# Patent

## Title
Aptamers for prion diagnostics and aptamer binding detection system

## Abstract

There is disclosed PrPSc aptamers. There is further disclosed PrPSc aptamers. There is further disclosed an infectious agent or neurodegenerative disease bifunctional aptamer comprising a first sequence component, and a second sequence component, wherein the first sequence component is a complement binding sequence component selected from the group consisting of SEQ ID NOs 1-89 and 92-96, each having a 5′ end and a 3′ end, wherein the second sequence component binds to a specific infectious agent, and wherein the second sequence component sequence is inserted into the first sequence component from 1 to 5 bases from the 5′ end.

## Background
CROSS REFERENCE TO RELATED APPLICATION
This patent application claims priority to U.S. Provisional Patent application 61/668,023 filed 4 Jul. 2012.


TECHNICAL FIELD
The present disclosure provides PrPSc aptamers. In particular, the disclosure provides protease resistant and nuclease resistant PrPSc aptamers. The PrPSc aptamers are used in diagnostic tests for determining the presence of PrPSc in CNS tissue or even from live animal blood samples. The present disclosure further provides an infectious agent bifunctional aptamer comprising a first sequence component, and a second sequence component. The present disclosure further provides a method for determining the presence of infectious prion PrPSc using an infectious agent bifunctional aptamer comprising a first sequence component, and a second sequence component. The present disclosure further provides a component for detecting the presence of an agent configured in a bifunctional aptamer comprising a first sequence component, and a second sequence component.
BACKGROUND
Transmissible spongiform encephalopathies (TSEs) are a heterogeneous group of fatal neurodegenerative disorders that occur in humans, ruminant herbivores, mink, and cats. Sheep scrapie is the prototype of this group. TSEs are characterized by deposition of prion proteins (also denoted as PrP-Scrapie or PrPSc, the infectious form of the proteins), in the central nervous system of affected individuals. Prions have been defined as small proteinaceous infectious particles which resist inactivation by procedures that modify nucleic acids. The term “prion” is a contraction of the words “protein” and “infection,” and prions are comprised largely if not exclusively of PrP-Sc molecules encoded by a PrP gene. Prion diseases are often called spongiform encephalopathies because of the post mortem microscopic or histopathologic appearance of the brain of an infected animal with large vacuoles in the cortex and cerebellum. Prion proteins are insoluble, protease-resistant glycoproteins resulting from post translational modification of normal mammalian glycoproteins (PrP-Cellular or PrP-C). Deposition of the prion protein, an abnormal isoform of a native cellular sialoglycoprotein, in the central nervous system is a reliable marker of TSE infection.
The most widely studied TSEs in food-producing animals include scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cattle (also known as “Mad Cow” disease), and chronic wasting disease (CWD) in mule deer and elk. Other TSEs in animals include transmissible mink encephalopathy (TME) in mink and feline spongiform encephalopathy (FSE) in cats. Prion diseases of humans have also been identified, which include: Creutzfeldt-Jakob Disease (CJD); Gerstmann-Straussler-Scheinker Syndrome (GSS); Fatal Familial Insomnia (FFI); Alper's Syndrome, and Kuru.
The transmissible agent in these diseases remains controversial. It appears that the scrapie isoform of the prion protein (PrP-Sc or PrPSc) is necessary for both the transmission and pathogenesis of the transmissible neurodegenerative diseases of animals and humans (see Prusiner, Science, 252, 1515-1522, 1991).
A new clinical version of CJD in humans is believed to be the result of transfer of BSE from cattle to humans. This finding led to the conclusion that variant CJD was caused by infection of humans with prions from BSE infected cattle. Furthermore, the incidence and timing of the appearance of variant CJD cases opened the possibility that a considerable number of humans, presently free of clinical symptoms, could be latent for the disease. Given that CJD might pass from human to human in infected blood, it can be assumed that humans infected with variant CJD contain the infectious agent in their blood. A potentially infectious species specific agent has been discovered in blood of humans with CJD, cattle with BSE, and sheep with scrapie. ELISA tests have been developed that detect these TSE specific proteins that are associated with PrPSc (“Prion associated proteins”) in the blood of animals and humans. Prion associated proteins are expressed in a disease specific manner in all subjects with clinical symptoms of BSE (“BSAS”), scrapie (“SCRAPAS”) and CJD (“CJD”). Prion associated proteins appear to have the chemical characteristics for binding to PrP-C and converting it to PrPSc, and thus are most likely the infectious agents in TSE diseases. Furthermore, expression of prion associated proteins in a subject is accompanied by expression of specific anti-prion associated protein endogenous antibody. Detecting this endogenous antibody in human blood and blood products is the basis of some TSE ELISA tests (U.S. Pat. No. 6,350,854).
The occurrence of novel transmissible spongiform encephalopathies in cattle in the United Kingdom and Europe, and in mule deer and elk in parts of the United States has emphasized the need for reliable diagnostic tests. Further, the epizootic of a TSE in cattle and its postulated relationship to a new variant of human Creutzfeldt Jakob Disease have increased public and scientific awareness of these relatively rare disorders, and have highlighted the need for preclinical detection of TSEs. Accordingly, sensitive immunohistochemical techniques and preclinical detection methods are necessary for the detection, surveillance, and control of TSEs.
Confirmation of TSEs is accomplished by postmortem microscopic or histological examination of brain tissue of suspected cases. Postmortem histopathologic diagnosis of the ruminant TSEs is based on the appearance of neuronal vacuolation, spongiform changes, gliosis, and astrocytosis. However, these can vary in intensity and anatomic location depending on the host species, the individuals, host genetics, stage of disease, and infectious source. Thus, diagnosis by histopathology alone may be equivocal in early cases and usually not possible in autolyzed tissue.
Monoclonal antibody 263K 3F4 (U.S. Pat. No. 4,806,627) detects PrPSc in hamsters and humans, and has received use in diagnostic assays and pathogenesis studies of human TSEs. Ante-mortem testing in humans with suspected CJD is performed by immunohistochemical and histologic examination of brain biopsies. Because brain biopsy in ruminant animals is not feasible, an alternative approach has been to biopsy selected lymph nodes.
Therefore, there exists a need for a practical, inexpensive, and more rapid method for detection of prion proteins, prion-associated proteins and peptides and/or their respective host antibodies in live animals or humans. In addition, there exists a need for sensitive diagnostic assays to detect prion and prion associated proteins and peptides and/or their respective host antibodies in animal tissues and animal by-products in a most rapid fashion to minimize quarantine time.
Aptamers are functional synthetic nucleic acids useful for high-affinity binding to targets (e.g., nucleic acids, proteins, and chemical compounds). Unlike naturally occurring nucleic acids, which transfer genetic information, aptamers are selected on the basis of their ability to specifically bind their ligand. The specificity of binding is defined in terms of the dissociation constant Kd of the aptamer for its ligand. Aptamers can have high affinity with Kd range similar to antibody (pM to nM) and specificity similar/superior to antibody (Tuerk and Gold, Science, 249:505, 1990; Ellington and Szostak, Nature, 346:818, 1990).
