RNA aptamers specific for α-synuclein protein fibril conformers

Abstract

The present invention relates to aptamers, characterized in that they have the ability to distinguish conformers of F-type α-Syn fibers from conformers of R-type α-Syn fibers of α-Syn (α-Syn) protein, and characterized in that they comprise a sequence specific for modified ribonucleic acid (RNA) having at least 85% identity with a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, preferably selected from SEQ ID NO:1 and SEQ ID NO:2. The present invention also relates to compositions or kits comprising at least one of these aptamers and their use in vitro. The present invention also relates to methods for the diagnosis of synucleinopathies, as well as for the stratification, monitoring, prognosis and evaluation of the efficacy of synucleinopathies, comprising the use of at least one aptamer and / or composition and / or kit as described above.

Description

[Technical field]

[0001] The present invention relates to the field of aptamers and neurodegenerative diseases, in particular synucleinopathies.

[0002] The present invention relates to an aptamer, characterized in that the aptamer has the ability to distinguish the conformational isomer (conformer) of F-type α-Syn fibers of α-synuclein (α-Syn) protein from the conformer of R-type α-Syn fibers, and is characterized in that the aptamer comprises a specific sequence for modified ribonucleic acid (modified RNA) having at least 85% identity to a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, preferably selected from SEQ ID NO:1 and SEQ ID NO:2.

[0003] The present invention further relates to compositions or kits comprising at least one of these aptamers, as well as to uses thereof.The present invention also relates to methods for diagnosing neurodegenerative diseases, as well as methods for stratifying, monitoring, prognosticating, and assessing the efficacy of synucleinopathic treatments, comprising the use of at least one of the above aptamers and / or the above compositions and / or kits. [Background technology]

[0004] The diagnosis of neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's, Creutzfeldt-Jakob or Lewy body dementia is based on the observation of cognitive, motor and sensory impairments experienced by patients. However, the symptoms of these different diseases are very similar, especially in the early stages of the disease, making their diagnosis difficult. In many cases, a definitive diagnosis can only be made when the disease has reached an advanced stage and the patient is suffering from the most severe symptoms (Gomez-Rio and al, 2016). It is therefore essential to develop new diagnostic methods to reliably distinguish between different neurodegenerative diseases (NDs). Moreover, improved diagnostics would allow patients to be offered a more reliable monitoring and prognosis, and the effectiveness of the applied treatments to be better evaluated.

[0005] Moreover, the motor symptoms associated with these diseases often appear rather late, at a time when neuronal damage is already very great. For example, it is estimated that 50% of dopaminergic neurons in the substantia nigra have already died by the time motor symptoms of Parkinson's disease appear (Cheng and al., 2010). Therefore, detecting the onset of ND before the appearance of motor symptoms is important, since treatment can be initiated early, when neurodegenerative phenomena are not yet widespread.

[0006] Two types of biomarkers have been specifically investigated for diagnosing ND: genetic and biochemical.

[0007] Genetic biomarkers can be alleles or mutations in the genome that have been identified as predisposing to a disease, and thus this type of marker can identify "at risk" populations that have a higher probability of developing ND.

[0008] Biochemical markers are biological molecules whose presence and / or quantity correlate with the progression of a disease. For example, Parkinson's disease is histologically characterized by the accumulation of α-synuclein (or α-synuclein or α-Syn) in Lewy bodies (Katsuno and al.). Thus, biochemical markers are considered features of the appearance and development of the disease, while genetic biomarkers are rather considered factors that cause the disease. Identifying biochemical markers allows for earlier detection of the appearance of the disease, more accurate diagnosis, easier tracking of the disease's progression, and even accurate evaluation of the effectiveness of treatment. Therefore, many efforts are currently being made to identify markers that are present in the patient's tissues or body fluids (blood, serum, cerebrospinal fluid, etc.) and that consist of a unique signature of each disease (Agrawal and Biswas, 2015; Beach, 2017).

[0009] The accumulation of protein aggregates in the central nervous system is a common feature of several progressive neurodegenerative disorders (e.g. Alzheimer's, Parkinson's, Huntington's and Creutzfeldt-Jakob diseases). The three-dimensional misfolding of certain proteins increases their tendency to bind together and communicate this misfolding to each other. The aggregated form of these proteins then gradually accumulates, disrupting the normal function of neurons and gradually leading to their death. Among the proteins that can form this type of aggregates, the α-synuclein protein, the tau protein, the huntingtintin, the β-amyloid peptide or the PrP protein are some of them.

[0010] It has been demonstrated that the pathological three-dimensional conformation of these proteins can be transmitted not only between cells, but also between individuals. This proves that certain proteins, called "prions", can be infectious agents, similar to viruses, bacteria and parasites. Although transmission from individual to individual has been demonstrated mainly for the PrP protein, recent studies have shown that other proteins involved in neurodegenerative diseases may have similar properties. These proteins include α-synuclein protein, tau protein, huntingtintin or beta amyloid peptide.

[0011] Alpha-synuclein (α-Syn) fibrils are found among protein aggregates in the brains of patients suffering from certain NDs grouped under the name "synucleinopathies". These diseases include, among others: Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Alpha-Syn fibrils are composed of thousands of misfolded protein repeats. Alpha-Syn fibrils form by sequential recruitment of the protein, following a propagation mechanism in which a misfolded Alpha-Syn protein transmits its incorrect conformation when it binds to another protein. Recently, several forms of Alpha-Syn fibrils with different structural conformations have been isolated, showing that they may cause different pathologies (Peelaerts and al., 2015; Rey and al., 2019). These different conformations are called conformers. Among these conformers, F-, R-, 91- and 65-type α-Syn fiber conformers can be mentioned (Bousset and al., 2013; Makky and al., 2016). The F-, 65- and 91-type α-Syn fiber conformers are cylindrical, the R-type fiber conformers are flat ribbon-like, and the 91- and 65-type fibers are twisted but with different helical pitches. Analysis by solid-state NMR and atomic force microscopy revealed structural differences between these four fiber conformers. Thus, the length of these fiber conformers is on average 1–2 μm, and the width is about 15–20 nm, with the F-type α-Syn fiber conformer being the narrowest and the 65-type fiber conformer being the widest. Their height is about 5–7 nm, with the R-type α-Syn fiber conformer being the lowest and the fiber conformer 65 being the highest. Finally, the 65- and 91-fiber conformers differ from the conformers of F- and R-type α-Syn fibers in that they undergo periodic changes in height. Other analyses also showed that the four fiber conformers have distinct mechanical properties.Thus, the F-type α-Syn fiber conformer is the stiffest, with bending strength measurements 4-fold greater than the R-type conformer and 2-fold greater than the 65- and 91-type conformers. α-Syn fiber conformers can also be distinguished from each other by analysis by proteolysis with proteinase K. Their structural differences result in different degradation profiles for each one conformer. These profiles are comparable to barcodes (or fingerprints) specific for each conformer (see example degradation profiles in Figure 1 (Landureau and al., 2021)). Moreover, the overall form of the protein in the F-type α-Syn fiber conformer is more resistant to proteolysis than the R-type conformer (Fenyi and al., 2021).

[0012] Various studies suggest that these different α-Syn fiber conformers could be specific markers for different synucleinopathies. A rat animal model demonstrated that injection of F- or R-type fiber conformers into the animal brain results in two distinct phenotypes. In particular, it was shown that F-type α-Syn fiber conformers cause greater motor impairment in this model, while R-type α-Syn fiber conformers cause more Lewy bodies and neuritic deposits (Peelaerts and al., 2015). More recent studies have shown that fiber conformers obtained from PD or SMA patients resemble R-type fiber conformers, whereas fiber conformers obtained from DLB patients resemble F-type fiber conformers (Van der Perren and al., 2020).

[0013] Therefore, it is essential to develop methods that can detect these markers efficiently, specifically, sensitively and reliably.

[0014] Several antibodies have been developed that can quantify α-Syn in plasma or cerebrospinal fluid (CSF) (WO2007 / 011907; Perrin and al., 2003; Vaikath and al., 2019). However, most of these antibodies are not specific for a particular conformation of the protein. As a result, these antibodies are not useful from a diagnostic point of view, since they can measure the protein but cannot measure the amount of the protein in the form of aggregates. New antibodies have been developed that specifically recognize the aggregated form of the protein compared to the monomeric form of the protein (Vaikath and al., 2015). However, these antibodies are not specific for a particular fiber type. Meanwhile, it is always difficult and costly to simultaneously quantify multiple biomarkers using multiple antibodies.

[0015] In this context, aptamers offer an advantageous possibility. Aptamers are nucleic acid structures with properties comparable to antibodies. They are generally obtained by a process of directed molecular evolution called SELEX (Systematic Evolution of Ligands by Exponential Enrichment). Since the discovery of SELEX in the 1990s, the use of aptamers has been experimentally validated in many applications (diagnostics, purification, therapy, etc.). Moreover, an anti-VEGF aptamer (Macugen®) has already been approved for sale as a treatment for macular degeneration of the eye.

[0016] Several studies have identified DNA aptamers that recognize α-Syn. One DNA aptamer (named M5-15) was selected against the monomeric form of α-Syn. However, this aptamer also nonspecifically binds to the oligomeric and fibrillar forms of α-Syn (Tsukakoshi et al., 2010). A second study yielded a second DNA aptamer (T-SO508) that was able to discriminate the oligomeric form of α-Syn from the monomeric and fibrillar forms. However, additional tests showed that this aptamer also recognized the oligomeric form of amyloid-β with the same degree of affinity (Kd ≈100 nM; Tsukakoshi et al., 2012). Aptamers F5R1 and F5R2 were selected by Zheng et al. in 2019 using DNA chemistry as well. These recognize α-Syn with kd of 2.4 and 3.07 nM, respectively (Zheng et al., 2018; Ren et al., 2019). Other aptamers in DNA chemistry have also been selected by Derosa and coworkers (WO201979887). None of these different aptamers have been evaluated for their ability to discriminate between different α-Syn fiber conformers.

[0017] Considering the importance of α-Syn fibril conformers, especially F- and R-type α-Syn fibril conformers, in the diagnosis of various neurodegenerative diseases, it is essential to develop molecules that can distinguish between the various α-Syn fibril conformers, especially F- and R-type α-Syn fibril conformers.

[0018] The present invention meets this need.

[0019] Disclosure of the Invention In the context of the present invention, the inventors have developed ribonucleic acid (RNA) aptamers that can specifically recognize certain conformers of α-Syn fibers, and have indeed selected and isolated RNA aptamers with different affinities for different α-Syn fiber conformers.

[0020] In particular, the inventors have surprisingly shown that the developed aptamer, unlike the DNA aptamers against α-Syn described in the prior art, is able to distinguish F-type α-Syn fibril conformers from R-type α-Syn fibril conformers.

[0021] The data notably show that the aptamer developed by the inventors has a strong affinity for the F-type α-Syn fibril conformer (dissociation constant K d On the other hand, unexpectedly, these aptamers have very low or zero affinity for R-type α-Syn fibril conformers. We also demonstrated that these aptamers recognize native (monomeric, non-fibrillar) α-Syn protein with at least 10-fold lower affinity. Surprisingly, we developed a method to effectively identify these fibril conformers by high-throughput sequencing using a mixture of these aptamers that is applicable to patient samples.

[0022] Data show that these aptamers are tools for specific and sensitive detection of different α-Syn fibrils.Thus, the present invention provides an effective and reliable method for diagnosing neurodegenerative diseases, a method for screening molecules, as well as a tool for research in the field of neurodegenerative diseases.

[0023] Thus, the present invention relates to an aptamer characterized by its ability to distinguish conformers of F-type α-syn fibrils from conformers of R-type α-syn fibrils of α-Syn (α-Syn) protein, characterized in that it comprises a specific sequence in a modified ribonucleic acid (RNA) having at least 85% identity with a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, preferably SEQ ID NO:1 and SEQ ID NO:2. According to a preferred embodiment, the RNA is modified to increase its resistance to RNA nucleases, preferably by modifying the ribose of a pyrimidine of the aptamer, carrying a fluorine atom on the 2' carbon position.

[0024] The present invention further relates to compositions or kits comprising at least one of these aptamers, as well as to their uses.The present invention also relates to methods for diagnosing neurodegenerative diseases, for stratifying, monitoring, prognosing and assessing the effectiveness of synucleinopathic treatments, comprising the use of at least one aptamer and / or the compositions and / or kits described above. Summary of the Invention

[0025] Detailed Description of the Invention Contents of the Invention The present invention relates to an aptamer characterized by having the ability to distinguish F-type α-Syn fiber conformers from R-type α-Syn fiber conformers of α-Syn protein (α-Syn), and characterized by comprising a specific sequence in a modified ribonucleic acid (RNA) having at least 85% identity to a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, preferably selected from SEQ ID NO:1 and SEQ ID NO:2.

[0026] According to one embodiment of the aptamer of the present invention, the dissociation constant K measured for the conformer of F-type α-Syn fibrils is d(F) teeth, a) The dissociation constant K measured for the R-type α-Syn fibril conformer d(R) lower, preferably at least 10 times lower; b) Dissociation constant K measured for α-Syn monomer d(Mono) lower, preferably at least two times lower; c) Dissociation constants, K, of random aptamers measured for F-type α-Syn fibril conformers. d(Random) lower, preferably at least two times lower; d) The dissociation constant K measured for the R-type α-Syn fibril conformer. d(R) lower, preferably at least 10-fold lower; and the dissociation constant K measured for the α-Syn monomer d(Mono) lower, preferably at least two times lower; e) The dissociation constant K measured for the R-type α-Syn fibril conformer d(R) lower, preferably at least 10-fold lower; and the dissociation constant K of the random aptamer measured for the conformer of the F-type α-Syn fiber d(Random) lower, preferably at least two-fold lower; or f) The dissociation constant K measured for the R-type α-Syn fibril conformer d(R) Lower, preferably at least 10-fold lower, than the dissociation constant K measured for α-Syn monomer d(Mono) lower, preferably at least 2-fold lower; and the dissociation constant K of the random aptamer measured for the conformer of the F-type α-Syn fiber d(Random) Lower, preferably at least two times lower.

[0027] According to one embodiment of the aptamer of the invention, at least one dissociation constant K d is as follows: a) The dissociation constant K measured for the F-type α-Syn fibril conformer d(F) is less than 15 nM, preferably less than 10 nM; and / or b) The dissociation constant K measured for the R-type α-Syn fibril conformer d(R)is greater than 100 nM, preferably greater than 150 nM.

[0028] The present invention further comprises: i. a modified RNA primer sequence having at least 85% identity, preferably at least 90% identity, preferably at least 95% identity, with a sequence selected from SEQ ID NO:29 and SEQ ID NO:30, located 5' of the specific sequence, preferably at the 5' end of said specific sequence, and / or ii. a modified RNA primer sequence having at least 85% identity, preferably at least 90% identity, preferably at least 95% identity, with a sequence selected from SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33, located 3' of the specific sequence, preferably at the 3' end of said specific sequence; The present invention relates to the aptamer, further comprising:

[0029] The present invention further relates to said aptamer comprising a modified RNA sequence having at least 85% identity to a sequence selected from SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40, preferably selected from SEQ ID NO:34 and SEQ ID NO:35.

[0030] The present invention also relates to a kit comprising at least one aptamer according to the present invention.

[0031] According to one embodiment of the kit according to the invention, the kit further comprises at least one additional aptamer selected from aptamers comprising a sequence specific for modified RNA having at least 85% identity to a sequence selected from SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28; The kit preferably further comprises an aptamer comprising a modified RNA random sequence.

[0032] According to one embodiment of the kit according to the invention, the at least one additional aptamer comprises i. a modified RNA primer sequence having at least 85% identity, preferably at least 90% identity, preferably at least 95% identity, with a sequence selected from SEQ ID NO:29 and SEQ ID NO:30, located 5' of the specific sequence, preferably at the 5' end of said specific sequence; and / or ii. a modified RNA primer sequence having at least 85% identity, preferably at least 90% identity, preferably at least 95% identity, with a sequence selected from SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33, located 3' of the specific sequence, preferably at the 3' end of said specific sequence; Further includes:

[0033] The present invention also relates to said kit, wherein the at least one additional aptamer is selected from an aptamer comprising a modified RNA sequence having at least 85% identity to a sequence selected from SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 and SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59 and SEQ ID NO:60.

[0034] According to one embodiment of the kit according to the invention, the kit comprises at least the following aptamer: - an aptamer comprising a sequence specific for a modified RNA having at least 85% identity to SEQ ID NO:1; - an aptamer comprising a sequence specific for a modified RNA having at least 85% identity to SEQ ID NO:2; - an aptamer comprising a sequence specific for a modified RNA having at least 85% identity to SEQ ID NO: 3; - an aptamer comprising a sequence specific for a modified RNA having at least 85% identity to SEQ ID NO: 8; - an aptamer comprising a sequence specific for a modified RNA having at least 85% identity to SEQ ID NO: 9; - an aptamer comprising a sequence specific for a modified RNA having at least 85% identity to SEQ ID NO: 10; - an aptamer comprising a sequence specific for a modified RNA having at least 85% identity to SEQ ID NO: 11; - an aptamer comprising a sequence specific for a modified RNA having at least 85% identity to SEQ ID NO: 12; - an aptamer comprising a sequence specific for a modified RNA having at least 85% identity to SEQ ID NO: 13; - an aptamer comprising a sequence specific for a modified RNA having at least 85% identity to SEQ ID NO: 14, - an aptamer comprising a sequence specific for a modified RNA having at least 85% identity to SEQ ID NO: 16; - an aptamer comprising a sequence specific for a modified RNA having at least 85% identity to SEQ ID NO: 17, and - an aptamer comprising a sequence specific for a modified RNA having at least 85% identity to SEQ ID NO: 18; The kit preferably further comprises an aptamer comprising a modified RNA random sequence.

