGrA, rGrArGrA, rGdArArA, rGdAdGrA, rArGdArGrArC, rGdUrGrA, rGdlrGrA, rUrGrCrU, mGdAmGmA, rGdArGmA, mGdArGmA, or rGdAmGmA, where d is a DNA residue, r is an RNA residue, and m is a 2'-O-methyl RNA residue.

9. The assay of claim 4, wherein the oligonucleotide sequence is selected from a group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9.

10. The assay of claim 1, wherein the cleavage product is detected by hybridizing with a probe.

11. The assay of claim 10, wherein the probe is conjugated to a ligand, a label or both.

12. The assay of claim 10, wherein the probe comprises SEQ ID NO:11.

13. The assay of claim 11, wherein the label is an ECL label.

14. The assay of claim 1, which further comprises measuring the activity of the enzyme in the presence of an antibody.

15. An assay for measuring the activity or amount of an enzyme which comprises correlating the amount of the cleavage product quantified according to claim 1 with a standard or control.

16. An assay for characterizing or identifying an unknown enzyme which comprises correlating the amount of the cleavage product quantified according to claim 1 with at least one activity profile for a known enzyme.

17. An assay for identifying substrates for an enzyme which comprises contacting the substrate with the enzyme and determining whether the enzyme hydro lyzes the substrate by detecting the formation of any cleavage products after exposure to a cleaving agent.

18. An assay for screening for an agent or a condition which has an effect on the activity of an enzyme on a substrate which comprises conducting the assay of claim 1 in the presence and absence of the agent or condition and observing any difference between the cleavage products.

19. A substrate for a N-glycosidase selected from the group consisting of an oligonucleotide in the form of a hairpin loop structure wherein the nucleotide sequence of the loop portion of the hairpin loop structure comprises GAAA, AGAGAC, GUGA, GIGA, or UGCU, and at least one base is an RNA residue or a 2'-O-methyl RNA residue; an oligonucleotide in the form of a hairpin loop structure wherein the nucleotide sequence of the loop portion of the hairpin loop structure comprises rGdArGrA, rGrArGrA, rGdArArA, rGdAdGrA, rArGdArGrArC, rGdUrGrA, rGdlrGrA, rUrGrCrU, mGdAmGmA, rGdArGmA, mGdArGmA, or rGdAmGmA, where d is a DNA residue, r is an RNA residue, and m is a 2'-O-methyl RNA residue;

SEQ ID NO: 1;

SEQ ID NO:3;

SEQ ID NO:4;

SEQ ID NO:5;

SEQ ID NO:6;

SEQ ID NO:7;

SEQ ID NO:8; and

SEQ ID NO:9.

20. A probe for detecting the substrate of claim 19.

21. The probe of claim 16, wherein the probe is Ru-ODN.