Many aptamers have a stem-loop structure in which the bases in the loop and the stem are intimately involved in interaction with the ligand. RNA aptamers have been isolated against the protease-sensitive, N-terminus of PrP (Weiss et al., J. Virol. 71:8790-8797, 1997) but these do not discriminate between PrPC and PrPSc and are sensitive to nucleases. Therefore, there is a need in the art to design and utilize aptamers for binding to specifically folded prions, specifically those prions that are infectious and disease-causing in animals/mammals, in order to prevent the transmission and spread of such diseases in the food supply. The present disclosure provides improved aptamers for detecting the presence of PrPSc where the aptamers are not sensitive to nucleases.
SUMMARY
The present disclosure provides PrPSc aptamers. In particular, the disclosure provides protease- and nuclease-resistant PrPSc aptamers. The PrPSc aptamers are used in diagnostic tests for determining the presence of PrPSc in CNS tissue or even from live animal blood samples. The present disclosure further provides an infectious agent bifunctional aptamer comprising a first sequence component, and a second sequence component, wherein the first sequence component is a complement binding sequence component selected from the group consisting of SEQ ID NOs 1-89 and 92-96, each having a 5′ end and a 3′ end, wherein the second sequence component binds to a specific infectious agent, and wherein the second sequence component sequence is inserted into the first sequence component from 1 to 5 bases from the 5′ end. The present disclosure provides a neurodegenerative disease bifunctional aptamer comprising a first sequence component, and a second sequence component, wherein the first sequence component is a complement binding sequence component selected from the group consisting of SEQ ID NOs 1-89 and 92-96, each having a 5′ end and a 3′ end, wherein the second sequence component binds to a specific infectious agent, and wherein the second sequence component sequence is inserted into the first sequence component from 1 to 5 bases from the 5′ end. The present disclosure provides a method for determining the presence of infectious prion PrPSc using an infectious agent bifunctional aptamer having comprising a first sequence component, and a second sequence component, wherein the first sequence component is a complement binding sequence component selected from the group consisting of SEQ ID NOs 1-89 and 92-96, each having a 5′ end and a 3′ end, wherein the second sequence component binds to a specific infectious agent, and wherein the second sequence component sequence is inserted into the first sequence component from 1 to 5 bases from the 5′ end. The present disclosure further provides a component for detecting the presence of an agent configured in a bifunctional aptamer comprising a first sequence component, and a second sequence component, wherein the first sequence component is a complement binding sequence component selected from the group consisting of SEQ ID NOs 1-89 and 92-96, each having a 5′ end and a 3′ end, wherein the second sequence component binds to an agent, and wherein the second sequence component sequence is inserted into the first sequence component from 1 to 5 bases from the 5′ end.
The present disclosure further provides an infectious agent bifunctional aptamer comprising a first sequence component, and a second sequence component, wherein the first sequence component is a complement binding sequence component selected from the group consisting of SEQ ID NOs 1-89 and 92-96, each having a 5′ end and a 3′ end, wherein the second sequence component binds to a specific infectious agent, and wherein the second sequence component sequence is inserted into the first sequence component from 1 to 5 bases from the 5′ end. Preferably, the infectious agent is selected from the group consisting of PrPC, PrPSc, Transmissible spongiform encephalopathies (TSEs), Creutzfeldt-Jacob disease (CJD), variant CJD (vCJD), bovine spongiform encepathy (BSE) and scrapie. Preferably, the infectious agent is PrPSc and the sequence of the second sequence component is SEQ ID NO. 90.
The present disclosure provides a neurodegenerative disease bifunctional aptamer comprising a first sequence component, and a second sequence component, wherein the first sequence component is a complement binding sequence component selected from the group consisting of SEQ ID NOs 1-89 and 92-96, each having a 5′ end and a 3′ end, wherein the second sequence component binds to a specific infectious agent, and wherein the second sequence component sequence is inserted into the first sequence component from 1 to 5 bases from the 5′ end. Preferably, the first sequence component is selected from the group consisting of SEQ ID NOs 1-10, 70 and 74. Preferably, the neurodegenerative disease is selected from the group consisting of Alzheimer's Disease, mild cognitive impairment, Parkinson's disease, and other neurodegenerative diseases. Preferably, the neurodegenerative disease is Alzheimer's Disease and the sequence of the second sequence component is SEQ ID NO. 91.
The present disclosure provides a method for determining the presence of infectious prion PrPSc using an infectious agent bifunctional aptamer having comprising a first sequence component, and a second sequence component, wherein the first sequence component is a complement binding sequence component selected from the group consisting of SEQ ID NOs 1-89 and 92-96, each having a 5′ end and a 3′ end, wherein the second sequence component binds to a specific infectious agent, and wherein the second sequence component sequence is inserted into the first sequence component from 1 to 5 bases from the 5′ end. The process for testing for PrPSc comprises:
(a) obtaining a reagent mix comprising microspheres, fibrinogen, prothrombin, Factor Va, and Factor X, and buffer;
(b) incubating separately for at least 5-20 minutes at about 10° C. to about 37° C. a sample for testing, a bifunctional aptamer and RVV-X activator;
(c) adding the reagent mix; and
(d) determining the presence of the infectious agent by observing clotting.
Preferably, the step of determining the presence of the infectious agent is determined by blood coagulation or by a spectrophotometer reading of an optical density (OD) at approximately 405 nm. Preferably, the reagent mix further comprises from about 500 nM to about 700 nM phospholipid vesicles (for example, a mixture of phosphatidylserine:phosphatidylcholine). Preferably, the amount of fibrinogen in the reagent mix is from about 150 nM fibrinogen to about 300 nM. Preferably, the amount of prothrombin in the reagent mix is from about 150 nM to about 300 nM. Preferably, the reagent mix further comprises polymer microspheres to aid in the visual determination of clotting. Most preferably, the polymer microspheres are made from polystyrene. Preferably, the method is conducted in a multiwell plate. Preferably, the buffer in the reaction mix is selected from the group consisting of phosphate buffer, PBS (phosphate buffered saline), IC buffer (imidazole-HCl, CaCl2), Heparin BSA buffer (Tris-HCl, NaCl, EDTA, PEG600, BSA), HEPES, TAE, isocitrate and combinations thereof.
The present disclosure further provides a component for detecting the presence of an agent configured in a bifunctional allosteric aptamer comprising a first sequence component, and a second sequence component, wherein the first sequence component is a complement binding sequence component selected from the group consisting of SEQ ID NOs 1-89 and 92-96, each having a 5′ end and a 3′ end, wherein the second sequence component binds to an agent, and wherein the second sequence component sequence is inserted into the first sequence component from 1 to 5 bases from the 5′ end.