[0035] The kit according to the invention preferably comprises, when said kit comprises a plurality of aptamers, said aptamers being: a) all contained in one composition, or b) distributed among several different compositions in separate containers (including cases where the aptamers are each contained in a different composition that is disposed in a separate container); It is characterized by:

[0036] The present invention further relates to said aptamer or said kit, wherein the RNA of said aptamer or all the aptamers of said kit has been modified to increase resistance to RNA nucleases, preferably wherein the ribose of the pyrimidines of said aptamer or all the aptamers of said kit carries a fluorine atom at the 2' carbon position.

[0037] The present invention also provides a) detecting the presence or absence of at least one conformer of F-type α-Syn fibrils in a biological sample; b) Determination of the amount of F-type α-Syn fibril conformers in biological samples; c) establishing molecular fingerprints of α-Syn fiber conformers in biological samples, preferably F- and R-type α-Syn fibers; d) screening for compounds / molecules capable of detecting and / or recognizing F-type α-Syn fiber conformers, preferably compounds / molecules capable of distinguishing F-type α-Syn fiber conformers from R-type α-Syn fiber conformers; or e) any combination of a) to d) The present invention relates to the in vitro use of at least one aptamer according to the present invention, at least one kit according to the present invention, or any combination thereof, for the treatment of a disease in which the patient is ill or has a medical condition.

[0038] The present invention provides an in vitro method for diagnosing a synucleinopathy in a subject having at least one symptom of a neurodegenerative disease, comprising: a) contacting a biological sample from said subject with at least one aptamer of the invention, at least one kit of the invention, or any combination thereof; b) detecting the presence or absence of at least one conformer of F-type α-syn fibers, quantifying conformers of F-type α-syn fibers, establishing molecular fingerprints of conformers of α-syn fibers (preferably F-type and R-type α-syn fibers) in a biological sample from said subject, or any combination thereof; and c) diagnosing the presence or absence of a synucleinopathy in said subject based on the result of step b); The present invention relates to a method comprising the steps of:

[0039] The present invention also provides an in vitro method for stratifying synucleinopathy, prognosing synucleinopathy, monitoring synucleinopathy, or assessing the efficacy of a synucleinopathy treatment in a subject suffering from synucleinopathy, comprising: a) contacting a biological sample from said subject with at least one aptamer of the present invention, at least one kit of the present invention, or a combination thereof; b) detecting the presence or absence of at least one conformer of F-form α-syn fibers in a biological sample from said subject; and quantifying the conformers of F-form α-syn fibers; Establishing molecular fingerprints of α-Syn fiber conformers (preferably F- and R-type α-Syn fibers), or a combination thereof; and c) stratifying synucleinopathy, prognosing synucleinopathy, monitoring synucleinopathy, or assessing efficacy of a synucleinopathy treatment in said subject based on step b); Including, The present invention relates to a method, wherein the synucleinopathy is preferably selected from Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), more preferably the synucleinopathy is DLB.

[0040] definition "α-Synuclein" or "α-Syn" or "α-syn" or "α-synuclein" or "a-synuclein" means a phosphoprotein of the synuclein family that is abundant in the human brain. It is also found in smaller amounts in heart, muscle and other tissues. In the brain, α-Syn is found primarily at the ends of nerve cells (neurons) at presynaptic terminals. The reference protein sequence for the human α-synuclein protein is the NCBI sequence referenced under P37840.1.

[0041] In physiological conditions, α-Syn protein exists in two forms: soluble and membrane-bound. In its soluble state in the cytosol, α-Syn has a disordered structure (Fauvet and al., 2012). In the presence of lipid membranes, the N-terminal part of α-Syn adopts an α-helical structure that is embedded within the lipid membrane (Eliezer and al., 2001). When bound to membranes of large diameter (at least 100 nm), α-Syn adopts the shape of a large, elongated helix (Trexler and Rhoades, 2009). Conversely, when interacting with small vesicles (hence high curvature), α-Syn adopts a structure consisting of two small α-helices (Chandra and al., 2003).

[0042] Upon abnormal folding, α-Syn can adopt conformations rich in β-sheets, referred to as “pathological” conformations. These pathological conformations have a strong tendency to form fibrillar aggregates (referred to as “fibrillar α-Syn” or “α-Syn fibrils”) that are found in the form of intracellular deposits in synucleinopathies (El-Agnaf and al., 1998a). “Fibrillar α-Syn” or “α-Syn fibrils” or “α-Syn fibrillar aggregates” or “α-Syn aggregates” refers to fibrils (or aggregates, or assemblies) consisting of a large number of misfolded α-Syn protein repeats (in particular, 25 to several hundred misfolded α-Syn protein repeats). Thus, it is an assembly of several copies (25 to several hundred) of misfolded α-Syn protein (with abnormal / pathological / non-native folding). These α-Syn fibrils are formed by the sequential recruitment of α-Syn proteins following a propagation mechanism in which misfolded α-Syn proteins transmit their incorrect conformation when they bind to other proteins. These insoluble fibrils are found in the Lewy bodies that characterize certain pathologies known as synucleinopathies, e.g., Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA).

[0043] Several forms of α-Syn fibrils with different structural conformations have been isolated and shown to induce different synucleinopathies. These different conformations are called α-Syn fibril conformers.

[0044] The α-Syn protein may be subject to one or more post-translational modifications, such as the addition of a functional group (e.g., acetylation, alkylation, biotinylation, carboxylation, glutamylation, glycylation, glycosylation, hydroxylation, isoprenylation, lipoylation, phosphopantetheinylation, phosphorylation, ribosylation, sulfation, selenylation, amidation, and any combination thereof), the addition of a peptide or protein group (provided that in the latter case, the protein to which it is added is not the α-Syn protein; the addition of a peptide or protein group is selected from, for example, ubiquitination, NEDDylation, SUMOylation, uronylation, and the like, and any combination thereof), a change in the chemical nature of an amino acid (e.g., selected from citrullination, deamidation, and the like, and any combination thereof), a structural change (e.g., selected from disulfide bridge formation, and the like), and any combination thereof.

[0045] "α-Syn conformer" or "α-Syn fiber conformer" refers to a particular structural / spatial arrangement (conformation) formed by aggregates of abnormally folded α-Syn protein (i.e., a particular structural arrangement adopted by α-Syn in fibrils). Various α-Syn fiber conformers have been identified, including F-, R-, 65- and 91-type α-Syn fiber conformers. The F-, 65- and 91-type α-Syn fiber conformers are cylindrical, the R-type fiber conformers are flat ribbon-like, and the 91- and 65-type fibers are twisted but with different helical pitches. Within these fibers, at least one copy of the α-Syn protein may contain one or more post-translational modifications as defined above.

[0046] “F fibers” or “F-type α-Syn fiber conformers” or “α-SynF-type fiber conformers” are those that generate nine bands during proteolysis by proteinase K (e.g., Figure 1 (Landureau and al., 2021)) and have a bending stiffness of approximately 5.8 10 -26 Nm 2 where α-Syn protein aggregates are cylindrical in shape.

[0047] "R fibers" or "R-type α-Syn fiber conformers" or "α-Syn R-type fiber conformers" refer to flattened ribbon-like aggregates of α-Syn protein. R-type fibers are flatter than F-type fibers. They form three bands during proteolysis by proteinase K (e.g., Figure 1 (Landureau and al., 2021)) and are approximately 1.4 x 10 -26 Nm 2 It has a bending stiffness of

[0048] By "65 fiber" or "65 fiber conformer" or "65α-Syn fiber conformer" is meant an assembly of α-Syn protein in a tightly twisted conformation. These conformations are approximately 2.710 -26 Nm 2 It has a bending stiffness of

[0049] "91 fiber", "91 fiber conformer" or "91α-Syn fiber conformer" refers to a gently twisted assembly of α-Syn protein that forms seven bands during proteolysis by proteinase K (e.g., Figure 1 (Landureau and al., 2021)) and has a length of approximately 2.410 nm. -26 Nm 2 It has a bending stiffness of

[0050] These different types of fibers can be distinguished using various molecular and structural analysis techniques known to those skilled in the art, such as solid-state NMR analysis, atomic force microscopy, electron microscopy, controlled proteolysis, diffraction of X-rays by fibers, and immobilization of ligands such as thioflavin T or antibodies. These techniques have shown, in particular, that the F, R, 65, and 91 fiber conformers have an average length of 1-2 μm and a width of about 15-20 nm, with the F-type α-Syn fiber conformer being the narrowest and the 65 fiber conformer being the widest. Their heights are on the order of 5-7 nm, with the R-type α-Syn fiber conformer being the lowest and the 65 type being the highest. Finally, the 65 and 91 fiber conformers have a periodic change in height, unlike the F-type and R-type α-Syn fiber conformers. Alternatively, in combination with molecular and structural analysis techniques, these different types of fiber conformers can be distinguished by their mechanical properties using different mechanical analysis techniques known to those skilled in the art, such as atomic force microscopy. In particular, these mechanical analyses demonstrated that each of the four types of fibers has distinct mechanical properties. Thus, the F-type α-Syn fiber conformer is the stiffest, with measured bending strengths four times greater than the R-type conformer and two times greater than the 65- and 91-fiber conformers. The α-Syn fiber conformers can also be distinguished from each other by their protease degradation profiles (in particular, the α-Syn fiber conformer can be distinguished from the other types of α-Syn fiber conformers). Their structural differences result in distinct degradation profiles for each conformer. These profiles are comparable to barcodes (or fingerprints) specific to each conformer (see example degradation profiles in Figure 1). Moreover, all forms of the protein in the F-type α-Syn fiber conformer are more resistant to proteolysis than the R-type conformer (Fenyi and al., 2021).

[0051] Within these fibres, at least one copy of the α-Syn protein may contain one or more post-translational modifications as defined above.

[0052] "α-Syn oligomer" or "α-Syn in oligomeric form" refers to an assembly containing several copies (2-24) of the α-Syn protein protein, presented in the form of a chain (i.e., an array of α-Syn proteins bound / associated with each other, containing 2-24 α-Syn proteins). α-Syn oligomers are distinguished from α-Syn fibers in that they contain a low number of α-Syn copies (Pierri et al., 2016). Thus, α-Syn oligomers contain 2 to about 20 copies of α-Syn protein. α-Syn oligomers are not α-Syn fibers in the sense of the present invention, since they contain less than 25 α-Syn monomers.

[0053] "α-Syn monomer" or "α-Syn in monomeric form" refers to a molecule of α-Syn protein that exists in a free form, i.e., that is not bound / associated with another molecule of α-Syn protein. Thus, an α-Syn monomer contains a single copy of α-Syn protein. Thus, an α-Syn monomer is distinguished from oligomers and aggregates / fibers of α-Syn protein. A single copy of α-Syn protein in a monomer may nevertheless contain one or more post-translational modifications, as described above.

[0054] By "aptamer" is meant an oligonucleotide (i.e. a segment of a nucleic acid chain) that adopts a three-dimensional structure that confers the ability to specifically bind to a given ligand (the ligand is referred to as a "target"), in particular a proteinaceous property. An aptamer is said to specifically bind to a target if it has essentially no affinity for compounds that are structurally unrelated to the target. Preferably, in the case of a protein target, a proteinaceous compound is said to have no structural relationship to the target according to the invention if the sequence identity between the target and the compound is less than 60%, preferably less than 70%, more preferably less than 80%. Preferably, according to the invention, an aptamer is a compound that specifically binds to a compound, in particular if the dissociation constant of the aptamer for the compound is less than 10 -6 If it is greater than mol / l, it is preferably 10 -7mol / l, it is said to have essentially no affinity for the compounds according to the invention. The dissociation constant can be determined, in particular, under standard conditions, using the Scatchard and Lineweaver Burk expressions well known to those skilled in the art.

[0055] Aptamers generally contain a few nucleotides to several tens of nucleotides, for example 15 to 100 nucleotides (preferably 20 to 90 nucleotides, more preferably 30 to 80 nucleotides, more preferably 40 to 70 nucleotides, more preferably 50 to 60 nucleotides). Aptamers are primarily produced synthetically using techniques known to those skilled in the art (such as chemical or enzymatic synthesis). Aptamers are generally selected / identified by a process of directed molecular evolution called SELEX (Systematic Evolution of Ligands by Exponential Enrichment).

[0056] Aptamer can contain at least one modified nucleotide (i.e., a nucleotide that is not a natural DNA or RNA nucleotide). These modified nucleotides can be used to increase the resistance of aptamer to degradation by nucleases. This is particularly advantageous for RNA aptamers, which are generally more sensitive to nucleases than DNA aptamers. The RNA that contains at least one modified nucleotide is called modified RNA.

[0057] Aptamers may also contain at least one additional group in addition to the nucleotides that make up their nucleic acid sequence, so that the nucleic acid of the aptamer can bind to at least one additional group.

[0058] "Modified RNA" refers to RNA that contains at least one modified nucleotide. Modified RNA is in particular RNA in which all or part of the nucleic acid backbone has been modified to be resistant to hydrolysis, in particular by the action of nucleases. RNA may be entirely modified (i.e. each nucleotide that constitutes it is modified) or partially modified (i.e. only some of the nucleotides that constitute it are modified). When RNA is partially modified, it is possible to choose to modify all or some of the purines and / or all or some of the pyrimidines.

[0059] Modifications of RNA (and / or nucleotides) are well known to those skilled in the art and are in particular selected from the following: modification of the OH function on the 2' carbon of ribose by methylation; replacement of the OH function on the 2' carbon of ribose by an O-methoxyethyl group; replacement of the OH function on the 2' carbon of ribose by an amino group; replacement of the OH function on the 2' carbon of ribose by a halogen, in particular fluorine; replacement of phosphodiester (PO) by phosphorothioate (PS), hence the name phosphorothioate backbone; use of Locked Nucleic Acid (LNA) type structures, i.e. formation of a methylene bridge to lock the ribose in a C3'-endo (N-type) conformation; use of Peptide Nucleic Acid (PNA) type structures, i.e. replacement of the sugar-phosphate backbone by a peptide-type backbone; and any combination of these.

[0060] "Additional group" means a chemical group of any type and nature that does not form part of the nucleic acid sequence of the aptamer. The additional group may be selected from, in particular, radioisotopes, organic molecules containing up to 100 carbon atoms, nanoparticles, proteins (in particular glycoproteins), carbohydrates, lipids, polynucleotides, and any combinations thereof. The additional group is preferably selected from detectable markers, pharmacological compounds, compounds that can modify the pharmacokinetic properties of the nucleic acid to which it is bound (such as polyethylene glycol (PEG)), and any combinations thereof.

[0061] The detectable marker may be of any type, in particular a fluorophore (e.g., fluorescein or luciferase), a radioisotope (especially one compatible with scintigraphy, e.g., 99m Tc), a label recognizable by an antibody (eg, c-Myc protein or a polyhistidine tag), an affinity tag (eg, biotin), an enzyme (eg, horseradish peroxidase), a contrast agent, and the like.

[0062] The pharmacological compound may also be of any type, particularly an anti-cancer chemotherapeutic agent (such as a cytostatic or cytological agent), an antibody, a toxin, a hormone, an enzyme, an antiviral compound, an antibiotic compound, an antifungal compound, an antibacterial compound, etc.

[0063] "Aptamer specific / target sequence" or "aptamer specific / target sequence" refers to a portion (section / region / part) of an aptamer sequence that is specific for the aptamer's ligand (target), i.e., a sequence that is unique to a particular aptamer. The aptamer specific sequence is different for each aptamer (and is therefore a variable sequence, as opposed to a constant sequence that may be present in an aptamer). Thus, the aptamer specific sequence is distinct from a primer sequence (or "constant" sequence, or "non-specific" sequence) that may also be present in the aptamer.

[0064] "Aptamer primer sequence" or "aptamer constant sequence" means a part (section / region / portion) of an aptamer's sequence that is present in all aptamers identified / selected by the same session (same run) of the selection method used (such as SELEX). This is generally the sequence of the primer used for the PCR step during SELEX. Thus, an aptamer generally contains two primer sequences, one 5' of the specific / target sequence and the other 3' of the specific / target sequence, allowing amplification by PCR. Thus, the primer sequence of an aptamer is not specific to the aptamer.

[0065] "Random sequence" means a sequence used as a control and randomly (randomly) designed by a person skilled in the art (i.e. the nucleotides that compose it are randomly (randomly) assembled). The random sequence preferably has the same length as the aptamer. The random sequence preferably has the same "constant" sequence ("primer"; preferably 3' and / or 5') as the aptamer, if applicable. In this case, the constant sequence frames the random sequence of the aptamer (hence the constant sequence is 5' and / or 3' of the random sequence of the random aptamer) to form a random aptamer having the same length as the sequence of the aptamer according to the present invention, whose complete sequence has a constant sequence (same or different, preferably same as the sequence of the random aptamer).