DESCRIPTION OF THE FIGURES
FIG. 1 shows a structural sequence of the first 55 bases to the complement cascade binding region (RVV-X Binding Region) of a preferred aptamer (SEQ ID NO. 74). This is also referred to as the first sequence component. This first sequence component of the preferred aptamer comprises two stems, a bulge, and two loop regions. The first stem region comprises bases 9-10 corresponding to bases 51-52. The second stem region comprises bases 17-29 and bases 38-48 and having a bulge (bases 22-25 and bases 42-43) contained therein. The first loop extends from bases 10-17 and 48-51 and the second loop is bases 29-38.
FIG. 2 shows a structural sequence of the 45 bases of the first sequence component of a preferred aptamer (SEQ ID NO. 97) comprising one stem, one bulge, and one loop region. The stem region comprises bases 7-19 and bases 28-38, having a bulge (bases 12-15 and bases 32-33) contained therein. The loop extends from bases 19-28.
FIG. 3 shows the prion binding region (PrPSc Binding Region) having 60 RNA bases of a preferred aptamer disclosed herein. This is also referred to as the second sequence component. This region of the preferred aptamer (SEQ ID NO. 90) comprises two loops and two short stems. The first loop is from bases 5-19 and the second loop is from bases 47-54. There is one stem from bases 3-5 corresponding to bases 19-21 and a second stem from bases 43-47 corresponding to bases 54-58.
FIG. 4 shows a second preferred embodiment fully formed bifunctional aptamer (SEQ ID NO. 98) having both a prion binding region (second sequence component) and a complement cascade binding region (first sequence component) and having five main loops, two bulges, and five stem regions.
FIG. 5 shows a third preferred embodiment fully formed aptamer (SEQ ID NO. 103) having both a prion binding region (second sequence component) and a complement cascade binding region (first sequence component) and having three main loops, two bulges, and three stem regions.
FIG. 6 shows a microwell plate with pictures and time points of reaction progression as described in Example 1 herein.
FIG. 7 shows the kinetics of the cascade described in Example 1. Percent transmittance through the in vitro cascade reaction mix was plotted as a function of time for several concentrations of RVV-X activator. Varying amounts of RVV-X (10 fmol, 1 fmol, 100 amol) were added to 100 μL of reaction mix and absorbance was monitored over time. The data was then transformed to % transmittance and plotted vs. t1/2, which were calculated: 12.6 min (10f), 21.2 min (1f), 36.3 min (100a).
FIG. 8 shows a Kaleidagraph illustration of a Kd(app) calculation for the aptamer SEQ ID NO. 103 to RVV-X; the gel shift is in FIG. 9B. Data were obtained from densitometry scanning using a BioRad FX Scanner and quantified with Quantity One software. Normalized data were plotted in Kaleidagraph and values for Kd (m1) and the Hill coefficient (m2) were calculated: Kd (m1 in legend)=63.86 nM, and Hill coefficient (m2)=1.0 (no cooperativity).
FIGS. 9A-C show examples of EMSA for monoclonal RNA aptamer sequences binding to RVV-X. RNA species used for gel shift are SEQ ID NOS. 98, 103, and 90 for FIGS. 9A, 9B, and 9C, respectively. Lanes 1-12 include 0.25 fmol end-labeled RNA with varying concentrations of RVV-X or BSA negative control protein. From left to right [RVV-X] (+RNA): 0, 8.66 nM to 4.44 μM (11 concentrations total), BSA (6 pmol/9 microL) (+RNA). For FIGS. 9A and 9B, lane 13 contains 2.22 μM RVV-X only. Note in FIG. 9C, lane 13 includes carry-over from lane 12.



DETAILED DESCRIPTION
The present disclosure provides a bifunctional RNA aptamer-based competitive homogeneous detection system having a readout by the naked eye. This became possible by coupling an RNA aptamer with a biochemical signal amplification cascade, BCC-MS. The signal amplification cascade resulted in the formation of the precipitate of polystyrene microspheres bound to clotted fibrin. In a preferred embodiment, the aptamer contains a domain that binds to RVV-X, thus inhibiting BCC. There is another domain on the aptamer which binds to an effector molecule, reversing the effect of the first domain. The latter domain of the aptamer is the only variable part of the detection system. Therefore, adjusting the detection system to a new effector molecule will involve only one molecular component, an aptamer to the effector molecule. The disclosed bifunctional aptamer provides features that can insert a detection system into a platform for on-site testing. Therefore, the presently disclosed bifunctional aptamer provides a commercial advantage of being field-deployable/on-site, relatively rapid to minimize the quarantine time for tested animals, and relatively inexpensive because no sophisticated laboratory equipment is necessary and the bifunctional aptamers can be manufactured by standard commercial nucleic acid synthesis techniques.
In one embodiment, a disclosed RNA aptamer (e.g., SEQ ID NOs. 1-89) selectively binds an activated or inactived form of the protease RVV-X. Preferably, the dissociation constant ranges from about 10 pM to about 100 nM. More preferably, the dissociation constant ranges from about 100 pM to about 100 nM, and can optionally comprise any value within the range, e.g. about 800 pM, about 900 pM, about 1 nM, about 2 nM, or about 5 nM.







TABLE 1





RNA Sequence Listing for first sequence component to RVV-X















RNA sequence of first component (SEQ ID NOs 1-89)








UAGCGACAAGGCGACAAGCAAUGACACAUUAACAGACCCUGGUUAGUGAACGAA
SEQ ID NO. 1


 


AAGGCGAGGUGCGACCCGCACGUGGCAUCUGAUAGCACAUGAAAAGGCACGUCA
SEQ ID NO. 2


 


UCUUCCAACGACCAUGCGGCGACAAGCGACUACAAGAGGGUACCCACGGACAGCA
SEQ ID NO. 3


 


UGGCGACGAGGCGGCAGGCAAUGACACAUUAGCAGACCCUGGUUAAUGAGCGAA
SEQ ID NO. 4


 


UAGCGACAAGGCGACAAGCAAUGACACAUUAACGGACCCUGGUUAAUGAACGAA
SEQ ID NO. 5


 


UGGCGGAUACUCUGCGAAGGGCGAACCCAACAUUUCGCACAGGACCGACUACUGCA
SEQ ID NO. 6


 


UGGCGGAUACUCUGCGAAGGGCGAACCCAACAUUUCGCACAGAACCGACUACUGCA
SEQ ID NO. 7


 


AAGCGACAAGGCGACAAGCAAUGACACAUUAACAGACCCUGGUUAAUGGACGAC
SEQ ID NO. 8


 


AGGCGACAAGGCGACAAGCAAUGUCACAUUAACAGACCCUGGUUAAUGAACGAA
SEQ ID NO. 9


 


UAGCGACAAGGCGACAGGCAAUGGUACAUUAACAGACCCUGGUUAAUGAACGAA
SEQ ID NO. 10


 


UAGCGACAAGGCGACGAGCAACGACACAUUAACAGACCCCGGUUAAUGAACGAA
SEQ ID NO. 11


 


UAGCGAGAAGGCGACAAGCAACGACACAUUAACAGACCCUGGUUAAUGGACGAA
SEQ ID NO. 12


 


UAGCGCCGAGGCGACAGGCGACGACACAUUAACAGACCCUGGUUAAUGAGUGAA
SEQ ID NO. 13


 


UAGCGGACACUCUGCGAAGGGCGAACCCAACAUUUCGCACGGAACCGACUACUACA
SEQ ID NO. 14


 


UCCGAUCUUCAUACCCGCGACCGGCGACAUUGUGACCGCAAAACCGGACAACCCC
SEQ ID NO. 15


 


UGGCGAUACUCUGCGAAGGGCGAACCCAACAUUUCGCACAGAACCGACUACUGCA
SEQ ID NO. 16


 


AAGGCGAGGCGCGACCCGCACGUGACAUCCGAUACCACGUGAAAAAGGCACGACA
SEQ ID NO. 17


 


UAGCGACAAGGCGACAAGCAACGACACAUUAACAGACCCUGGUUAAUGAACGAA
SEQ ID NO. 18


 


UAGCGACAAGGCGACAGGCAACGACGCAUUAACAGACCCUGGUUGAUGAACGAA
SEQ ID NO. 19


 


UAGCGACAAGGCGACAGGCAAUGACACAUUAAUAGACCCUGGUUAAUGAACGAA
SEQ ID NO. 20


 


UAGCGACAAGGCGACGAGCAAUGGCACAUUAACAGACCCUGGUUAAAGAACGGG
SEQ ID NO. 21


 


UAGCGACAAGGCGGCAAGCAACGACGCAUUAACAGACCCUGGUUAAUGAAUGAA
SEQ ID NO. 22


 


UAGCGACGGUGCGACAAGCAAUGGCACAUUAACAGACCCUGGUUAAUGAACGAA
SEQ ID NO. 23


 


UAGCGACUAGGCGACAAGCAAUGACACAUUAACAGACCCUGGUUAAUGAACGAA
SEQ ID NO. 24


 


UAGGCGAGGCGCGACCCGCACGUGACAUCUGAUAGCACGUGAAAAGACACUACA
SEQ ID NO. 25


 


UGAUUGGAAUUCUUGGGGCCGAGCGACCCGGCCGUGUAUGAGACAAGAUUACUCC
SEQ ID NO. 26


 


UGGAGUUUGAUUGCGACCGGGGCGACACCCACAAAGGCGACAAAAUCAUUCACAC
SEQ ID NO. 27


 


UGGCGACAAGGCGACAAGCAAUGACACAUUAACAGACCCUGGUUAAUGAACGUA
SEQ ID NO. 28


 


UGGCGGAUACUCUGCGAAGGGCGAACCCAACAUUUCGCACAGAACCGGCUACUCCA
SEQ ID NO. 29


 


UGGCGGCUACUCUGCGAAGGGCGAACCCAACAUUUCGCACAGAACCGACUACUGCA
SEQ ID NO. 30


 


UUGCUCGUACCCUGGGAGCAAAGACCUGAUCAGACCCAACAGAUCUAACAAGCA
SEQ ID NO. 31


 


AAGCGACGAGGCGACAAGCAAUGACACAUUAACAGACCCUGGUUAAUGGACGAA
SEQ ID NO. 32


 


ACGGUGGCGCGGGCGGACCCAAAAUGACGCCACAAAGAAGGCAACACAGAAAACA
SEQ ID NO. 33


 


ACGGUGGCGCGGGCGGACCCAAAAUGACGCCACAAAGAAGGCAACACAGAAACA
SEQ ID NO. 34


 


CAGCGACAAGGCGACAAGCAAUAGACACGUUAACAGACACUGGUUAAUGAACGA
SEQ ID NO. 35


 


CAGCGACGAGGCGACAGGCAACGACACAUUAACAGACCCCGGUUAAUGAGCGAA
SEQ ID NO. 36


 


CUGCGACAAGGCGACAAGCAACGACACAUUAACAGGCCCUGGUUAAUGAACGGA
SEQ ID NO. 37


 


GACAGUAUUUGCGGGGCAAGGGCGCGACAACAAACACAAGUACAGAAAAGGCUA
SEQ ID NO. 38


 


GAGCGACGAGGCGUCAAGCAAUGACACAUUAACAGACCCUGGCUAAUGAAUGAA
SEQ ID NO. 39


 