[0066] "Ability to distinguish / distinguish between at least two conformers of fibrillar α-synuclein (α-Syn) protein" refers to a property that allows a molecule (e.g., an aptamer) to bind to at least one α-Syn fiber conformer (such as the F-type, R-type, 91-type, and 61-type fiber conformers) with significantly higher affinity (i.e., lower dissociation constant) than to at least one other α-Syn fiber conformer. In particular, a molecule can distinguish between at least two α-Syn fiber conformers if it has a significantly stronger affinity for one α-Syn fiber conformer compared to the other α-Syn fiber conformer. In other words, a molecule can distinguish between at least two α-Syn fiber conformers if its binding ability to one α-Syn fiber conformer is significantly greater than its binding ability to at least one other α-Syn fiber conformer. For example, a molecule can distinguish between at least two α-Syn fiber conformers when the dissociation constant for one α-Syn fiber conformer is significantly lower than the dissociation constant for at least one other α-Syn fiber conformer.

[0067] The molecule is capable of distinguishing at least an F-type α-syn fibril conformer of α-Syn protein from an R-type α-syn fibril conformer of α-Syn protein, particularly if the F-type α-syn fibril conformer has a significantly higher affinity than the R-type α-syn fibril conformer. Thus, "ability to distinguish / distinguish an F-type α-syn fibril conformer of α-Syn protein from an R-type α-syn fibril conformer" means that a molecule has a property that allows it to bind to an F-type α-syn fibril conformer with significantly higher affinity (i.e., with a lower dissociation constant) than to an R-type α-syn fibril conformer. For example, the molecule can distinguish conformers of F-form α-Syn fibrils from conformers of R-form α-Syn fibrils if its average (measured / calculated) dissociation constant for conformers of F-form α-Syn fibrils is significantly lower than its average (measured / calculated) dissociation constant for conformers of R-form α-Syn fibrils. In particular, the molecule can distinguish conformers of F-form α-Syn fibrils from conformers of R-form α-Syn fibrils if the molecule can bind to conformers of F-form α-Syn fibrils but not to conformers of R-form α-Syn fibrils.

[0068] In particular, the molecule can further distinguish the F-type α-syn fiber conformer of the α-Syn protein from other fiber conformers of the α-Syn protein (such as the 65 fiber conformer and the 91 fiber conformer in addition to the R-type α-Syn fiber conformer); and / or the molecule can further distinguish the F-type α-Syn fiber conformer of the α-Syn protein from other forms of the α-Syn protein (such as α-Syn monomers and / or α-Syn oligomers). The above definition of the term "ability of an α-Syn protein to distinguish / distinguish F-type α-Syn fibril conformers from R-type α-Syn fibril conformers" applies mutatis mutandis to the ability to distinguish F-type α-Syn fibril conformers from 65 α-Syn fibril conformers, the ability to distinguish F-type α-Syn fibril conformers from 91 α-Syn fibril conformers, the ability to distinguish F-type α-Syn fibril conformers from α-Syn monomers, and the ability to distinguish F-type α-Syn fibril conformers from α-Syn oligomers.

[0069] "Equilibrium dissociation constant" or "K d " or "Kd" refers to a constant that allows the evaluation of the affinity between two molecules (for example, between an aptamer and a fiber of the α-Syn protein). This affinity is based on the nature, shape and number of physicochemical interactions (electrostatic interactions, hydrogen bonds, van der Waals interactions, hydrophobic forces) between the two molecules. The lower the Kd value, the higher the binding affinity between the two molecules. Kd is expressed in units of M (mol / l) and is often expressed in nM or pM.

[0070] Several techniques for determining / measuring dissociation constants are well known to those skilled in the art, such as ELISA, gel retardation test, filtration, chromatography, thermophoresis, "pull-down" test, equilibrium dialysis, analytical ultracentrifugation, surface plasmon resonance (SPR), spectroscopic test, isothermal titration calorimetry (ITC), nitrocellulose membrane filtration, etc. In particular, the equilibrium dissociation constant can be determined under standard conditions using the Scatchard and Lineweaver Burk expressions well known to those skilled in the art.

[0071] "Neurodegenerative diseases" or "ND" refers to pathologies whose primary cause is the death of neurons. NDs are a group of pathologies with highly diverse clinical manifestations that have in common that they are slowly progressive chronic diseases characterized by the dysfunction and progressive death of neurons (Gao and Hong, 2008). The impairments caused by this neurodegeneration can be motor, cognitive or sensory disorders. These impairments worsen as the disease progresses, making it increasingly difficult for the patient. The frequency of NDs increases significantly with age. However, due to the increasing aging population, the number of people affected by NDs has increased significantly in recent decades and is expected to continue to increase steadily in the future (Heemels, 2016). For example, the World Health Organization (WHO) currently estimates the number of people with dementia worldwide at 50 million, and predicts that it will reach 250 million by 2050 (World Alzheimer Report, 2019; Naqvi, 2017). NDs are extremely disabling conditions that cause a progressive loss of autonomy for patients.

[0072] The best known NDs are: Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease and amyotrophic lateral sclerosis (ALS), including, inter alia, the synucleinopathies.

[0073] "Synucleinopathies" or "α-synucleinopathies" or "alpha-synucleinopathies" refers to neurodegenerative diseases characterized by abnormal accumulation of α-Syn protein aggregates in neurons, nerve fibers or glial cells. α-Synucleinopathies are chronic and progressive pathologies manifested by movement disorders, cognitive impairment and behavioral changes. Synucleinopathies include Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). These three diseases have in particular the occurrence of behavioral disorders of paradoxical sleep, dysautonomia and asymmetric parkinsonism. Synucleinopathies also include rare conditions such as various neuroaxonal dystrophies.

[0074] "Diagnosis" means the identification / determination of a disease or the absence of a disease in a subject. Diagnosis includes, for example, the search for the cause (etiology) and effects (symptoms) of a disease using various tools, in particular based on observations and / or measurements. In the case of neurodegenerative diseases, diagnostic tools include the observation of cognitive, motor and sensory disorders experienced by the patient, the detection / quantification of genetic and / or biochemical biomarkers. Genetic biomarkers can be genomic alleles or mutations identified as predisposing to ND. This type of marker allows the identification of "at risk" populations that are more likely to develop ND. Biochemical markers are biological molecules whose presence and / or quantity correlate with the progression of the disease (e.g., accumulation of α-synuclein in synucleinopathies).

[0075] "Stratification" means separating / classifying subjects into subgroups according to the severity / severity of the disease. The different subgroups include in particular a subgroup of healthy subjects and different subgroups of subjects affected by the disease, classified according to the development / progression stage of the disease. It is also possible to stratify subjects according to the type of symptoms. The stage of development and symptoms can be determined based on observations and / or measurements performed with different tools. In the case of neurodegenerative diseases, the stratification tools include tools that are also used for diagnosis.

[0076] "Prognosis" refers to prediction / determination / assessment of the risk of disease progression in a subject. Prognosis includes, in particular, the assessment of the possibility of future development, improvement or cure of a subject's condition. Prognosis can be determined based on observations and / or measurements carried out using various tools. In the case of neurodegenerative diseases, prognostic tools also include tools used for diagnosis or stratification of subjects.

[0077] "Monitoring" means the determination / assessment of the progression of a disease in a subject. Monitoring can be based on observation and / or measurement, and is performed with different tools and at different time intervals. The intervals can be regular or irregular. The frequency depends on the disease and also on the stage of progression of the disease. It can be on the order of a few days (e.g., for severe / progressive / critical stage disease, and / or for rapidly progressing disease, and / or for exacerbation phase) to several years (e.g., for early, mild or moderate stage disease, and / or for slowly progressing disease). In the case of neurodegenerative diseases, monitoring tools include tools used for the diagnosis or prognosis of the disease, or even for stratification of controls.

[0078] "Evaluation of the effectiveness of treatment" refers to the determination of the clinical condition of the subject that has been treated. The treatment may be prophylactic, for example, in the case of a predisposition to a disease, or therapeutic, for example, in the case of a diagnosed disease. The effectiveness of treatment can be evaluated, for example, by determining the condition of the subject at different time intervals. In particular, the condition of the subject can be evaluated before the first intake of the treatment, and then at regular (or irregular) time intervals after this first intake (for example, after each new intake of the treatment). By comparing the condition of the subject evaluated at these different intervals, possible changes can be identified. In the case of a curative treatment, an improvement in the patient's condition, the absence of deterioration, or a deterioration that is less than the condition expected in the absence of treatment indicates that the treatment is effective, whereas a deterioration in the patient's condition that is at least equal to the condition expected in the absence of treatment indicates that the treatment is not effective. In the case of preventive treatment, the absence of expected disease appearance or the later and / or less severe than expected disease appearance in the absence of treatment indicates that the treatment is effective, and the earlier and more severe than expected disease appearance in the absence of treatment indicates that the treatment is ineffective. The condition of the patient can be evaluated based on the observations and / or measurements carried out using various tools. In the case of neurodegenerative diseases, the tools for evaluating the effectiveness of the treatment include those used for the diagnosis, prognosis or monitoring of the disease, or for stratifying controls.

[0079] "Stage of a neurodegenerative disease", "stage of progression of a neurodegenerative disease" or "stage of advancement of a neurodegenerative disease" means a stage of a neurodegenerative disease that is determined by the severity of the symptoms that a subject is suffering from and the impact / consequences on the subject's lifestyle and / or quality of life. There are four stages. For example:

[0080] - Stage 1 (or first stage), which is referred to as mild disease or mild stage.

[0081] - Stage 2 (or stage 2), which is referred to as moderate disease or moderate stage.

[0082] - Stage 3 (or stage 3), which is referred to as severe disease or severe stage.

[0083] - Stage 4 (or stage 4), which is referred to as very severe disease or the very severe stage. At this stage, quality of life is significantly impaired.

[0084] By "worsening" or "worsening phase" is meant a period of time during which clinical signs of a neurodegenerative disease increase in a subject suffering from a neurodegenerative disease.

[0085] "Subject" or "patient" refers to a human individual or a non-human animal. The subject is, for example, a human or animal that may suffer from a neurodegenerative disease or that suffers from such a disease. The subject is preferably a human. The subject may be a child (a human subject under 16 years of age) or an adult (a human subject over 16 years of age). "Healthy subject" refers to a subject that is not affected by the disease. In the context of the present invention, a healthy subject is preferably a subject that is not affected by any neurodegenerative disease, more preferably a subject that is not affected by any disease. "Reference subject" refers to a subject that suffers from a known neurodegenerative disease (particularly a synucleinopathic disease, in particular Parkinson's disease (PD), dementia with Lewy bodies (DLB) or multiple system atrophy (MSA)) at a known stage.

[0086] A "biological sample" or "sample" from a subject refers to a whole organ or tissue or a part thereof, a fluid or a fraction thereof, a cell or a cellular component obtained from the subject, as well as homogenates, lysates or extracts prepared therefrom. In particular, a "biological sample" or "sample" is preferably any tissue (preferably a part or fraction thereof) that may contain neurons and / or α-Syn protein, including, in non-limiting embodiments, a sample of the central nervous system (CNS), e.g., a brain sample or a spinal cord sample, salivary gland, digestive system (e.g., colon), cerebrospinal fluid, plasma, blood, etc.

[0087] The biological sample may have been previously obtained by any technique known in the art. These techniques include, for example, surgery (e.g., stereotactic surgery, etc.), puncture, excision, resection, biopsy. "Resection" refers to a surgical procedure consisting of cutting (resecting) a more or less wide or deep part of tissue, preferably a tissue abnormality or growth. Resection is performed to remove and / or analyze a cancer or a suspicious tumor. Resection is performed to remove and / or analyze a cancer or a suspicious tumor. The term "biopsy" as used herein refers to a sample of cells or tissue taken for analysis. Several types of biopsy techniques are known and practiced in the art. The most common types include (1) incisional biopsy, in which only a sample of tissue is taken; (2) excision biopsy (or surgical biopsy), which is a therapeutic and diagnostic operation by completely removing the tumor mass; and (3) needle biopsy, in which a tissue sample is taken using a thick or thin needle. There are other types of biopsy, such as smear or curettage, which can also be used to take a sample. Thus, the sample may be, for example, an explant, resection, biopsy, etc. The sample is preferably taken by a minimally invasive procedure, such as stereotactic surgery.

[0088] "Identity" or "sequence identity" refers to the exact sequence match between two polypeptides or amino acids, or between two nucleic acid molecules or oligonucleotides. The percentage of identity referred to in the context of the present invention is determined after optimal global alignment of the sequences being compared, and may therefore include one or more additions, deletions, truncations and / or substitutions. This percentage of identity can be calculated by any sequence analysis method well known to those skilled in the art. The percentage of identity is determined after global alignment over the entire length of the sequences being compared. In addition to manual work, it is also possible to determine the global sequence alignment using the Needleman and Wunsch (1970) algorithm.

[0089] In particular, for nucleotide sequences, the comparison of sequences can be carried out using any software known to those skilled in the art, such as, for example, the Needle software. The parameters used are in particular as follows: "Gap Open" equal to 10.0, "Gap Extend" equal to 0.5, and EDNAFULL matrix (EMBOSS version in NCBI NUC4.4).

[0090] In the case of amino acid sequences, the comparison of sequences can be carried out using any software known to those skilled in the art, such as, for example, the Needle software. The parameters used are in particular: "Gap Open" equal to 10.0, "Gap Extend" equal to 0.5, BLOSUM62 matrix.

[0091] By way of example, "at least 80% sequence identity" as used herein refers to, among others, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0092] Aptamers In the context of the present invention, the inventors have developed modified ribonucleic acid (RNA) aptamers that can specifically recognize certain conformers of α-Syn fibers, and indeed selected and isolated modified RNA aptamers with different affinities for different α-Syn fiber conformers.

[0093] In particular, the inventors have surprisingly shown that the developed aptamer, unlike the DNA aptamers against α-Syn described in the prior art, is able to distinguish F-type α-Syn fibril conformers from R-type α-Syn fibril conformers.

[0094] In particular, the data show that the aptamer developed by the present inventors has a strong affinity for the F-type α-Syn fibril conformer (dissociation constant K d On the other hand, unexpectedly, these aptamers have very low or no affinity for the conformers of R-type α-Syn fibrils, as well as for the conformers of 65 and 91 α-Syn fibrils. We also demonstrated that these aptamers recognize the native (monomeric, non-fibrillar form) α-Syn protein with at least 10-fold lower affinity. Surprisingly, we developed a method using a mixture of these aptamers, allowing us to effectively identify these fibril conformers by high-throughput sequencing applicable to patient samples.

[0095] The data show that these aptamers are tools for the specific and sensitive detection of different α-Syn fibrils, therefore the present invention provides an effective and reliable method for the diagnosis of neurodegenerative diseases, a molecular screening method, and a research tool in the field of neurodegenerative diseases.

[0096] Aptamers have several advantages: 1) they have recognition affinity and specificity for their targets comparable to antibodies; 2) because they are oligonucleotides, they can be used in a number of molecular biology techniques (quantitative PCR, ChIP, rolling circle amplification, high-throughput sequencing, etc.); 3) they are easy to synthesize or amplify in vitro; 4) they can be easily conjugated to a large number of compounds; 5) they have low immunogenicity; 6) they do not suffer from denaturation problems during storage; 7) they are resistant to temperature changes; and 8) they are significantly cheaper than antibodies.

[0097] The present invention therefore relates to an aptamer, characterized in that it has the ability to distinguish between at least two fiber conformers of alpha-synuclein (alpha-Syn) protein and which comprises, or essentially consists of, or consists of a sequence specific for a modified ribonucleic acid (RNA) having at least 85% identity with a sequence selected from SEQ ID NO:1 (specific sequence of aptamer N30), SEQ ID NO:2 (specific sequence of aptamer N124), SEQ ID NO:3 (specific sequence of aptamer N3), SEQ ID NO:4 (specific sequence of aptamer 4F02), SEQ ID NO:5 (specific sequence of aptamer 4F03), SEQ ID NO:6 (specific sequence of aptamer F124) and SEQ ID NO:7 (specific sequence of aptamer P65); preferably selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:5; more preferably selected from SEQ ID NO:1 and SEQ ID NO:2.

[0098] In particular, the present invention relates to an aptamer characterized by having the ability to distinguish conformers of F-type α-Syn fibers of α-Syn (α-Syn) protein from R-type α-Syn fibers, characterized in that it comprises, consists essentially of, or consists of a target / sequence specific for a modified ribonucleic acid (RNA) having at least 85% identity with a sequence selected from SEQ ID NO:1 (specific sequence of aptamer N30), SEQ ID NO:2 (specific sequence of aptamer N124), SEQ ID NO:3 (specific sequence of aptamer N3), SEQ ID NO:4 (specific sequence of aptamer 4F02), SEQ ID NO:5 (specific sequence of aptamer 4F03), SEQ ID NO:6 (specific sequence of aptamer F124) and SEQ ID NO:7 (specific sequence of aptamer P65); preferably selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:5; more preferably selected from SEQ ID NO:1 and SEQ ID NO:2.