GCUGAGGGCGGCGACCAGUACAUGCAGCGACAAAUGUACACACAAGCGACGAAAA
SEQ ID NO. 40


 


UACCUUAUUCCGCCCCCGCUGCCCUGGACGUGGAGACUCUGAAACUCCAGCUAU
SEQ ID NO. 41


 


UAGCAACAAGGCGACAGGCAAUGACACAUUAACAGAACCUGGUUAAAGAACGAA
SEQ ID NO. 42


 


UAGCAACAAGGCGACUAGCAACGACACAUUAACAGGCCCUGGUUAAUGGACGAA
SEQ ID NO. 43


 


UAGCAACAAGGCGAUAAGCAAUGGCACAUUAACUGGCCCUGGUUAAUGAACGAA
SEQ ID NO. 44


 


UAGCGACAAGGCGACAAAGCAAUGACACAUUAACGGACCCUGGUUAAUGAACGAA
SEQ ID NO. 45


 


UAGCGACAAGGCGACAAGCAACGACACAUUAACAGACCCUGGCUAAUGACGAAA
SEQ ID NO. 46


 


UAGCGACAAGGCGACAAGCAACGACACAUUAACAGACCCUGUUAAUGAACGAAA
SEQ ID NO. 47


 


UAGCGACAAGGCGACAAGCAACGACACAUUAACGGACCCUGGUUAAUGGACGAA
SEQ ID NO. 48


 


UAGCGACAAGGCGACAAGCAACGACACGUUAACAGACCCUGGUUAAUGAACGAA
SEQ ID NO. 49


 


UAGCGACAAGGCGACAAGCAAUGACACAUUAACAGACCCUGGUUAAUGAACGAA
SEQ ID NO. 50


 


UAGCGACAAGGCGACAAGCAAUGACACAUUAACAGACCCUGGUUAAUGAACGAG
SEQ ID NO. 51


 


UAGCGACAAGGCGACAAGCAAUGACACAUUAAUAGACCCUGGUUAAUGGACGAA
SEQ ID NO. 52


 


UAGCGACAAGGCGACAAGCAAUGACCCAUUAACAGGCCCUGGUUAAUCAACGAA
SEQ ID NO. 53


 


UAGCGACAAGGCGACAAGCAAUGGCACAUUAACAGACCCUGGUUAACGAACGAA
SEQ ID NO. 54


 


UAGCGACAAGGCGACAAGCAAUGGCACAUUGACAGACCCUGGUUAAUGAGAGAA
SEQ ID NO. 55


 


UAGCGACAAGGCGACAAGCGAUGACACAUUAACAGGCCCUGGUUAAUGAAUGAA
SEQ ID NO. 56


 


UAGCGACAAGGCGACAGGCAAUGACACAUUAAAUAGACCCUGGUUAAUGAACGAA
SEQ ID NO. 57


 


UAGCGACAAGGCGACAGGCAAUGACACAUUAACAGACCCUGGUUAAUGAACGAA
SEQ ID NO. 58


 


UAGCGACAAGGCGACAGGCAAUGACACAUUAACAGACCCUGGUUAAUGAAUGAA
SEQ ID NO. 59


 


UAGCGACAAGGCGACAGGCAAUGACACAUUAACAGGCCCUGGUUAAUGAACGAA
SEQ ID NO. 60


 


UAGCGACAAGGCGACAGGCAAUGACACAUUAGCGGACCCUGGUUAAUGAACGAA
SEQ ID NO. 61


 


UAGCGACAAGGCGACAGGCAAUGCCUCAUUAGCAGACCCUGGUUAAUGAACAAA
SEQ ID NO. 62


 


UAGCGACAAGGCGACGAGCAAAUGGCACAUUAACAGACCCUGGUUAAUGAACGAAA
SEQ ID NO. 63


 


UAGCGACAAGGCGACGGGCAAUGACCCAUUAACAGACCCUGGUUAAUGAACGAA
SEQ ID NO. 64


 


UAGCGACAAGGCGGCAGGCAAUAACACAUUAACAGACCCCGGUUAAUGAACGAA
SEQ ID NO. 65


 


UAGCGACAAGGCGGCGAGCAAUGACACAUUAACGGACCCUGGUUAAUGAACAAA
SEQ ID NO. 66


 


UAGCGACUAGGCGACAAGCAAUGACACAUUAACAGACCCUGGUUAAAUCGAACGAA
SEQ ID NO. 67


 


UAGCGACUAGGCGACGGGCAAUGACGCAUUAACAGGCCCUGGUUAAUGAACAGA
SEQ ID NO. 68


 


UAGGCGAGGCGCGACCCGCGCGGGACAUCUGAUAGCACGUGAAAAAUGGCACAACG
SEQ ID NO. 69


 


UAGGCGAGGCGCGACCCGCGCGUGACAUCUGAUAGCACGUGAAAAGGCACGACA
SEQ ID NO. 70


 


UAGGCGAGGCGGGACCCGCACGUGACAUCUGAUAGCACGUGAAAAGGCACGACAA
SEQ ID NO. 71


 


UCAAUAAAUGGCAGACCUGAUGCUGCGGGCGUAAGGCAUAGCGACCAACAUUCU
SEQ ID NO. 72


 


UCCCCGAUACUGCGACCAACAGAUUACCAGGGCGAACAGCGACCGAGCAACAAUG
SEQ ID NO. 73


 


UCCUCAAAGCGACCGACCUUUGCCUAAACAGCUGAUGGUUUACAAAGGAAGCACG
SEQ ID NO. 74


 


UCCUUCCCCAAUGCGACACCCCAGCAAGGCGACAGCUGGCCAGGCGACAAACAAAA
SEQ ID NO. 75


 


UCGCGACAAGGCGACGAGCAAUGGCACAUUAACAGACCCUGGUUAAUGAACGAA
SEQ ID NO. 76


 


UCUGAGGGCGGCGGCCAGUACAUGCAGCGACAAAAUGUACACACAAGCGACAAAA
SEQ ID NO. 77


 


UCUGGCGAGGGCGGCUAGGGGACACAGCGUAGUCUGAUGACGCAGAGCAAUCUAA
SEQ ID NO. 78


 


UGGCGAAGACCCGAACACCCUGAGCUGUUUAAAGGCGACGACGCAGCGACGAGCC
SEQ ID NO. 79


 


UGGCGAAGACCCGAUCACCCUGAGCUGUUUAAAGGCGACGACGCAGCGACGAGCC
SEQ ID NO. 80


 


UGGCGACAAGGCGACAAAGCAAUGACACAUUAACAGACCCUGGUUAAUGAACGUA
SEQ ID NO. 81


 


UGGCGACAAGGCGACAGGCAAUGAACACAUUAACGGACCCUGGUUAAUGAACGAA
SEQ ID NO. 82


 


UGGCGACAAGGCGACAGGCAAUGACACAUUAACGGACCCUGGUUAAUGAACGAA
SEQ ID NO. 83


 


UGGCGGAUACGCUGCGAAGGGCGAACCCAACAUUUCGCACAGAGCCGACUACUGCC
SEQ ID NO. 84


 


UGGCGGAUACUCUGCGAAGGGCGAACACAACAUUUCGCACAGAACCGACUACUGCA
SEQ ID NO. 85


 


UGGCGGAUACUCUGCGAAGGGCGAACCCAACAUCUCGCACAGAACCGACUACUGCG
SEQ ID NO. 86


 


UGGCGGAUACUCUGCGAAGGGCGAACCCAACGUUUCGCACAGAACCGACUACUGCG
SEQ ID NO. 87


 


UUGCUCAUACCCUGAGAGCAAAGAUCUGAUCAGACCCAACAGAUCUAGCAAGCAU
SEQ ID NO. 88


 


UUGGUGGCGCGGGCGAACCCAAAAUGACGCCACAAAGAAGACAAUACAGGAAGCA
SEQ ID NO. 89


 







RNA Sequence of second component








GGGAGACAAGACUAGACGCUCAACUACGAACUCAUGACACAAGGAUGCAAUCUCAUCCCG
SEQ ID NO. 90


 


UUUACCGUAAGGCCUGUCUUCGUUUGACAGCGGCUUGUUGACCCUCACACUUUGUACCUG
SEQ ID NO. 91


 


CUGCCAA









RNA was synthesized by in vitro transcription using a Durascribe Transcription kit (Epicentre Biotechnologies) according to manufacturer instructions. RNA molecules were resuspended in IC buffer (50 mM imidazole-HCl, 3 mM CaCl2, pH 7.8), heated to 95° C. and cooled slowly over the course of 2 hrs to room temp, then moved to ice to achieve final secondary structure for use in a diagnostic assay. In a preferred embodiment, each of the pyrimidine bases in sequences SEQ ID NOS. 1-108 are 2′ fluoro-modified. (2′-F-C, U-RNA).
The disclosed aptamer sequences are further modified. SEQ ID NO 97 recognizes RVV-X—involved in detection readout. SEQ ID NO 90 recognizes PrPSc. For example, by specifically modifying the regions provided in SEQ ID NO 97 and SEQ ID NO 90 herein. Sequence modification separately modifies SEQ ID NO. 97 while holding the region of SEQ ID NO. 104 steady. Alternatively, one can separately modify SEQ ID NO. 90 while holding the region of SEQ ID NO. 97 steady. Alternatively, several bases are removed to achieve minimal aptamer binding both RVV-X and PrPSc or insertion of bases, such as removal of: SEQ ID NO. 106 in SEQ ID NO. 97. Further and alternatively still one can remove bases at the 3′ or 5′ ends of SEQ ID NO. 106, or SEQ ID NO. 90, or SEQ ID NO. 97 either singly or up to 10 consecutive bases. Further and alternatively still, one can remove bases within SEQ ID NO. 106, or SEQ ID NO. 90, or SEQ ID NO. 97 either singly or up to 60 consecutive bases (and sets of 10). Or within the SEQ ID NO. 103 containing secondary structure base-pairing, removal of base pairs entails removal of bases at a 5′end and removal of the 3′ complementary base to which it base-pairs, including, for example, insertion of: bases at 3′ or 5′ ends of SEQ ID NO. 106, or SEQ ID NO. 90, or SEQ ID NO. 104 either singly or up to 10 consecutive bases. The present disclosure further provides for insertion of bases within SEQ ID NO. 106, or SEQ ID NO. 90, or SEQ ID NO. 97 either singly or up to 60 consecutive bases. Within SEQ ID NO. 103 containing secondary structure base-pairing, removal of base pairs entails removal of bases at a 5′end and removal of the 3′ complementary base to which it base-pairs. Table 4 lists the truncations of SEQ ID 74 (from Table 3) as fusions with SEQ ID NO. 90 as the second sequence component, constructed in the same manner listed above. Alternately, SEQ ID NO. 91 may be used in place of SEQ ID NO. 90.
Example 1
This example illustrates reaction times of an in vitro blood coagulation cascade. Time points were taken at 3 minute intervals after 20 seconds of plate shaking with photographic documentation using a Canon EOS XSI (50 mm fixed lens, ISO 100, (f-stop) f/11.0, 15 second exposure.) Serial dilutions of RVV-X Snake Venom protease activator (Haematologic Technologies, VT) were prepared in IC buffer (50 mM imidazole-HCl, 3 mM CaCl2, pH 7.8); the amount of activator listed on the Y-axis is in a 1 μL sample volume. 95 μL reaction buffer is added to 1 μL RVV-X, and shaken initially for 30 seconds. Then, the reaction was monitored over 90 minutes at room temperature and various time points were photographed. Control was either IC Buffer only (1 μL, top lane) or 100 fmol non-acetylated BSA (Sigma Aldrich) (bottom row).
In FIG. 6, the reaction times for initial phase transition of the BCC reaction upon addition of RVV-X were: 10 fmol, 13 min; 1 fmol, 16 min; 100 amol, 22-25 minutes; 10 amol, 37 minutes. The negative control (no RVV-X) did not react within the observed time frame. FIG. 7 includes data taken using a microplate reader at 405 nm and graphed in Microsoft Excel.
Table 2 shows the data from a BCC reaction that was prepared according to Example 1. Data were taken at two or three minute time points on a Synergy H1 Hybrid Reader (BioTek) using Gen5 data analysis software. Time points (t1/2) were calculated using Excel Solver and filtered to remove outliers. Assay CV was <5% (little noise).







TABLE 2







Average t1/2 of BCC reaction (filtered data).