[0099] According to a preferred embodiment, the aptamer comprises, essentially comprises or consists of a sequence specific for a modified RNA having at least 86% identity to a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7; preferably selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:5; more preferably selected from SEQ ID NO:1 and SEQ ID NO:2. Preferably, the aptamer comprises, consists essentially of, or consists of a sequence specific for a modified RNA having at least 87% identity, more preferably at least 88% identity, more preferably at least 89% identity, more preferably at least 90% identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, more preferably at least 99% identity to a sequence specific for a modified RNA selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7; preferably selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:5; more preferably, to a sequence specific for a modified RNA selected from SEQ ID NO:1 and SEQ ID NO:2. Particularly preferably, the aptamer comprises, consists essentially of, or consists of a sequence specific for a modified RNA selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7; preferably selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:5; more preferably selected from SEQ ID NO:1 and SEQ ID NO:2.

[0100] The sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 are shown in Table 1 below.

[0101] [Table 1]

[0102] According to one embodiment, the aptamer according to the invention is also capable of distinguishing F-type α-Syn fibers from α-Syn monomers. According to an alternative embodiment or combination, the aptamer according to the invention is also capable of distinguishing F-type α-Syn fiber conformers from 65α-Syn fibers and / or 91α-Syn fiber conformers. According to an alternative embodiment or combination, the aptamer according to the invention is also capable of distinguishing F-type α-Syn fiber conformers from α-Syn oligomers. Thus, according to one embodiment, the aptamer according to the invention is capable of distinguishing F-type α-Syn fiber conformers from R-type α-Syn fibers, as well as 65α-Syn fiber conformers, 91α-Syn fiber conformers, α-Syn oligomers and α-Syn monomers. Thus, according to an advantageous embodiment, the aptamer according to the invention is specific for F-type α-Syn fiber conformers. In one embodiment, the aptamer according to the invention has no affinity for the R-type α-Syn fiber conformer, as well as the 65α-Syn fiber conformer, the 91α-Syn fiber conformer and the α-Syn monomer.

[0103] In a preferred embodiment, the dissociation constant K of the aptamer according to the invention, measured for the conformer of the F-type α-Syn fiber, is d(F) is the dissociation constant K measured for the conformer of R-type α-Syn filaments. d(R), more preferably at least 10-fold lower, more preferably at least 11-fold lower, more preferably at least 12-fold lower, more preferably at least 13-fold lower, more preferably at least 14-fold lower, more preferably at least 15-fold lower, more preferably at least 20-fold lower, more preferably at least 25-fold lower, more preferably at least 30-fold lower, more preferably at least 40-fold lower, more preferably at least 50-fold lower, more preferably at least 100-fold lower, more preferably at least 200-fold lower, more preferably at least 300-fold lower, more preferably at least 400-fold lower, more preferably at least 500-fold lower, more preferably at least 600-fold lower, more preferably at least 700-fold lower, more preferably at least 800-fold lower, more preferably at least 900-fold lower, more preferably at least 1000-fold lower. According to a particularly preferred embodiment, the affinity of the aptamer according to the invention for the conformer of R-type α-Syn fibers is greater than the dissociation constant K d(R) is so low that it cannot be measured / determined using conventional methods for determining dissociation constants (such as those listed in the definitions section above).

[0104] According to an alternative preferred embodiment, or in combination with the above preferred embodiment, the dissociation constant K of the aptamer according to the invention measured on the conformer of the F-type α-Syn fiber is d(F) is the dissociation constant K measured for the α-Syn monomer d(Mono), preferably at least 2-fold lower, preferably at least 3-fold lower, preferably at least 4-fold lower, preferably at least 5-fold lower, preferably at least 6-fold lower, preferably at least 7-fold lower, preferably at least 8-fold lower, preferably at least 9-fold lower, more preferably at least 10-fold lower, more preferably at least 11-fold lower, even more preferably at least 12-fold lower, more preferably at least 13-fold lower, more preferably at least 14-fold lower, more preferably at least 15-fold lower, more preferably at least 20-fold lower, more preferably at least 25-fold lower, more preferably at least 30-fold lower, more preferably at least 40-fold lower, more preferably at least 50-fold lower, more preferably at least 100-fold lower, more preferably at least 200-fold lower, more preferably at least 300-fold lower, more preferably at least 400-fold lower, more preferably at least 500-fold lower, more preferably at least 600-fold lower, more preferably at least 700-fold lower, more preferably at least 800-fold lower, more preferably at least 900-fold lower, more preferably at least 1000-fold lower. According to a particularly preferred embodiment, the affinity of the aptamer according to the invention for α-Syn monomers is d(mono) is so low that it cannot be measured / determined using conventional methods for determining dissociation constants (such as those listed in the definitions section above).

[0105] According to an alternative preferred embodiment, or in combination with one or more of the above preferred embodiments, the dissociation constant K of the aptamer according to the invention, measured on the conformer of the F-type α-Syn fiber, is d(F) is the dissociation constant K measured for the 65α-Syn fiber conformer d(65), preferably at least 2-fold lower, preferably at least 3-fold lower, preferably at least 4-fold lower, preferably at least 5-fold lower, preferably at least 6-fold lower, preferably at least 7-fold lower, preferably at least 8-fold lower, preferably at least 9-fold lower, more preferably at least 10-fold lower, more preferably at least 11-fold lower, more preferably at least 12-fold lower, more preferably at least 13-fold lower, more preferably at least 14-fold lower, more preferably at least 15-fold lower, more preferably at least 20-fold lower, more preferably at least 25-fold lower, more preferably at least 30-fold lower, more preferably at least 40-fold lower, more preferably at least 50-fold lower, more preferably at least 100-fold lower, more preferably at least 200-fold lower, more preferably at least 300-fold lower, more preferably at least 400-fold lower, more preferably at least 500-fold lower, more preferably at least 600-fold lower, more preferably at least 700-fold lower, more preferably at least 800-fold lower, more preferably at least 900-fold lower, more preferably at least 1000-fold lower. d(65) is so low that it cannot be measured / determined using conventional methods for determining dissociation constants (such as those listed in the definitions section above).

[0106] According to an alternative preferred embodiment, or in combination with one or more of the above embodiments, the dissociation constant K of the aptamer according to the invention, measured on the conformer of the F-type α-Syn fiber, is d(F) is the dissociation constant K measured for the α-Syn fiber conformer d(91), preferably at least 2-fold lower, preferably at least 3-fold lower, preferably at least 4-fold lower, preferably at least 5-fold lower, preferably at least 6-fold lower, preferably at least 7-fold lower, preferably at least 8-fold lower, preferably at least 9-fold lower, more preferably at least 10-fold lower, more preferably at least 11-fold lower, more preferably at least 12-fold lower, more preferably at least 13-fold lower, more preferably at least 14-fold lower, more preferably at least 15-fold lower, more preferably at least 20-fold lower, more preferably at least 25-fold lower, more preferably at least 30-fold lower, more preferably at least 40-fold lower, more preferably at least 50-fold lower, more preferably at least 100-fold lower, more preferably at least 200-fold lower, more preferably at least 300-fold lower, more preferably at least 400-fold lower, more preferably at least 500-fold lower, more preferably at least 600-fold lower, more preferably at least 700-fold lower, more preferably at least 800-fold lower, more preferably at least 900-fold lower, more preferably at least 1000-fold lower. d(91) is so low that it cannot be measured / determined using conventional methods for determining dissociation constants (such as those listed in the definitions section above).

[0107] According to an alternative preferred embodiment, or in combination with one or more of the above embodiments, the dissociation constant K of the aptamer according to the invention, measured on the conformer of the F-type α-Syn fiber, is d(F) is the dissociation constant K of a random aptamer (i.e., an aptamer comprising a random sequence of modified RNA, preferably as defined in the "Compositions and Kits" section below), measured on the conformer of the F-type α-Syn fiber. d(Random)lower (preferably significantly lower), preferably at least 2-fold lower, preferably at least 3-fold lower, preferably at least 4-fold lower, preferably at least 5-fold lower, preferably at least 6-fold lower, preferably at least 7-fold lower, preferably at least 8-fold lower, preferably at least 9-fold lower, more preferably at least 10-fold lower, more preferably at least 11-fold lower, more preferably at least 12-fold lower, more preferably at least 13-fold lower, more preferably at least 14-fold lower, more preferably at least 15-fold lower, more preferably at least 20-fold lower, more preferably at least 25-fold lower, more preferably at least 30-fold lower, more preferably at least 40-fold lower, more preferably at least 50-fold lower, more preferably at least 100-fold lower, more preferably at least 200-fold lower, more preferably at least 300-fold lower, more preferably at least 400-fold lower, more preferably at least 500-fold lower, more preferably at least 600-fold lower, more preferably at least 700-fold lower, more preferably at least 800-fold lower, more preferably at least 900-fold lower, more preferably at least 1000-fold lower. In a particularly preferred embodiment, the affinity of a random aptamer for a conformer of F-type α-Syn fiber is determined by a dissociation constant K d(random) is so low that it cannot be measured / determined using conventional methods for determining dissociation constants (such as those listed in the definitions section above).

[0108] Advantageously, the dissociation constant K for the conformer of the F-type α-Syn fiber is determined, in particular using the conventional methods for determining dissociation constants (such as those listed in the definitions section above). d(F)is less than 50 nM, more preferably less than 40 nM, more preferably less than 30 nM, more preferably less than 25 nM, more preferably less than 20 nM, more preferably less than 18 nM, more preferably less than 16 nM, more preferably less than 14 nM, more preferably less than 13 nM, more preferably less than 12 nM, more preferably less than 11 nM, more preferably less than 10 nM, more preferably less than 9 nM, more preferably less than 8 nM, more preferably less than 7 nM.

[0109] Advantageously, the dissociation constant K for the conformer of the R-type α-Syn fiber is determined, in particular using the conventional methods for determining dissociation constants (such as those listed in the definitions section above). d(R) is greater than 100 nM, more preferably greater than 150 nM, more preferably greater than 200 nM, more preferably greater than 300 nM, more preferably greater than 400 nM, more preferably greater than 500 nM, more preferably greater than 600 nM, more preferably greater than 700 nM, more preferably greater than 800 nM, more preferably greater than 900 nM, more preferably greater than 1000 nM. According to a particularly preferred embodiment, the affinity of the aptamer according to the invention for the conformer of R-type α-Syn fibers is determined by the dissociation constant K d(R) is so low that it cannot be measured / determined using conventional methods for determining dissociation constants (such as those listed in the definitions section above).

[0110] According to a preferred embodiment, the aptamer according to the invention has at least one of the following dissociation constants K d Having: a) Dissociation constant K measured for the F-type α-Syn fibril conformer d(F) is less than 15 nM, preferably less than 10 nM; and / or b) The dissociation constant K measured for the R-type α-Syn fibril conformer d(R) is greater than 100 nM, preferably greater than 500 nM.

[0111] Dissociation constant of the aptamer, K dis measured, for example, using conventional methods for determining dissociation constants, preferably selected from those listed in the definitions section above, more preferably by filtration, even more preferably by filtration onto a nitrocellulose membrane.

[0112] According to a preferred embodiment, the aptamer according to the invention comprises: i. a primer sequence 5' of a modified RNA having at least 85% identity, preferably at least 86% identity, preferably at least 87% identity, preferably at least 88% identity, preferably at least 89% identity, preferably at least 90% identity, preferably at least 91% identity, preferably at least 92% identity, preferably at least 93% identity, preferably at least 94% identity, preferably at least 95% identity, preferably at least 96% identity, preferably at least 97% identity, preferably at least 98% identity, preferably at least 99% identity, preferably 100% identity, to a sequence selected from SEQ ID NO:29 (primer sequence P72 of aptamer N in 5') and SEQ ID NO:30 (primer sequence P73 of aptamers 4F, F, R and P in 5'), located 5' of said specific sequence, preferably at the 5' end of said specific sequence; and / or ii. a primer sequence 3' of a modified RNA having at least 85% identity, preferably at least 86% identity, preferably at least 87% identity, preferably at least 88% identity, preferably at least 89% identity, preferably at least 90% identity, preferably at least 91% identity, preferably at least 92% identity, preferably at least 93% identity, preferably at least 94% identity, preferably at least 95% identity, preferably at least 96% identity, preferably at least 97% identity, preferably at least 98% identity, preferably at least 99% identity, preferably 100% identity with SEQ ID NO: 31 (primer PiRT at 3'), SEQ ID NO: 32 (sequence G of 24 nts at 3') and SEQ ID NO: 33 (PiRT-G at 3'; combination of primer PiTR + sequence G), which is located at the terminal 3' of said specific sequence, Further includes:

[0113] According to embodiment i. above, the aptamer is in the form of "primer 5'-specific sequence" (5'-3' direction of the modified RNA sequence). According to embodiment ii. above, relating to the primer of SEQ ID NO: 31, the aptamer is in the form of "specific sequence-primer 3'" (5'-3' direction of the modified RNA sequence). According to embodiment i. in combination with embodiment ii. above, relating to the primer of SEQ ID NO: 31, the aptamer is in the form of "primer 5'-specific sequence-primer 3'" (5'-3' direction of the sequence of the modified RNA), the latter form being referred to hereinafter as "complete aptamer sequence" (or "combined aptamer sequence").

[0114] The sequences of SEQ ID NO:29 to SEQ ID NO:33 are shown in Table 2 below.

[0115] [Table 2]

[0116] According to a particularly advantageous embodiment, the aptamer according to the invention comprises or essentially consists of or consists of the complete sequence of a modified RNA having at least 85% identity with a sequence selected from SEQ ID NO:34 (complete sequence of aptamer N30 with primers 5' and 3' but without sequence G), SEQ ID NO:35 (complete sequence of aptamer N124), SEQ ID NO:36 (complete sequence of aptamer N3), SEQ ID NO:37 (complete sequence of aptamer 4F02), SEQ ID NO:38 (complete sequence of aptamer 4F03), SEQ ID NO:39 (complete sequence of aptamer F124) and SEQ ID NO:40 (complete sequence of aptamer P65), preferably selected from SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37 and SEQ ID NO:38; more preferably, it comprises or essentially consists of or consists of the complete sequence of a modified RNA having at least 85% identity with a sequence selected from SEQ ID NO:34 and SEQ ID NO:35.

[0117] According to a preferred embodiment, the aptamer comprises, consists essentially of, or consists of the complete sequence of a modified RNA having at least 86% identity to a sequence selected from SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40; preferably selected from SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37 and SEQ ID NO:38; more preferably selected from SEQ ID NO:34 and SEQ ID NO:35. Preferably, the aptamer comprises, consists essentially of, or consists of the complete sequence of a modified RNA having at least 87% identity, more preferably at least 88% identity, more preferably at least 89% identity, more preferably at least 90% identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, more preferably at least 99% identity to a fully modified RNA sequence selected from SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:40; preferably selected from SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37 and SEQ ID NO:38; more preferably, SEQ ID NO:34 and SEQ ID NO:35. Particularly preferably, the aptamer comprises, consists essentially of, or consists of the complete sequence of a modified RNA selected from SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40; preferably selected from SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37 and SEQ ID NO:38; more preferably, selected from SEQ ID NO:34 and SEQ ID NO:35.

[0118] SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40 are shown in Table 3 below.

[0119] [Table 3]

[0120] The aptamer according to the present invention may further comprise, at 3' of the complete sequence "primer 5'-specific sequence-primer 3'" defined above, preferably located at the 3' end of the aptamer, a primer sequence 3' of a modified RNA having at least 85% identity, preferably at least 86% identity, preferably at least 87% identity, preferably at least 88% identity, preferably at least 89% identity, preferably at least 90% identity, preferably at least 91% identity, preferably at least 92% identity, preferably at least 93% identity, preferably at least 94% identity, preferably at least 95% identity, preferably at least 96% identity, preferably at least 97% identity, preferably at least 98% identity, preferably at least 99% identity, preferably 100% identity to SEQ ID NO: 32. According to this embodiment, the aptamer is in the form of "primer 5'-specific sequence-primer 3'-sequence G" (5'-3' direction of the modified RNA sequence), which is hereinafter referred to as "complete aptamer sequence G" (combined aptamer sequence G).

[0121] In a preferred embodiment, the RNA of the aptamer of the present invention (i.e., any of the above aptamers including the specific sequence of the aptamer, the primer 5' and / or 3' sequence, the complete sequence of the aptamer, and the complete sequence G of the aptamer) is modified to increase its resistance to RNA nucleases. Advantageously, the RNA of the aptamer according to the invention is modified by at least one modification selected from the following: modification of the OH function of the 2' carbon of ribose by methylation; replacement of the OH function of the 2' carbon of ribose by an O-methoxyethyl group; replacement of the OH function of the 2' carbon of ribose by an amino group; replacement of the OH function of the 2' carbon of ribose by a halogen, in particular fluorine; replacement of phosphodiester (PO) by a phosphorothioate (PS) group (hence the name phosphorothioate backbone); use of a locked nucleic acid (LNA) type structure, i.e. the formation of a methylene bridge to lock the ribose in a C3'-endo (N-type) conformation; use of a peptide nucleic acid (PNA) type structure, i.e. the replacement of the sugar-phosphate backbone by a peptide type backbone; and any combination of these. The RNA of the aptamer according to the invention is preferably modified by at least one modification selected from the following: modification of the OH functional group of the 2' carbon of the ribose by methylation; substitution of the OH functional group of the 2' carbon of the ribose by an O-methoxyethyl group; substitution of the OH functional group of the 2' carbon of the ribose by an amino group; substitution of the OH functional group of the 2' carbon of the ribose by a halogen, in particular fluorine; and any combination of these. According to a particularly preferred embodiment of the aptamer according to the invention, the ribose of the pyrimidine carries a fluorine atom at the 2' carbon.

[0122] Thus, preferably, the modified RNA of the aptamer according to the present invention is an RNA in which the ribose of the pyrimidine nucleotide carries a fluorine atom at the 2' carbon position, and preferably an RNA in which the ribose of the purine nucleotide is unchanged (it is therefore a 2'fluoropyrimidine RNA (2'F-PyRNA)).

[0123] According to one embodiment, the aptamer according to the invention further comprises at least one additional group as defined in the "Definitions" section above. The additional group can be added to any nucleotide of the aptamer. The additional group is preferably located at the 3' end of the aptamer, or at the 5' end of the aptamer, or at both the 3' and 5' ends of the aptamer.

[0124] Compositions and Kits We developed an aptamer with strong affinity for the F-type α-Syn fibril conformer (dissociation constant K d On the other hand, unexpectedly, these aptamers have very low or no affinity for R-type α-Syn fibril conformers as well as 65α-Syn and 91α-Syn fibrils. We also demonstrated that these aptamers recognize the native (monomeric, non-fibrillar form) α-Syn protein with at least 10-fold lower affinity. Surprisingly, we developed a method using a mixture of these aptamers that allows for the effective discrimination of these fibril conformers by high-throughput sequencing applicable to patient samples.

[0125] Thus, the present invention relates to a composition comprising or essentially consisting of at least one aptamer (as defined above) according to the invention.

[0126] The present invention further relates to a kit comprising or essentially consisting of at least one aptamer (as defined above) according to the invention.

[0127] According to one embodiment, the composition or kit comprises SEQ ID NO:8 (specific sequence of aptamer N0), SEQ ID NO:9 (specific sequence of aptamer N1), SEQ ID NO:10 (specific sequence of aptamer N2), SEQ ID NO:11 (specific sequence of aptamer N4), SEQ ID NO:12 (specific sequence of aptamer N5), SEQ ID NO:13 (specific sequence of aptamer N15), SEQ ID NO:14 (specific sequence of aptamer N20), SEQ ID NO:15 (specific sequence of aptamer N37), SEQ ID NO:16 (specific sequence of aptamer N62) and SEQ ID NO:17 (specific sequence of aptamer N73), SEQ ID NO:18 (specific sequence of aptamer N164), SEQ ID NO:19 (specific sequence of aptamer 4F01), SEQ ID NO:20 (specific sequence of aptamer N21), SEQ ID NO:22 (specific sequence of aptamer N23), SEQ ID NO:23 (specific sequence of aptamer N24), SEQ ID NO:24 (specific sequence of aptamer N25), SEQ ID NO:25 (specific sequence of aptamer N26), SEQ ID NO:26 (specific sequence of aptamer N27), SEQ ID NO:27 (specific sequence of aptamer N28), SEQ ID NO:28 (specific sequence of aptamer N29), SEQ ID NO:30 (specific sequence of aptamer N31), SEQ ID NO:31 (specific sequence of aptamer N32), SEQ ID NO:32 (specific sequence of aptamer N33), SEQ ID NO:33 (specific sequence of aptamer N34), SEQ ID NO:34 (specific sequence of aptamer N35), SEQ ID NO:35 (specific sequence of aptamer N36), SEQ ID NO:36 (specific sequence (specific sequence for aptamer 4F04), SEQ ID NO:21 (specific sequence for aptamer 4F05), SEQ ID NO:22 (specific sequence for aptamer R01), SEQ ID NO:23 (specific sequence for aptamer R02), SEQ ID NO:24 (specific sequence for aptamer R03), SEQ ID NO:25 (specific sequence for aptamer R04), SEQ ID NO:26 (specific sequence for aptamer R05), SEQ ID NO:27 (specific sequence for aptamer R84) and SEQ ID NO:28 (specific sequence for aptamer P91); The composition or kit preferably further comprises at least one aptamer comprising a modified RNA random sequence.

[0128] In a preferred embodiment, the additional aptamer comprises, consists essentially of, or consists of a sequence specific for a modified RNA having at least 86% identity to a sequence selected from SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28. Preferably, said further aptamer has at least 87% identity, more preferably at least 88% identity, more preferably at least 89% identity to a sequence selected from SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28. In one embodiment, the nucleic acid sequence comprises, consists essentially of, or consists of a sequence specific for a modified RNA having a sequence identical to, or more preferably at least 90% identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, and more preferably at least 99% identity. Particularly preferably, said additional aptamer comprises, consists essentially of or consists of a sequence specific for a modified RNA selected from SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28.

[0129] SEQ ID NOs:8 to 28 are shown in Table 4 below.

[0130] [Table 4-1] [Table 4-2]

[0131] According to one embodiment, at least one additional aptamer of the composition or kit according to the invention comprises i. a primer sequence 5' of a modified RNA having at least 85% identity, preferably at least 86% identity, preferably at least 87% identity, preferably at least 88% identity, preferably at least 89% identity, preferably at least 90% identity, preferably at least 91% identity, preferably at least 92% identity, preferably at least 93% identity, preferably at least 94% identity, preferably at least 95% identity, preferably at least 96% identity, preferably at least 97% identity, preferably at least 98% identity, preferably at least 99% identity, preferably 100% identity, to a sequence selected from SEQ ID NO:29 (primer sequence P7 of aptamer N in 5') and SEQ ID NO:30 (primer sequence P73 of aptamers 4F, F, R and P in 5'), located 5' of said specific sequence, preferably at the terminal 5' of said specific sequence; and / or ii. a primer sequence 3' of a modified RNA having at least 85% identity, preferably at least 86% identity, preferably at least 87% identity, preferably at least 88% identity, preferably at least 89% identity, preferably at least 90% identity, preferably at least 91% identity, preferably at least 92% identity, preferably at least 93% identity, preferably at least 94% identity, preferably at least 95% identity, preferably at least 96% identity, preferably at least 97% identity, preferably at least 98% identity, preferably at least 99% identity, preferably 100% identity, with a sequence selected from SEQ ID NO: 31 (primer PiRT at 3'), SEQ ID NO: 32 (sequence G of 24 nts at 3') and SEQ ID NO: 33 (PiRT-G at 3'; combination of primer PiTR + sequence G), preferably located at the terminal 3' of said specific sequence, Further includes:

[0132] According to embodiment i. above, the additional aptamer is in the form of "primer 5'-specific sequence of additional aptamer" (5'-3' direction of the modified RNA sequence). According to embodiment ii. above, relating to the primer of SEQ ID NO: 31, the additional aptamer is in the form of "specific sequence of additional aptamer-primer 3'" (5'-3' direction of the modified RNA sequence). According to embodiment i. in combination with embodiment ii. above, relating to the primer of SEQ ID NO: 31, the additional aptamer is in the form of "primer 5'-specific sequence of additional aptamer-primer 3'" (5'-3' direction of the modified RNA sequence), the latter form being referred to hereinafter as "complete sequence of additional aptamer" (or combined sequence of additional aptamer).

[0133] The sequences of SEQ ID NO:29 to SEQ ID NO:33 are shown in Table 2 above.

[0134] According to a particularly advantageous embodiment, the at least one additional aptamer of the composition or kit according to the invention is selected from the group consisting of SEQ ID NO: 41 (complete sequence of aptamer N0 with primers 5' and 3' or without sequence G), SEQ ID NO: 42 (complete sequence of aptamer N1), SEQ ID NO: 43 (complete sequence of aptamer N2), SEQ ID NO: 44 (complete sequence of aptamer N4), SEQ ID NO: 45 (complete sequence of aptamer N5), SEQ ID NO: 46 (complete sequence of aptamer N15), SEQ ID NO: 47 (complete sequence of aptamer N20), SEQ ID NO: 48 (complete sequence of aptamer N37), SEQ ID NO: 49 (complete sequence of aptamer N62) and SEQ ID NO: 50 (complete sequence of aptamer N73), SEQ ID NO: 51 (complete sequence of aptamer N164). the complete sequence of aptamer R84), SEQ ID NO: 51 (complete sequence of aptamer R84), SEQ ID NO: 52 (complete sequence of aptamer 4F01), SEQ ID NO: 53 (complete sequence of aptamer 4F04), SEQ ID NO: 54 (complete sequence of aptamer 4F05), SEQ ID NO: 55 (complete sequence of aptamer R01), SEQ ID NO: 56 (complete sequence of aptamer R02), SEQ ID NO: 57 (complete sequence of aptamer R03), SEQ ID NO: 58 (complete sequence of aptamer R04), SEQ ID NO: 59 (complete sequence of aptamer R05), SEQ ID NO: 60 (complete sequence of aptamer R84) and SEQ ID NO: 61 (complete sequence of aptamer P91).

[0135] According to a preferred embodiment, the at least one additional aptamer comprises, consists essentially of, or consists of the complete sequence of a modified RNA having at least 86% identity to a sequence selected from SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60 and SEQ ID NO:61. Preferably, said further aptamer has at least 87% identity, more preferably at least 88% identity, more preferably at least 90% identity, to a fully modified RNA sequence selected from SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60 and SEQ ID NO:61. or consisting essentially of, or consisting of a fully modified RNA sequence having 89% identity, more preferably at least 90% identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, more preferably at least 99% identity.Particularly preferably, said at least further aptamer comprises, consists essentially of or consists of a fully modified RNA sequence selected from SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60 and SEQ ID NO:61.

[0136] The sequences of SEQ ID NOs:41 to 61 are shown in Table 5 below.

[0137] [Table 5-1] [Table 5-2]

[0138] At least one additional aptamer of the composition or kit according to the invention may further comprise, in the 3' of the complete sequence "primer 5'-additional aptamer specific sequence-primer 3'" as defined above, a primer sequence 3' of a modified RNA having at least 85% identity, preferably at least 86% identity, preferably at least 87% identity, preferably at least 88% identity, preferably at least 89% identity, preferably at least 90% identity, preferably at least 91% identity, preferably at least 92% identity, preferably at least 93% identity, preferably at least 94% identity, preferably at least 95% identity, preferably at least 96% identity, preferably at least 97% identity, preferably at least 98% identity, preferably at least 99% identity, preferably 100% identity to SEQ ID NO: 32, preferably located at the 3' end of said additional aptamer. According to this embodiment, the additional aptamer is in the form of "primer 5'-additional specific aptamer sequence-primer 3'-sequence G" (5'-3' direction of the modified RNA sequence), which is hereinafter referred to as "additional aptamer complete sequence G" (or additional aptamer combination sequence G).

[0139] According to a preferred embodiment, the RNA of at least one aptamer according to the invention of the composition according to the invention or of the kit according to the invention has been modified to increase its resistance to RNA nucleases. Advantageously, the RNA of at least one aptamer according to the invention of the composition or kit according to the invention (preferably the RNA of all aptamers according to the invention of the composition or kit according to the invention) is modified by at least one modification selected from the following: modification of the OH function of the 2' carbon of the ribose by methylation; replacement of the OH function of the 2' carbon of the ribose by an O-methoxyethyl group; replacement of the OH function of the 2' carbon of the ribose by an amino group; replacement of the OH function of the 2' carbon of the ribose by a halogen, in particular fluorine; replacement of the phosphodiester (PO) by a phosphorothioate (PS) group (hence the name phosphorothioate backbone); use of a locked nucleic acid (LNA) type structure, i.e. the formation of a methylene bridge to lock the ribose in a C3'-endo (N-type) conformation; use of a peptide nucleic acid (PNA) type structure, i.e. the replacement of the sugar-phosphate backbone by a peptide type backbone; and any combination of these.

[0140] The RNA of at least one aptamer according to the invention of the composition or kit according to the invention (preferably the RNA of all aptamers according to the invention of the composition or kit according to the invention) is preferably modified by at least one modification selected from the following: modification of the OH functional group of the 2' carbon of ribose by methylation; substitution of the OH functional group of the 2' carbon of ribose by an O-methoxyethyl group; substitution of the OH functional group of the 2' carbon of ribose by an amino group; substitution of the OH functional group of the 2' carbon of ribose by a halogen, in particular fluorine; and any combination thereof. According to a particularly preferred embodiment, the RNA of at least one aptamer according to the invention of the composition or kit according to the invention (preferably the RNA of all aptamers according to the invention of the composition or kit according to the invention) is modified so that the ribose of the pyrimidine nucleotide carries a fluorine atom at the 2' carbon. Thus, preferably, the modified RNA of the aptamer according to the present invention of the composition or kit according to the present invention (preferably the modified RNA of all aptamers according to the present invention of the composition or kit according to the present invention) is an RNA in which the ribose of the pyrimidine nucleotide carries a fluorine atom at the 2' carbon position, preferably in which the ribose of the purine nucleotide is unchanged (hence it is 2'fluoro-pyrimidine RNA (2'F-PyRNA)).

[0141] According to a preferred embodiment, the RNA of at least one additional aptamer of the composition or kit according to the invention has been modified to increase its resistance to RNA nucleases. Advantageously, the RNA of at least one additional aptamer of the composition or kit according to the invention (preferably the RNA of all additional aptamers of the composition or kit according to the invention) is modified by at least one modification selected from the following: modification of the OH function of the 2' carbon of the ribose by methylation; replacement of the OH function of the 2' carbon of the ribose by an O-methoxyethyl group; replacement of the OH function of the 2' carbon of the ribose by an amino group; replacement of the OH function of the 2' carbon of the ribose by a halogen, in particular fluorine; replacement of a phosphodiester (PO) by a phosphorothioate (PS) group (hence the name phosphorothioate backbone); use of a locked nucleic acid (LNA) type structure, i.e. the formation of a methylene bridge to lock the ribose in a C3'-endo (N-type) conformation; use of a peptide nucleic acid (PNA) type structure, i.e. the replacement of the sugar-phosphate backbone by a peptide type backbone; and any combination thereof.

[0142] The RNA of at least one additional aptamer of the composition or kit according to the invention (preferably the RNA of all additional aptamers of the composition or kit according to the invention) is preferably modified by at least one modification selected from the following: modification of the OH functional group of the 2' carbon of ribose by methylation; substitution of the OH functional group of the 2' carbon of ribose by an O-methoxyethyl group; substitution of the OH functional group of the 2' carbon of ribose by an amino group; substitution of the OH functional group of the 2' carbon of ribose by a halogen, in particular fluorine; and any combination thereof. According to a particularly preferred embodiment, the RNA of at least one additional aptamer of the composition or kit according to the invention (preferably the RNA of all additional aptamers of the composition or kit according to the invention) is modified so that the ribose of the pyrimidine nucleotide carries a fluorine atom at the 2' carbon. Thus, preferably, the modified RNA of the additional aptamer of the composition or kit according to the invention (preferably the modified RNA of all additional aptamers of the composition or kit according to the invention) is an RNA in which the ribose of its pyrimidine nucleotides carries a fluorine atom at the 2' carbon position, preferably an RNA in which the ribose of the purine nucleotides is unchanged (it is therefore a 2'fluoro-pyrimidine RNA (2'F-PyRNA)).

[0143] Advantageously, the RNA of all the additional aptamers of the composition or kit according to the invention is modified as defined above. Particularly preferably, the RNA of at least one additional aptamer of the composition or kit according to the invention (preferably all the additional aptamers of the composition or kit according to the invention) is modified in the same way as the RNA of at least one aptamer of the composition or kit according to the invention (preferably all the aptamers of the composition or kit according to the invention).

[0144] According to a preferred embodiment, the composition or kit according to the invention comprises, or consists essentially of, or consists of at least the following aptamers: - an aptamer comprising a sequence specific for a modified RNA having at least 85% identity with SEQ ID NO: 1 (specific sequence of aptamer N30), - an aptamer comprising a sequence specific for modified RNA having at least 85% identity with SEQ ID NO: 2 (specific sequence of aptamer N124), - an aptamer comprising a sequence specific for modified RNA having at least 85% identity with SEQ ID NO: 3 (specific sequence of aptamer N3), - an aptamer comprising a sequence specific for modified RNA having at least 85% identity with SEQ ID NO: 8 (specific sequence of aptamer N0), - an aptamer comprising a sequence specific for a modified RNA having at least 85% identity with SEQ ID NO: 9 (specific sequence of aptamer N1), - an aptamer comprising a sequence specific for modified RNA having at least 85% identity with SEQ ID NO: 10 (specific sequence of aptamer N2), - an aptamer comprising a sequence specific for modified RNA having at least 85% identity with SEQ ID NO: 11 (specific sequence of aptamer N4), - an aptamer comprising a sequence specific for modified RNA having at least 85% identity with SEQ ID NO: 12 (specific sequence of aptamer N5), - an aptamer comprising a sequence specific for a modified RNA having at least 85% identity with SEQ ID NO: 13 (specific sequence of aptamer N15), - an aptamer comprising a sequence specific for modified RNA having at least 85% identity with SEQ ID NO: 14 (specific sequence of aptamer N20), - an aptamer comprising a sequence specific for modified RNA having at least 85% identity with SEQ ID NO: 16 (specific sequence of aptamer N62), - an aptamer comprising a sequence specific for a modified RNA having at least 85% identity with SEQ ID NO: 17 (specific sequence of aptamer N73), and - an aptamer comprising a sequence specific for a modified RNA having at least 85% identity with SEQ ID NO: 18 (specific sequence of aptamer N164); The kit or composition preferably further comprises at least one aptamer comprising a random sequence of modified RNA.