RVV-X (per






100 μL reaction)
Avg t1/2 (min)
St dev
CV (%)
N (sample size)





  10 f
12.40
0.26
2.1
3



15.17
0.57
3.7
3


  1f
20.80
0.42
2.0
2



28.47
1.20
4.2
3


100a
36.57
0.74
2.0
3



42.90
0.14
0.3
2









Example 2
This example illustrates how Kd values of monoclonal aptamers to RVV-X are determined. Electrophoretic mobility (gel) shift assays (EMSA) were performed on the RVV-X aptamer candidates (Tables 3-5) to measure Kd values. Approximately 0.25 fmol 32P-end-labeled RNA was incubated with varying concentrations of RVV-X (0 nM and 8.66 nM-4.44 μM, 11 concentrations total) in a 9 μL total reaction volume for 1 hr at room temperature. Samples were mixed with 1.5 μL 6× glycerol loading buffer (Affymetrix) and electrophoresed on a 7% non-denaturing polyacrylamide gel in 0.5×TBE for 40 minutes at 120V. The gel was then dried and exposed to film for 2 hrs or overnight. Film was developed and scanned. Bands were quantified by densitometry and Kd calculated by fitting data to a non-linear regression curve using a One Site-Specific Binding with Hill Slope model with Kaleidagraph. BSA (660 nM) and Human alpha-Thrombin (alpha-Thr) (4.4 μM, Haematologic Technologies) were used as negative controls. In this example, aptamer sequences Flfus74 (SEQ ID NO. 98) and Fus74—10—5 (SEQ ID NO. 103) were titrated against varying concentrations of RVV-X for Kd assessment (FIGS. 9A-B). The Kds were measured as 60 and 63 nM, respectively. Kd values for selected monoclonals are in Tables 3-5. Gel-shift of B-end (SEQ ID NO. 90) to the control protein alpha-Thr is illustrated in FIG. 9C. The Kd for this aptamer-protein set is 4224±483.6 nM. The measured Kd s of SEQ ID NO. 90 to RVV-X and PrP were 170.2±2.9 nM and 44.3±34.0 nM, respectively.







TABLE 3







Kd determinations of select RVV-X first part sequences.


Binding kinetics to RVV-X.








Sequence
Kd (nM)





UAGCGACAAGGCGACAAGCAAUGACACAUUAACAGACCCUGGUUAGUGAAC
 30


GAA SEQ ID NO. 1



 


AAGGCGAGGUGCGACCCGCACGUGGCAUCUGAUAGCACAUGAAAAGGCACG
 23.04


UCA SEQ ID NO. 2



 


UCUUCCAACGACCAUGCGGCGACAAGCGACUACAAGAGGGUACCCACGGAC
 94.7


AGCA SEQ ID NO. 3



 


UGGCGACGAGGCGGCAGGCAAUGACACAUUAGCAGACCCUGGUUAAUGAGC
158.4


GAA SEQ ID NO. 4



 


UGGCGGAUACUCUGCGAAGGGCGAACCCAACAUUUCGCACAGGACCGACUA
 19


CUGCA SEQ ID NO. 6



 


UCCUCAAAGCGACCGACCUUUGCCUAAACAGCUGAUGGUUUACAAAGGAAG
 75


CACG SEQ ID NO. 74



 


GGAGG CCAAC UAAUA ACGCC AGAAC UAUAG GAAUC CCAUG AAGCG
131 ± 19.7


AGCGA GAAUU SEQ ID NO. 107






A “Scrambled” sequence (SEQ ID NO. 107) is a randomized sequence of SEQ ID NO. 1 and was used as a negative control for the following studies.






Example 3
This example provides a series of bifunctional fusion aptamers of the present disclosure using SEQ ID NO. 74 as the first sequence component.
Table 4 below lists truncations of SEQ ID NO. 74. The truncated sequences for the first sequence component, SEQ ID NO. 74, are the fusion SEQ ID NOs 93-96. The species are labeled according to the region from which bases are truncated: “5 from 3′ “means 5 bases were removed from the 3′ end (in this list, from SEQ ID. NO. 74).







TABLE 4







Truncations of SEQ ID NO. 74 from RNA aptamer selection to RVV-X.











Size
Kd



Species
(nt)
(nM)
Sequence













74 alone
55
75
UCCUCAAAGCGACCGACCUUUGCCUAAACAGCUGAUGGUUUA





CAAAGGAAGCACG SEQ ID NO. 74


 


5 from 3′
50
351.7 ±
UCCUCAAAGCGACCGACCUUUGCCUAAACAGCUGAUGGUUUA




 48.4
CAAAGGAA SEQ ID NO. 92


 


5 from 5′
50
  374 ±
AAAGCGACCGACCUUUGCCUAAACAGCUGAUGGUUUACAAAG




 12.7
GAAGCACG SEQ ID NO. 93


 


5 from both
45
  426 ±
AAAGCGACCGACCUUUGCCUAAACAGCUGAUGGUUUACAAAG




261.6
GAA SEQ ID NO. 94


 


10 from 3′
45
301.2 ±
UCCUCAAAGCGACCGACCUUUGCCUAAACAGCUGAUGGUUUA




 33.6
CAA SEQ ID NO. 95


 


10 from 5′
45
250
GACCGACCUUUGCCUAAACAGCUGAUGGUUUACAAAGGAAGC





ACG SEQ ID NO. 96









Table 5 includes the list of bifunctional fusion aptamers for monoclonal aptamer using SEQ ID NO. 74 as the first sequence component and SEQ ID NO. 90 as the second sequence component to form bifunctional fusion aptamers. These are labeled to describe the components of the aptamer: “Fus74—5—3” indicates the first sequence component is aptamer sequence 93 (SEQ ID NO. 93) and the second sequence component for all listed fusion aptamers in Table 5 is SEQ ID NO. 90. The construction of these fusion aptamers is described in Example 5.