[0145] Indeed, the inventors have shown that such an assortment of aptamers can be used to obtain a specific molecular fingerprint (characteristic signature or characteristic profile) for a biological sample containing different conformers of α-Syn protein, which can be used for the diagnosis, prognosis, stratification or monitoring of neurodegenerative diseases, in particular synucleinopathies.

[0146] According to a particularly preferred embodiment, the composition or kit according to the invention has at least 85% identity, preferably at least 86% identity, preferably at least 87% identity, preferably at least 88% identity, preferably at least 89% identity, preferably at least 90% identity, preferably at least 92% identity, with the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18. comprising, alternatively consisting essentially of, or consisting of aptamers (i.e., an assortment / mixture of aptamers) comprising sequences specific for modified RNAs having at least 91% identity, preferably at least 92% identity, preferably at least 93% identity, preferably at least 94% identity, preferably at least 95% identity, preferably at least 96% identity, preferably at least 97% identity, preferably at least 98% identity, preferably at least 99% identity, preferably 100% identity; The kit or composition preferably further comprises at least one aptamer comprising a random sequence of modified RNA.

[0147] According to one embodiment, at least one aptamer of the assortment of aptamers of a composition or kit according to the invention is i. a primer sequence 5' of a modified RNA having at least 85% identity, preferably at least 86% identity, preferably at least 87% identity, preferably at least 88% identity, preferably at least 89% identity, preferably at least 90% identity, preferably at least 91% identity, preferably at least 92% identity, preferably at least 93% identity, preferably at least 94% identity, preferably at least 95% identity, preferably at least 96% identity, preferably at least 97% identity, preferably at least 98% identity, preferably at least 99% identity, preferably 100% identity, to a sequence selected from SEQ ID NO:29 (primer sequence P72 of aptamer N in 5') and SEQ ID NO:30 (primer sequence P73 of aptamers 4F, F, R and P in 5'), located 5' of said specific sequence, preferably at the terminal 5' of said specific sequence; and / or ii. a primer sequence 3' of a modified RNA having at least 85% identity, preferably at least 86% identity, preferably at least 87% identity, preferably at least 88% identity, preferably at least 89% identity, preferably at least 90% identity, preferably at least 91% identity, preferably at least 92% identity, preferably at least 93% identity, preferably at least 94% identity, preferably at least 95% identity, preferably at least 96% identity, preferably at least 97% identity, preferably at least 98% identity, preferably at least 99% identity, preferably 100% identity, with a sequence selected from SEQ ID NO: 31 (primer PiRT at 3'), SEQ ID NO: 32 (24 nts sequence G at 3', see page 128 of the paper) and SEQ ID NO: 33 (PiRT-G at 3'; combination of primer PiTR + sequence G), preferably located at the terminal 3' of said specific sequence, Further includes:

[0148] Thus, according to a particularly advantageous embodiment, the composition or kit according to the invention comprises at least a sequence which has at least 85% identity, preferably at least 86% identity, preferably at least 87% identity, preferably at least 88% identity, preferably at least 89% identity, preferably at least 90% identity, preferably at least 91% identity, with the sequences SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51. preferably comprising, or consisting essentially of, or consisting of an aptamer (i.e. an assortment / mixture of aptamers) comprising the complete sequence of a modified RNA having at least 91% identity, preferably at least 92% identity, preferably at least 93% identity, preferably at least 94% identity, preferably at least 95% identity, preferably at least 96% identity, preferably at least 97% identity, preferably at least 98% identity, preferably at least 99% identity, preferably 100% identity; The kit or composition preferably further comprises at least one aptamer comprising a random sequence of modified RNA.

[0149] At least one aptamer from the assortment of aptamers of a composition or kit according to the invention may further comprise, in the 3' of the complete sequence "primer 5'-specific aptamer assortment sequence-primer 3" defined above, preferably located at the end 3' of the aptamer of said assortment, a primer sequence 3' of a modified RNA having at least 85% identity, preferably at least 86% identity, preferably at least 87% identity, preferably at least 88% identity, preferably at least 89% identity, preferably at least 90% identity, preferably at least 91% identity, preferably at least 92% identity, preferably at least 93% identity, preferably at least 94% identity, preferably at least 95% identity, preferably at least 96% identity, preferably at least 97% identity, preferably at least 98% identity, preferably at least 99% identity, preferably 100% identity to SEQ ID NO: 32. According to this embodiment, the assortment aptamer is in the form of "primer 5'-specific aptamer assortment sequence-primer 3'-sequence G" (5'-3' direction of the modified RNA sequence), which is hereinafter referred to as "complete assortment aptamer sequence G" (or combination assortment aptamer sequence G).

[0150] According to one embodiment, the composition or kit according to the invention further comprises at least one aptamer (aptamer referred to as "random aptamer") that comprises, consists essentially of or consists of a random sequence of modified RNA having at least 85% identity, preferably at least 86% identity, preferably at least 87% identity, preferably at least 88% identity, preferably at least 89% identity, preferably at least 90% identity, preferably at least 91% identity, preferably at least 92% identity, preferably at least 93% identity, preferably at least 94% identity, preferably at least 95% identity, preferably at least 96% identity, preferably at least 97% identity, preferably at least 98% identity, preferably at least 99% identity, preferably 100% identity to a sequence selected from SEQ ID NO: 62 (Scr-1) and SEQ ID NO: 63 (Scr-2), preferably SEQ ID NO: 63.

[0151] Table 6 shows the sequences of SEQ ID NO:62 and SEQ ID NO:63.

[0152] [Table 6]

[0153] According to a preferred embodiment, the RNA of at least one random aptamer of the composition or kit according to the invention is modified to increase its resistance to RNA nucleases. Advantageously, the RNA of at least one random aptamer of the composition or kit according to the invention (preferably the RNA of all random aptamers of the composition or kit according to the invention) is modified by at least one modification selected from the following: modification of the OH function of the 2' carbon of ribose by methylation; replacement of the OH function of the 2' carbon of ribose by an O-methoxyethyl group; replacement of the OH function of the 2' carbon of ribose by an amino group; replacement of the OH function of the 2' carbon of ribose by a halogen, in particular fluorine; replacement of phosphodiester (PO) by a phosphorothioate (PS) group (hence the name phosphorothioate backbone); use of a locked nucleic acid (LNA) type structure, i.e. the formation of a methylene bridge to lock the ribose in a C3'-endo (N-type) conformation; use of a peptide nucleic acid (PNA) type structure, i.e. the replacement of the sugar-phosphate backbone by a peptide type backbone; and any combination thereof.

[0154] At least one random aptamer RNA of the composition or kit according to the present invention (preferably all random aptamer RNAs of the composition or kit according to the present invention) is preferably modified by at least one modification selected from the following: modification of the OH functional group of the 2' carbon of ribose by methylation; substitution of the OH functional group of the 2' carbon of ribose by an O-methoxyethyl group; substitution of the OH functional group of the 2' carbon of ribose by an amino group; substitution of the OH functional group of the 2' carbon of ribose by a halogen, particularly fluorine; and any combination thereof. According to a particularly preferred embodiment, at least one random aptamer RNA of the composition or kit according to the present invention (preferably all random aptamer RNAs of the composition or kit according to the present invention) is modified so that the ribose of the pyrimidine nucleotide carries a fluorine atom at the 2' carbon. Thus, preferably, the modified RNA of the random aptamer of the composition or kit according to the present invention is an RNA in which the ribose of the pyrimidine nucleotide carries a fluorine atom at the 2' carbon position, and preferably an RNA in which the ribose of the purine nucleotide is unchanged (hence it is 2'fluoro-pyrimidine RNA (2'F-PyRNA)).

[0155] Advantageously, the RNA of all random aptamers of the composition or kit according to the invention is modified as defined above.Particularly preferably, the RNA of at least one random aptamer of the composition or kit according to the invention (preferably, all random aptamers of the composition or kit according to the invention) is modified in the same manner as the RNA of at least one aptamer of the composition or kit according to the invention and / or at least one additional aptamer (as defined above) of the composition or kit according to the invention (preferably, all aptamers of the composition or kit according to the invention and / or all additional aptamers of the composition or kit according to the invention).

[0156] According to one embodiment, the kit according to the invention, when the kit comprises a plurality of aptamers, said aptamers are a) all contained in one composition, or b) distributed among several different compositions in separate containers (including the case where the aptamers are each contained in a different composition that is disposed in a separate container).

[0157] According to one embodiment, the kit according to the invention further comprises instructions for use.

[0158] The composition or kit according to the present invention may further comprise an excipient (e.g., selected from carriers, solvents, diluents, adjuvants, dispersion media, coating agents, antibacterial and antifungal agents, absorbents, and any combination thereof), a buffer (e.g., selected from Tris, Hepes, phosphate, sodium, and any combination thereof), a divalent ion solution (e.g., selected from magnesium ion, calcium, sodium, potassium, and any combination thereof), an enzyme (e.g., selected from DNA polymerase, RNA polymerase, reverse transcriptase, ligase, and any combination thereof), a nucleotide, and the like, and any combination thereof. In the case of a kit, the excipient, buffer, divalent ion solution, enzyme, nucleotide, and the like may each be contained in a different composition arranged in a separate container, may be mixed in one or more pairs in different compositions arranged in a separate container, or may all be contained in the same composition. They may also be mixed with one or more aptamers of the kit.

[0159] Non-limiting examples of excipients include water, NaCl, saline, saccharide solutions (e.g., glucose, trehalose, sucrose, dextrose, etc.), lactated Ringer's, alcohol, oil, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, and the like (see, e.g., the latest edition of Remington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott, Williams & Wilkins).

[0160] The composition or kit according to the invention may further comprise compounds necessary for the amplification (in vitro) of F-type and / or R-type α-Syn fiber conformers. Amplification of fiber conformers can be carried out by any technique known to those skilled in the art. Such techniques include, in particular, the protein misfolding circular amplification (PMCA) method described, for example, in Fenyi et al., 2019. Thus, the composition or kit according to the invention may further comprise a buffer suitable for the amplification of α-Syn fiber conformers (e.g., selected from Tris, Tris-HCl, Hepes, KCl, and any combination thereof), and / or α-Syn monomers, etc.

[0161] use In the context of the present invention, we have developed modified ribonucleic acid (RNA) aptamers that can distinguish the conformer of F-type α-Syn fibers from other α-Syn and α-Syn monomers.

[0162] The data in particular show that these aptamers are tools for the specific and sensitive detection of different α-Syn fibrils. Thus, the present invention provides aptamers that are not only useful for carrying out molecular screening, but also tools for research in the field of neurodegenerative diseases.

[0163] Thus, the present invention provides a) detecting the presence or absence of at least one F-type α-Syn fiber conformer in a biological sample; b) quantification of F-type α-Syn fibril conformers (i.e., determination of their amount) in biological samples; c) establishing molecular fingerprints of α-Syn fiber conformers in biological samples, preferably F- and R-type α-Syn fibers; d) screening for compounds / molecules capable of detecting and / or recognizing conformers of F-type α-Syn fibers, preferably compounds / molecules capable of distinguishing conformers of F-type α-Syn fibers from conformers of R-type α-Syn fibers; or e) any combination of a) to d); 2. In vitro use of at least one aptamer according to the present invention, at least one composition according to the present invention, at least one kit according to the present invention, or any combination thereof, for:

[0164] a) The in vitro use for detecting the presence or absence of at least one F-type α-Syn fibril conformer in a biological sample preferably comprises the following steps: 1. contacting a biological sample with at least one aptamer according to the invention, at least one composition according to the invention, at least one kit according to the invention, or any combination thereof; 2. detecting the presence or absence of immobilized aptamer in the sample; 3. Determining the amount and / or presence or absence of at least one α-Syn conformer, preferably an F-type α-Syn fibril conformer, in the biological sample.

[0165] Detection methods which can be used include, inter alia, ELISA techniques, gel retardation tests, filtration (especially on nitrocellulose membranes), chromatography, thermophoresis, "pull-down" tests, equilibrium dialysis, analytical ultracentrifugation, surface plasmon resonance (SPR), spectroscopic tests, isothermal titration calorimetry (ITC), PCR, quantitative PCR, sequencing (especially high-throughput sequencing), DNA chips, etc.

[0166] b) The in vitro use for quantification (i.e., determination of the amount) of conformers of F-α-Syn fibers in a biological sample preferably comprises the following steps: 1. contacting a biological sample with at least one aptamer according to the invention, at least one composition according to the invention, at least one kit according to the invention, or any combination thereof; 2. Quantifying the amount of aptamer immobilized in the sample; 3. Determining the amount of at least one α-Syn conformer, preferably an F-type α-Syn fibril conformer, in the biological sample.

[0167] Quantitative methods that can be used include, inter alia, ELISA techniques, gel retardation tests, filtration (especially on nitrocellulose membranes), chromatography, thermophoresis, "pull-down" tests, equilibrium dialysis, analytical ultracentrifugation, surface plasmon resonance (SPR), spectroscopic tests, isothermal titration calorimetry (ITC), PCR, quantitative PCR, high-throughput sequencing, DNA chips, etc.

[0168] c) The in vitro use of α-Syn fiber conformers, preferably α-Syn F- and R-type α-Syn fibers to establish molecular fingerprints (i.e., characteristic signatures or characteristic profiles) preferably comprises the following steps: 1. contacting a biological sample with at least one aptamer according to the invention, at least one composition according to the invention, at least one kit according to the invention, or any combination thereof; 2. Quantifying the amount of aptamer immobilized in the sample; 3. To establish molecular fingerprints of α-Syn fiber conformers of α-Syn in biological samples, preferably F-type and R-type α-Syn fibers.

[0169] Methods for establishing molecular fingerprints of α-Syn conformers, in particular of F-type α-Syn fibrils, consist in particular of ELISA techniques, gel retardation tests, filtration (in particular nitrocellulose membrane filtration), chromatography, thermophoresis, "pull-down" tests, equilibrium dialysis, analytical ultracentrifugation, surface plasmon resonance (SPR), spectroscopic tests, isothermal titration calorimetry (ITC), PCR, quantitative PCR, high-throughput sequencing, DNA chips, etc.

[0170] In a situation of use for establishing a molecular fingerprint, preferably a composition or kit according to the invention is selected which comprises at least two aptamers, in particular a composition or kit which comprises an assortment of aptamers as defined above.

[0171] d) The in vitro use for screening of compounds / molecules capable of detecting and / or recognizing conformers of F-type α-Syn fibrils preferably comprises the following steps: 1. contacting at least one compound / molecule with a sample of F-type α-Syn fibril conformers and at least one aptamer according to the invention, at least one composition according to the invention, at least one kit according to the invention, or any combination thereof; 2. Quantifying the amount of aptamer immobilized in the sample; 3. Based on the quantification of step 2, determining the ability of the compound to bind to a conformer of F-type α-Syn fibrils (and thus induce a loss of aptamer affinity).

[0172] Quantitative methods that can be used include, inter alia, ELISA techniques, gel retardation tests, filtration (especially on nitrocellulose membranes), chromatography, thermophoresis, "pull-down" tests, equilibrium dialysis, analytical ultracentrifugation, surface plasmon resonance (SPR), spectroscopic tests, isothermal titration calorimetry (ITC), PCR, quantitative PCR, high-throughput sequencing, DNA chips, etc.

[0173] According to one embodiment, the in vitro uses according to the invention (i.e., the above uses for a) detecting the presence or absence of at least one F-type α-Syn fiber conformer in a biological sample, b) quantifying F-type α-Syn fiber conformers in a biological sample, c) establishing a molecular fingerprint of α-Syn fiber conformers in a biological sample, d) screening for compounds / molecules capable of detecting and / or recognizing F-type α-Syn fiber conformers, and any combination thereof) further comprise step 1') performed prior to step 1) [amplifying (in vitro) the F-type and / or R-type α-Syn fiber conformers or a sample of F-type α-Syn fiber conformers in a biological sample]. The amplification of fiber conformers can be made by any technique known to the skilled person. Such techniques include in particular the protein misfolding circular amplification (PMCA) method, as described, for example, in Fenyi et al., 2019.

[0174] method The inventors have shown that the aptamers according to the invention can be used as tools for the diagnosis, prognosis, stratification or monitoring of neurodegenerative diseases, in particular synucleinopathies, or as tools for assessing the efficacy of treatments.

[0175] The data show in particular that by using an assortment of different aptamers, it is possible to obtain a specific molecular fingerprint (characteristic signature or profile) for biological samples containing different conformers of the α-Syn protein, which can distinguish neurodegenerative diseases, especially synucleinopathies, from one another.

[0176] Accordingly, the present invention provides an in vitro method for diagnosing a synucleinopathy in a subject having at least one symptom of a neurodegenerative disease, comprising: a) contacting a biological sample of said subject (designated sample (A)) with at least one aptamer according to the invention, at least one composition according to the invention, at least one kit according to the invention, or any combination thereof; b) detecting the presence or absence of at least one F-type α-syn fiber conformer, quantifying F-type α-syn fiber conformers, establishing a molecular fingerprint of α-syn fiber conformers (preferably F-type and R-type α-syn fibers) in a biological sample from said subject, or any combination thereof; and c) diagnosing the presence or absence of a synucleinopathy in said subject based on the result of step b); The present invention relates to a method comprising the steps of:

[0177] According to a preferred embodiment, the subject is diagnosed with a synucleinopathy if the presence of at least one F-type α-Syn fibril conformer is detected and / or if a molecular fingerprint characteristic of a synucleinopathy is obtained.

[0178] According to one embodiment, the method further comprises a step a') performed prior to step a) of amplifying (in vitro) conformers of F- and / or R-type α-Syn fibers in a biological sample from said subject, designated sample (A). Amplification of fiber conformers can be performed by any technique known to the skilled artisan. Such techniques include, in particular, the protein misfolding cyclic amplification (PMCA) method, as described, for example, in Fenyi and al., 2019.

[0179] According to an alternative embodiment or in combination with the above-mentioned embodiment, the method further comprises a step b') performed between steps b) and c) of detecting the presence or absence of at least one conformer of F-type α-Syn fibrils, quantifying conformers of F-type α-Syn fibrils, establishing molecular fingerprints of conformers of α-Syn fibrils (preferably F-type and R-type α-Syn fibrils of α-Syn), or any combination thereof, in one or more biological samples of the reference subject. The reference subjects preferably comprise at least one reference subject suffering from a synucleinopathic disease and optionally at least one healthy reference subject. As a sample from a reference subject suffering from a synucleinopathic disease, in particular a sample from a subject suffering from Parkinson's disease (PD), dementia with Lewy bodies (DLB) or multiple system atrophy (MSA) can be used. In step b'), a plurality of samples from reference subjects suffering from different synucleinopathies can be used. According to this embodiment, the method may further comprise a step b'') between steps b') and c) of comparing the detected F-type α-Syn fiber conformers, the quantified F-type α-Syn fiber conformers, and / or the obtained molecular fingerprints of the F-type α-Syn fiber conformers in steps b) and b'). In this case, step c) comprises diagnosing the subject for synucleinopathy based on the comparison of step b''). If the comparison of step b') is comparable to the result of step b') for a reference sample of a reference subject suffering from a synucleinopathy (different reference samples allow a more accurate diagnosis between synucleinopathy in the same principle), the subject is diagnosed as suffering from a synucleinopathy, and if it is comparable to the result of step b') for a reference sample of a healthy reference subject, the subject is diagnosed as not suffering from a synucleinopathy.

[0180] Thus, the method according to the invention may comprise the above mentioned steps a'), a), b) and c), or the above mentioned steps a), b), b') and c), or the above mentioned steps a'), a), b), b') and c), or the above mentioned steps a), b), b'), b'') and c), or the above mentioned steps a'), a), b), b'), b'') and c).

[0181] The present invention provides an in vitro method for stratifying synucleinopathy, prognosing synucleinopathy, monitoring synucleinopathy, or assessing the efficacy of a synucleinopathy treatment in a subject suffering from synucleinopathy, comprising: a) contacting a biological sample of said subject (designated sample (A)) with at least one aptamer according to the invention, at least one composition according to the invention, at least one kit according to the invention, or any combination thereof; b) detecting the presence or absence of at least one F-type α-syn fiber conformer, quantifying F-type α-syn fiber conformers, establishing a molecular fingerprint of α-syn fiber conformers (preferably F-type and R-type α-syn fibers) in a biological sample from said subject, or a combination thereof; and c) stratifying synucleinopathy, prognosing synucleinopathy, monitoring synucleinopathy, or assessing efficacy of a synucleinopathy treatment in said subject based on step b); Including, The present invention relates to a method, wherein the synucleinopathy is preferably selected from Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), more preferably the synucleinopathy is DLB.

[0182] According to a preferred embodiment, detection of the presence of at least one F-type α-Syn fibril conformer and / or obtaining a molecular fingerprint specific for a synucleinopathy indicates that the synucleinopathy is worsening, the prognosis for the synucleinopathy is negative, the synucleinopathy is progressing, or treatment for the synucleinopathy is ineffective or poorly effective.

[0183] According to one embodiment, the method further comprises a step a') performed prior to step a) of amplifying (in vitro) conformers of F- and / or R-type α-Syn fibers in a biological sample from said subject, designated sample (A). Amplification of fiber conformers can be performed by any technique known to the skilled artisan. Such techniques include in particular the protein misfolding cyclic amplification (PMCA) method, as described, for example, in Fenyi and al., 2019.

[0184] According to an alternative embodiment, or in combination with the above-mentioned embodiment, the in vitro prognosis and / or stratification method further comprises a step b') between steps b) and c) of detecting the presence or absence of at least one conformer of F-type α-Syn fibers in one or more biological samples of the reference subject, quantifying the conformers of F-type α-Syn fibers, establishing a molecular fingerprint of α-Syn fibers (preferably F-type and R-type α-Syn fibers), or any combination thereof. The reference subject preferably comprises at least one subject suffering from a synucleinopathy with a known stratified prognosis / stage / level (preferably at least one reference subject suffering from a synucleinopathy with a known stratified prognosis / stage / level, the synucleinopathy being the same in the reference subject and the test / prognosis subject), and optionally at least one healthy reference subject.

[0185] As samples from reference subjects suffering from synucleinopathies, in particular samples from subjects suffering from Parkinson's disease (PD), dementia with Lewy bodies (DLB) or multiple system atrophy (MSA) can be used. In step b'), several samples from reference subjects suffering from different synucleinopathies can be used. In step b'), several samples from reference subjects suffering from the same synucleinopathies, but with different known stratification prognoses / stages / levels, can be used. According to this embodiment, the method can further comprise a step b'' between steps b') and c) of comparing the detected conformers of F-type α-Syn fibers, the quantified conformers of F-type α-Syn fibers and / or the molecular fingerprints of the conformers of F-type α-Syn fibers obtained in steps b) and b'). In this case, step c) comprises a prognosis and / or stratification of synucleinopathies in the subject based on the comparison in step b''). If the comparison of step b') shows that the result of step b') is comparable to the result obtained in step b') for the reference sample of the reference subject, the subject has a comparable prognosis and / or is at a comparable stratification stage / level to the reference subject suffering from the same synucleinopathic disease with known stratification prognosis / stage / level (with different reference samples prognosis between different stages and / or more accurate stratification may be performed in the same principle). If the result of step b) shows that there is a higher detection of F-fibrils, a higher amount of F-type α-Syn fibrils or a molecular fingerprint enriched in F-fibrils is obtained than that obtained in step b') for the reference sample of the reference subject or healthy reference subject, the subject is stratified with a more negative prognosis and / or a more advanced (more severe) stratification stage / level than the reference subject suffering from the same synucleinopathic disease with known stratification prognosis / stage / level or healthy reference subject.Conversely, if the results of step b) show lower detection of F-fibrils, a lower amount of F-type α-Syn fibrils or a poorer molecular footprint of F-fibrils than obtained in step b') for the reference sample of the reference subject, then the subject is stratified as having a better prognosis and / or a less advanced (less severe) stratification stage / level than a reference subject suffering from the same synucleinopathy with a known stratification prognosis / stage / level.

[0186] Thus, the method according to the invention may comprise the above mentioned steps a'), a), b) and c), or the above mentioned steps a), b), b') and c), or the above mentioned steps a'), a), b), b') and c), or the above mentioned steps a), b), b'), b'') and c), or the above mentioned steps a'), a), b), b'', b'''') and c).

[0187] According to an alternative embodiment, or in combination with the above embodiment, the in vitro method for synucleinopathy stratification, synucleinopathy prognosis, synucleinopathy monitoring, or evaluation of the efficacy of a synucleinopathy treatment in a subject suffering from synucleinopathy further comprises a step b') performed between steps b) and c) of detecting the presence or absence of at least one F-type α-Syn fiber conformer, quantifying the F-type α-Syn fiber conformer, establishing a molecular fingerprint of α-Syn fiber conformers (preferably F-type and R-type α-Syn fibers), or any combination thereof, in a second biological sample of the subject to be tested (referred to as sample (B)). Said second sample (B) is preferably obtained / taken after sample (A), for example during a second visit (sample (A) being subsequently obtained during a first visit), preferably said sample (B) is obtained / taken at least 24 hours after sample (A), more preferably at least 48 hours after sample (A), more preferably at least 72 hours after sample (A), more preferably at least 7 days after sample (A), more preferably at least 10 days after sample (A), more preferably at least 15 days after sample (A), more preferably at least 1 month after sample (A), more preferably ...24 hours after sample (A), more preferably at least 24 hours after sample (A), more preferably at least 24 hours after sample (A), more preferably at least 24 hours after sample (A), more preferably at least 24 hours after sample (A), more preferably at least 24 hours after sample (A), more preferably at least 24 hours after sample (A), more preferably at least 24 hours after sample (A), more preferably at least 24 hours after sample (A), more preferably at least 24 hours after sample (A), more preferably at least 24 hours after sample (A), more preferably at least 24 hours after sample (A), more preferably at is obtained at least 2 months after sample (A), more preferably at least 3 months after sample (A), more preferably at least 4 months after sample (A), more preferably at least 5 months after sample (A), more preferably at least 6 months after sample (A), more preferably at least 7 months after sample (A), more preferably at least 8 months after sample (A), more preferably at least 9 months after sample (A), more preferably at least 10 months after sample (A), more preferably at least 11 months after sample (A), more preferably at least 12 months after sample (A).More preferably, the sample (B) is obtained between 7 days and 6 months after the sample (A), more preferably, the sample (B) is obtained between 10 days and 5 months, more preferably, between 15 days and 4 months, more preferably, between 21 days and 3 months, more preferably, between 30 days and 60 days, more preferably, between 40 days and 50 days after the sample (A). According to this embodiment, the method may further comprise a step b'') performed between steps b') and c) of comparing the detected F-form α-Syn fibril conformers, quantified F-form α-Syn fibril conformers, and / or obtained molecular fingerprints of F-form α-Syn fibril conformers in step b (thus for the subject sample (A)) and step b') (thus for the subject sample (B)). In this case, step c) comprises stratifying synucleinopathy in the subject, prognosing synucleinopathy, monitoring synucleinopathy, evaluating the efficacy of a treatment for synucleinopathy based on the comparison in step b''). If the comparison in step b') shows that the results of step b) are comparable to those obtained in step b'), the subject is stratified as being at a stable / comparable stratification stage / level, the subject has a stable / comparable prognosis, the synucleinopathy in the subject is barely progressing (stable) or the treatment for synucleinopathy is reasonably effective.

[0188] If the results of step b) show a higher detection of F-fibrils, a higher amount of F-type α-Syn fibrils, or a molecular fingerprint rich in F-fibrils than those obtained in step b'), the subject is stratified as being at a more advanced (more severe) stratification stage / level, the subject's prognosis is more negative, the subject's synucleinopathy is progressing (worsened, aggravated), or the treatment of synucleinopathy is ineffective or less effective. Conversely, if the results of step b) show a lower detection of F-fibrils, a lower amount of F-type α-Syn fibrils, or a lower molecular fingerprint of F-fibrils than those obtained in step b'), the subject is stratified as being at a less advanced stage / level stratification (less severe), the subject's prognosis is good, the subject's synucleinopathy is improving (it will be less severe and regressing), or the treatment of synucleinopathy is effective.

[0189] The method according to the above-mentioned embodiment may also comprise a step a') carried out before step a) of amplifying (in vitro) conformers of F- and / or R-type α-Syn fibers in sample (A) and / or sample (B). Amplification of fiber conformers can be carried out by any technique known to the skilled artisan. Such techniques include, in particular, the protein misfolding cyclic amplification (PMCA) method, as described, for example, in Fenyi and al., 2019.

[0190] Thus, the method according to the invention may comprise the above mentioned steps a'), a), b) and c), or the above mentioned steps a), b), b') and c), or the above mentioned steps a'), a), b), b') and c), or the above mentioned steps a), b), b'), b'') and c), or the above mentioned steps a'), a), b), b'), b'') and c).

[0191] The following examples are intended to illustrate the invention and are not to be construed as limiting. [Brief description of the drawings]

[0192] [Figure 1] Representative examples of barcodes / molecular fingerprints of F-type α-Syn fiber conformers (left panel), R-type α-Syn fiber conformers (middle panel) and 91-type α-Syn fiber conformers (right panel) obtained after controlled proteolysis with proteinase K (PK) at different times in minutes (1, 5 or 15 min, as indicated at the top of each panel; Landureau and al., 2021 ).

[0193] [Diagram 2] Results of screening 28 aptamer candidates against the F, R, 65, and 91 strands of α-Syn and their associated random sequences: Ratio between the amount of oligonucleotide that remains bound on the nitrocellulose membrane to the amount of associated random sequence (Scr1-G or Scr2-G) that remains bound. The graph shows the ratio between the amount of oligonucleotide that remains bound on the nitrocellulose membrane to the amount of associated random sequence (Scr1-G or Scr2-G) that remains bound.

[0194] [Diagram 3] Measurement of the affinity of aptamer N30-G for F- or R-type α-Syn fibrils.

[0195] [Figure 4] Measurement of the affinity of aptamer N30-G for “amyloid-β” type fibrils or fibril P110 of α-Syn or α-Syn monomers.

[0196] [Diagram 5] Measurement of the affinity of aptamer N124-G for F- or R-type α-Syn fibrils.

[0197] [Figure 6] Measurement of the affinity of aptamer N124-G for “amyloid-β” type fibrils or fibril P110 of α-Syn or α-Syn monomers.

[0198] [Figure 7] Detection of F-type α-Syn fibrils by aptamer N30-G after separation on an Sp6 column.

[0199] [Figure 8] Representative example of molecular fingerprinting of conformer presence by analysis of aptamer frequency in a mixture by high-throughput sequencing.

[0200] [Figure 9] Molecular fingerprinting of the presence of F or R conformers in the medium by sequencing the evolution of the frequency of 15 oligonucleotides. A mixture of 14 aptamers (designated N) and a control sequence (designated Scr-2 (scramble 2)) was incubated in medium containing F or R fiber conformers (conditions Fn1-Fn3 and Rn1-Rn3, respectively) or in a fiber-free environment (conditions 0n1-0n4). The figure shows the evolution of the frequency of each aptamer in the mixture compared to its initial frequency in the mixture. This analysis allows revealing a specific signature of each conformer. EXAMPLES

[0201] Example 1: Design and development of specific α-Syn conformational aptamers Materials and methods 1.1.1. SELEX

[0202] Several SELEX studies were performed on different α-synuclein fiber conformers, specifically those designated "F" and "R" conformers. F fiber conformers have a cylindrical appearance, whereas R fiber conformers are ribbon-like. In rodents, injection of F or R fiber conformers, respectively, has been shown to induce two different forms of synucleinopathies: Parkinson's disease and multiple system atrophy.

[0203] In RNA chemistry, we selected aptamers in which all pyrimidines were modified at the 2' position of the ribose with a fluorine group, a chemical modification known to significantly increase the resistance of RNA to degradation by ribonucleases (RNAses).

[0204] During these SELEXes, a counter-selection step was performed to ensure that the recognition specificity of the aptamers was directed to a particular conformational form and that the aptamers had little affinity for the monomeric form of α-Syn. For example, to select aptamers specific for the F fiber conformer, 10 15 A library of different oligonucleotides was preincubated with either the monomeric or R-fiber conformers. Only oligonucleotides with no affinity for these conformations were used for selection against the F-fiber conformer. The aim of this type of counterselection was to isolate aptamers with affinity for the F-fiber conformer but not for other forms of the protein.

[0205] The library samples were analyzed by high-throughput sequencing. From these SELEX, 28 candidates were selected (Table 7 below). The affinity of these candidates was screened by filtration through nitrocellulose.

[0206] 1.1.2. Affinity testing of candidate aptamers 10 nM of each candidate aptamer (hybridized to P32 radiolabeled oligonucleotides by heat shock) was incubated with 1 μM of α-Syn fiber conformer in a solution of Ts1X (10 mM HEPES (pH 7.6), 150 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2) containing 0.1% Igepal, and ssDNA was added in a ratio of 5-1 to oligonucleotide (i.e., at a level of 1.7 μg / ml). After 30 min of incubation at 37 °C, 25 μL of each mixture was deposited in triplicate on a nitrocellulose membrane and then filtered. Two washes with Ts1X were then performed. The amount of oligonucleotide retained on the nitrocellulose membrane was quantified by exposing the membrane to a photostimulable phosphorus screen.