TABLE 5





SEQ





ID
Fusion apt (seq
Size



NO
74)
(nt)
Sequence







 97
Full Length
115
UCCUCGGGAGACAAGACUAGACGCUCAACUACGAACUCA





UGACACAAGGAUGCAAUCUCAUCCCGAAAGCGACCGACC





UUUGCCUAAACAGCUGAUGGUUUACAAAGGAAGCACG





SEQ ID NO. 97


 


 98
5 from 3′ end
110
UCCUCGGGAGACAAGACUAGACGCUCAACUACGAACUCA





UGACACAAGGAUGCAAUCUCAUCCCGAAAGCGACCGACC





UUUGCCUAAACAGCUGAUGGUUUACAAAGGAA SEQ ID





NO. 98


 


 99
5 from 5′ end
110
AAAGCGGGAGACAAGACUAGACGCUCAACUACGAACUCA





UGACACAAGGAUGCAAUCUCAUCCCGGACCGACCUUUGC





CUAAACAGCUGAUGGUUUACAAAGGAAGCACG SEQ ID





NO. 99


 


100
5 from both
105
AAAGCGGGAGACAAGACUAGACGCUCAACUACGAACUCA



ends

UGACACAAGGAUGCAAUCUCAUCCCGGACCGACCUUUGC





CUAAACAGCUGAUGGUUUACAAAGGAA SEQ ID NO. 100


 


101
10 from 3′ end
105
UCCUCGGGAGACAAGACUAGACGCUCAACUACGAACUCA





UGACACAAGGAUGCAAUCUCAUCCCGAAAGCGACCGACC





UUUGCCUAAACAGCUGAUGGUUUACAA SEQ ID NO. 101


 


102
10 from 5′ end
105
GACCGGGGAGACAAGACUAGACGCUCAACUACGAACUCA





UGACACAAGGAUGCAAUCUCAUCCCGACCUUUGCCUAAA





CAGCUGAUGGUUUACAAAGGAAGCACG SEQ ID NO. 102


 


 90
Second
 59
GGGAGACAAGACUAGACGCUCAACUACGAACUCAUGACA



sequence

CAAGGAUGCAAUCUCAUCCC SEQ ID NO. 90



component-





BSE









Example 4
This example illustrates various second sequence components that can be used to form bifunctional aptamers that can measure PrPSc.







TABLE 6





Fusion aptamers of Table 3 first part sequences.















SEQ ID NO. 96 (45-mer):


5′ GACCGACCUUUGCCUAAACAGCUGAUGGUUUACAAAGGAAGCACG 3′


 


SEQ ID NO. 103 (40-mer):


5′ ACCUUUGCCUAAACAGCUGAUGGUUUACAAAGGAAGCACG 3′


It should be noted that SEQ ID NO. 103 is


SEQ ID NO. 96 with the removal of five bases


from the 5′ end.


 


SEQ ID NO. 90 (60-mer):


5′ GGGAGACAAGACUAGACGCUCAACUACGAACUCAUGACACAAGGAU


GCAAUCUCAUCCCG 3′


 


SEQ ID NO. 103 (105-mer):


5′ GACCGGGGAGACAAGACUAGACGCUCAACUACGAACUCAUGACACAA


GGAUGCAAUCUCAUCCCGACCUUUGCCUAAACAGCUGAUGGUUUACAAAG


GAAGCACG 3′


 


SEQ ID NO. 104 (105-mer):


5′ GGGAGACAAGACUAGACGCUCAACUACGAACUCAUGACACAAGGAUG


CAAUCUCAUCCCG GACCGACCUUUGCCUAAACAGCUGAUGGUUUACAAA


GGAAGCACG 3′


 


(5-MER):


5′ GACCG 3′


 


SEQ ID NO. 105 (105- MER):


5′ GACCGACCUUUGCCUAAACAGCUGAUGGUUUACAAAGGAAGCACG


GGGAGACAAGACUAGACGCUCAACUACGAACUCAUGACACAAGGAUGC


AAUCUCAUCCCG 3′









Example 5
The aptamer shown in FIG. 5 (SEQ ID NO. 103) was constructed by:
1) Adding SEQ ID NO. 90 to the 3′ end of SEQ ID NO 105.
2) Inserting SEQ ID NO. 103 to the 3′ end of SEQ ID NO. 90.
The bifunctional fused aptamer of (SEQ ID NO. 104) was constructed by:
1) Adding SEQ ID NO. 96 to the 3′ end of SEQ ID NO 90.
The bifunctional fused aptamer of (SEQ ID NO 106) was constructed by:
1) Adding SEQ ID NO 90 to the 3′ end of SEQ ID NO 97.

## Claims

1. An infectious agent bifunctional aptamer comprising a first sequence component, and a second sequence component, wherein the first sequence component is a complement binding sequence component selected from the group consisting of SEQ ID NOs 1-14, 16-25, 28-30, 32, 36, 37, 39, 42-44, 46, 48-56, 58-62, 64-68, 70, 71, 74, 76, and 83-87 and SEQ ID NOs 90-96, each having a 5′ end and a 3′ end, wherein the second sequence component binds to a specific infectious agent, and wherein the second sequence component sequence is inserted into the first sequence component from 1 to 5 bases from the 5′ end of the first sequence component.

2. The infectious agent bifunctional allosteric aptamer of claim 1, wherein the infectious agent is selected from the group consisting of PrPC, PrPSc, Transmissible spongiform encephalopathies (TSEs), Creutzfeldt-Jacob disease (CJD), variant CJD (vCJD), bovine spongiform encepathy (BSE), Chronic Wasting Disease (CWD), and scrapie.

3. The infectious agent bifunctional aptamer of claim 1, wherein the infectious agent is PrPSc and the sequence of the second sequence component is SEQ ID NO. 90.

4. A neurodegenerative disease bifunctional aptamer comprising a first sequence component, and a second sequence component, wherein the first sequence component is a complement binding sequence component selected from the group consisting of SEQ ID NOs 1-1-14, 16-25, 28-30, 32, 36, 37, 39, 42-44, 46, 48-56, 58-62, 64-68, 70, 71, 74, 76, and 83-87 and SEQ ID NOs 90-96, each having a 5′ end and a 3′ end, wherein the second sequence component binds to a specific infectious agent, and wherein the second sequence component sequence is inserted into the first sequence component from 1 to 5 bases from the 5′ end.

5. The neurodegenerative disease bifunctional aptamer of claim 4, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's Disease, mild cognitive impairment, Parkinson's disease, and other neurodegenerative diseases.

6. The neurodegenerative disease bifunctional aptamer of claim 4, wherein the neurodegenerative disease is Alzheimer's Disease and the sequence of the second sequence component is SEQ ID NO. 91.