[0207] 1.1.3. Determination of the dissociation constants of candidate aptamers and comparison with prior art aptamers The affinity of the aptamers was evaluated using a second method that measures the amount of complex formed by varying the concentration of the aptamer while keeping the concentration of the target constant. During these measurements, the target concentration is assumed to be very high compared to the aptamer concentration, so it is possible to neglect the amount of target bound to the aptamer compared to the amount of free target. If the interaction between the candidate and the target is of the "specific" type, the curve of the amount of complex formed as a function of the concentration of the candidate initially present should be hyperbolic and show saturation. The affinity constants, Bmax and Kd, of the aptamer for the target can then be calculated.

[0208] The affinity of the candidate aptamers was also compared with aptamers described in the literature as being selected against α-syn. Aptamers M5-15 and T-SO508 were selected by Tsukagoshi et al. in 2010 and 2012, respectively (Tsukagoshi et al., 2010, 2012). These DNA chemistry aptamers were selected to recognize α-syn monomers and oligomers, respectively. T-SO508, which recognizes α-Syn oligomers with a kd of 68 nM, was also shown to have affinity for Aβ40 oligomers (kd of 25 nM). Aptamers F5R1 and F5R2 were also selected by DNA chemistry and were selected by Zheng et al. in 2019 (Zheng and al., 2018; Ren and al., 2019). These recognize α-Syn with a kd of 2.4 and 3.07 nM, respectively. Finally, we also tested the DNA chemistry aptamer Tau 3146. This aptamer was isolated by a rapid method called "Non-SELEX" in which three consecutive rounds of selection were performed against the monomeric isoform Tau 441 without amplification between selections (Lisi and al., 2018). The aptamer Tau 3146 was shown to be able to bind to the isoforms Tau 441, Tau 381, Tau 352, and Tau 383 with kds of 13±3nM, 116±6nM, 84±6nM, and 49±4nM, respectively.

[0209] Since these aptamers are all of DNA chemistry, we need to compare random sequences of comparable length with the same chemistry. We selected a DNA sequence used as a PCR primer in the laboratory as a control. This primer, named "Scr_DNA", has a size of 87 nucleotides and is comparable to the sequence of the DNA aptamer selected from the literature (Table 7 below).

[0210] [Table 7]

[0211] The affinity of the candidate and literature aptamers was measured against different "amyloid" type fibrils (Tau1N3R and Aβ40 fibrils) in addition to the F and R α-syn fibril conformers. We also measured the affinity of the candidate aptamers against α-Syn fibril "P110", which was made from an α-Syn protein truncated at amino acid number 110 (thus missing 30 amino acids of the C-terminal domain).

[0212] 10 nM of oligonucleotide (aptamer) (hybridized by heat shock to SpG-LNA radiolabeled with P32 for RNA2'F chemical oligonucleotides or to SpG-LNA directly labeled with P32 for DNA oligonucleotides) is given to 250 nM of protein in a solution of Ts1X containing 0.1% Igepal, ssDNA is added at a ratio of 5-1 to oligonucleotide (i.e., 0.135 μg / ml to 17.325 μg / ml). After 30 min of incubation at 37 °C, 25 μL of each mixture is deposited on a nitrocellulose membrane and then filtered. Two washes with Ts1X are then performed. The amount of oligonucleotide retained on the nitrocellulose membrane is quantified by exposing the membrane to a photostimulable phosphorus screen. K d and B. max was calculated using Prism software using a nonlinear regression model that assumes only one binding site on the target for the candidate and analyzes the total affinity (specific and nonspecific) of the candidate for the target.

[0213] Measurements were performed in triplicate (three independent experiments on different days) for F- and R-type α-Syn fibrils and in duplicate for other fibrils (Tau1N3R, Aβ40 fibrils, P110 fibrils) and α-Syn monomers.

[0214] 1.1.4. Sp6 column affinity test Oligonucleotides radiolabeled with P32 (by hybridization to SpG-LNA labeled with P32 in the case of 2'F-PyRNA sequences or to SpG-LNA directly labeled in the case of DNA sequences) were mixed with 250 nM of protein (F fiber or α-Syn monomer) in Ts1X, Igepal 0.1%. After 30 min of incubation at 37 °C, 25 μL of the mixture was deposited on an Sp6 column whose buffer had been changed beforehand to Ts1X. After a first centrifugation of the deposit, the column was washed twice with Ts1X. Finally, the column was eluted by washing with a 2% SDS solution. Fractions were then deposited on 24-well plates and exposed for several hours on a photostimulable phosphor screen. Exposure analysis allowed the quantification of each fraction for each condition.

[0215] 1.1.5. Molecular fingerprinting by aptamer frequency analysis of mixtures by high-throughput sequencing (FootBal-Seq) A mixture of 14 aptamers (designated N) and a control sequence (designated Scr-2 (scrambled 2)) was incubated in medium containing F-fiber or R-fiber conformers (conditions Fn1–Fn3 and Rn1–Rn3, respectively) or in a fiber-free environment (conditions 0n1–0n4). The mixture was filtered through an exclusion column, and the sequences retained on the column after three washes were eluted with 2% SDS. After phenol-chloroform extraction, the oligonucleotide mixture was amplified by RT-PCR. During this step, the sequences were extended by "adapter" sequences, allowing high-throughput sequencing. After purification by agarose gel electrophoresis, the mixture was sequenced by high-throughput sequencing.

[0216] 1.2. Results 1.2.1. Aptamers selected by Selex Table 8 below lists the sequences of successful candidates.

[0217] [Table 8-1] [Table 8-2]

[0218] 1.2.2. Affinity of candidate aptamers to different α-Syn conformers Figure 2 shows that aptamers N3, N30, N124, 4F01, 4F02, 4F03, 4F05, F124 and P65 remain bound to the F-form α-syn fibril conformer in much greater amounts than the random sequences. In particular, candidates N3-G, N30-G and N124-G bind on average 3.5-fold more to F-form α-syn fibrils than Scr2-G.

[0219] Our data further show that aptamers N3, N30, N124, 4F01, 4F02, 4F03, 4F05, F124, and P65 remain bound to the F-form α-Syn fibril conformer in much greater amounts than the other conformers (R, 91, and 61; Figure 2). These results indicate that aptamers N3, N30, N124, 4F01, 4F02, 4F03, 4F05, F124, and P65 can distinguish the F-form α-Syn fibril conformer from other α-Syn conformers.

[0220] 1.2.3. Determination of the dissociation constants of candidate aptamers and comparison with prior art aptamers Representative results from each affinity test are shown in Figures 3 and 4 for aptamer N30 and in Figures 5 and 6 for aptamer N124. If an aptamer is determined to bind significantly more to the protein than the associated random sequence, the K calculated by Prism is d and B. max The calculated values ​​are shown in the graph.

[0221] Tables 9 to 12 below show the calculated K for each aptamer and each protein from all duplicate or triplicate experiments performed, by fiber type. d and B. max This is a summary of the average values.

[0222] [Table 9]

[0223] [Table 10]

[0224] [Table 11]

[0225] [Table 12]

[0226] The coefficient of variation is shown as a percentage and is the ratio between the standard deviation of the value and its mean. The "Reproducibility" column indicates the number of experiments in which the binding between the aptamer and the target was measured. The symbol "-" means that the experiment was not performed. If the binding of the tested aptamer was not significantly greater than that of the associated random sequence, the term "no binding" is indicated.

[0227] The data show that affinity measurements for F-fibers are highly reproducible: aptamers N30-G, N124-G, T-SO508 and F5R1 bind to F-type α-Syn fibers with a Kd of less than 10 nM in each experiment. Aptamers T-SO508 and F5R1 also bind to R-type α-Syn fibers in each experiment, but aptamers N30-G and N124-G do not show significantly higher binding to R-type α-Syn fibers compared to the associated random sequence. Aptamers M5-15, F5R2 and Tau 3146 do not bind to either F or R-type α-Syn fibers (no significantly higher binding than the associated random sequence).

[0228] Thus, the data show that among the aptamers tested, only aptamers N30-G and N124-G are able to distinguish F-fibers from R-fibers.

[0229] Prior art aptamers either recognize the two fibers with comparable affinity (aptamers T-S0508 and F5R1) or fail to recognize either R-type or F-type α-Syn fibers (M5-15, F5R2 and Tau 3146).

[0230] The aptamers T-SO508 and F5R1 bind to the K d The B for R fiber is low. max is high (a factor 2 difference). However, d A two-fold difference in is not sufficient to reliably distinguish between the two types of fibers.

[0231] Figures 4 and 6 further show that aptamers N30 and N124 have no affinity for α-Syn monomers.

[0232] Similarly, data reveal that aptamers N30 and N124 do not significantly recognize amyloid-β fibrils (FIGS. 4 and 6, Table 9).

[0233] All these data indicate that aptamers N30 and N124 have a highly specific affinity for Fα-Syn fibrils: in fact, they are able to distinguish, in a reproducible and specific manner, Fα-Syn fibrils not only from Rα-Syn fibrils but also from α-Syn monomers and other types of fibrils, such as amyloid-β fibrils.

[0234] 1.2.4. Sp6 column affinity test The affinity of aptamer N30-G for F-fiber conformers or monomers was tested by filtration on an exclusion column (Biorad Sp6). The performance of aptamer N30-G was compared to that of aptamer T-SO508. Sequences Scr2-G and Scr_DNA were used as controls.

[0235] The results of the detection test are shown in Figure 7. The data show that these results are reproducible. When the oligonucleotides were applied to F-type α-Syn fibers, it was observed that 16.7% of N30-G, 2.1% of Scr2-G, 3.9% of T-SO508, and 3.0% of Scr_DNA were eluted in the fraction washed from the Sp6 column with 2% SDS. This means that N30-G has about 8 times better retention on F-type α-Syn fibers than the related random sequences and 4 times better than T-SO508. These results further confirm the ability of N30-G to recognize F-type fibers and also show its superiority compared to T-SO508.

[0236] The data show that when N30-G was applied to monomers, only 1.1% of the oligonucleotides were eluted in the 2% SDS elution fraction, which means that N30-G recognizes F-type α-Syn fibers approximately 16 times better than monomers. Interestingly, the fraction eluted with 2% SDS was similar when T-SO508 was applied to F-type α-Syn fibers and monomers (3.9% and 3.8%, respectively). These results indicate that T-SO508 does not distinguish between F-type α-Syn fibers and monomers.

[0237] 1.2.5. Obtaining diagnostic molecular fingerprints by FootBal-Seq We developed a new diagnostic method using a mixture of aptamers to diagnose the presence of fiber conformers in the medium (Figure 8). 14 of the 28 aptamers developed were selected and mixed in equimolar amounts as well as a control sequence. This mixture was incubated in a medium containing no fiber or fiber conformer F or fiber conformer R. The mixture was then deposited on an Sp6 exclusion column. After amplification and purification, the mixture was sequenced by high-throughput sequencing. The proportion of each sequence in the mixture is compared to the starting proportion.

[0238] In Figure 9, the evolution of the frequency of each aptamer in the mixture is shown compared to its initial frequency in the mixture. This analysis can reveal specific signatures of each conformer. For example, N30, N73 and N15 are strongly enriched for the F-fiber conformer compared to other sequences, while sequences N30, N124, N5, N73 and N15 are enriched for the R-fiber conformer.

[0239] Thus, the data highlight specific signatures for F- and R-fiber conformers (Figure 9).

[0240] These results were confirmed on patient samples. These data confirm that such a mixture of aptamers can be used to obtain a signature from a patient's biological sample, which allows the diagnosis of neurodegenerative diseases. This mixture makes it possible, among other things, to determine whether a patient suffers from a synucleinopathic disease or another type of neurodegenerative disease, which can also be determined by a similar test.

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Claims

**Claim 1** An aptamer, characterized by having the ability to distinguish the conformer of the F-type α-Syn fiber of α-Syn protein (α-Syn) from the conformer of the R-type α-Syn fiber, and comprising a sequence specific to a modified ribonucleic acid (RNA) having at least 85% identity with a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, preferably selected from SEQ ID NO: 1 and SEQ ID NO:

2. **Claim 2** The dissociation constant K of the aptamer measured for the conformer of the F-type α-Syn fiber d(F) is a) Dissociation constant K measured for the conformer of R-type α-Syn fiber d(R) Lower, preferably at least 10-fold lower; b) Dissociation constant K measured for the α-Syn monomer d(Mono) Lower, preferably at least two-fold lower; c) Dissociation constant K of random aptamers measured for conformers of F-type α-Syn fibers d(Random) Lower, preferably at least 2-fold lower; d) Dissociation constant K measured for the conformer of R-type α-Syn fibers d(R) Lower, preferably at least 10-fold lower; and the dissociation constant K measured for the α-Syn monomer d(Mono) Lower, preferably at least 2-fold lower; e) Dissociation constant K measured for the conformer of R-type α-Syn fibers d(R) Lower, preferably at least 10-fold lower; and the dissociation constant K of a random aptamer measured for the conformer of F-type α-Syn fibers d(Random) Lower, preferably at least 2-fold lower; or f) Dissociation constant K measured for the conformer of R-type α-Syn fibers d(R) Lower, preferably at least 10-fold lower than the dissociation constant K measured for the α-Syn monomer d(Mono) Lower, preferably at least 2-fold lower than; and the dissociation constant K of the random aptamer measured for the conformer of F-type α-Syn fibers d(Random) Lower, preferably at least 2-fold lower than, the aptamer according to claim 1 **Claim 3** At least one of the following dissociation constants K d The aptamer according to claim 1, having: a) The dissociation constant K measured for the conformer of the F-type α-Syn fiber d(F) is less than 15 nM, preferably less than 10 nM; and / or b) Dissociation constant K measured for the conformer of R-type α-Syn fiber d(R) is greater than 100 nM, preferably greater than 150 nM. **Claim 4** i. A modified RNA primer sequence having at least 85% identity, preferably at least 90% identity, preferably at least 95% identity, with a sequence selected from SEQ ID NO: 29 and SEQ ID NO: 30, preferably located at the 5'-end of the specific sequence, at the 5'-end of the specific sequence; and / or ii. A modified RNA primer sequence having at least 85% identity, preferably at least 90% identity, preferably at least 95% identity, with a sequence selected from SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 33, preferably located at the 3'-end of the specific sequence, at the 3'-end of the specific sequence, the aptamer according to claim 1, further comprising. **Claim 5** The aptamer according to claim 1, comprising a modified RNA sequence having at least 85% identity with a sequence selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40, preferably selected from SEQ ID NO: 34 and SEQ ID NO:

35. **Claim 6** A kit comprising at least one aptamer according to claim 1. **Claim 7** The kit according to claim 6, further comprising at least one additional aptamer selected from aptamers comprising a sequence that has at least 85% identity with a modified RNA selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28; Preferably, the kit further comprises an aptamer comprising a random sequence of modified RNA.

8. The at least one additional aptamer is i. a modified RNA primer sequence having at least 85% identity, preferably at least 90% identity, preferably at least 95% identity, with a sequence selected from the group consisting of SEQ ID NO: 29 and SEQ ID NO: 30, located 5' of the specific sequence, preferably at the 5' end of the specific sequence; and / or ii. a modified RNA primer sequence having at least 85% identity, preferably at least 90% identity, preferably at least 95% identity, with a sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, located 3' of the specific sequence, preferably at the 3' end of the specific sequence, The kit according to claim 7, further comprising.

9. The at least one additional aptamer is selected from aptamers comprising a modified RNA sequence having at least 85% identity with a sequence selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60, the kit according to claim 6.

10. - an aptamer comprising a sequence specific for a modified RNA having at least 85% identity with SEQ ID NO: 1, - an aptamer comprising a sequence specific for a modified RNA having at least 85% identity with SEQ ID NO: 2, - An aptamer comprising a sequence specific for a modified RNA having at least 85% identity with SEQ ID NO: 3, - An aptamer comprising a sequence specific for a modified RNA having at least 85% identity with SEQ ID NO: 8, - An aptamer comprising a sequence specific for a modified RNA having at least 85% identity with SEQ ID NO: 9, - An aptamer comprising a sequence specific for a modified RNA having at least 85% identity with SEQ ID NO: 10, - An aptamer comprising a sequence specific for a modified RNA having at least 85% identity with SEQ ID NO: 11, - An aptamer comprising a sequence specific for a modified RNA having at least 85% identity with SEQ ID NO: 12, - An aptamer comprising a sequence specific for a modified RNA having at least 85% identity with SEQ ID NO: 13, - An aptamer comprising a sequence specific for a modified RNA having at least 85% identity with SEQ ID NO: 14, - An aptamer comprising a sequence specific for a modified RNA having at least 85% identity with SEQ ID NO: 16, - An aptamer comprising a sequence specific for a modified RNA having at least 85% identity with SEQ ID NO: 17, and - An aptamer comprising a sequence specific for a modified RNA having at least 85% identity with SEQ ID NO: 18, The kit according to claim 6, comprising at least; Preferably, a kit further comprising an aptamer comprising a random sequence of modified RNA.

11. When the kit comprises a plurality of aptamers, the aptamers are a) all contained in one composition, or b) distributed in several different compositions in separate containers (including the case where each aptamer is contained in a different composition placed in a separate container), the kit according to claim 6.

12. The RNA of the aptamer or all of the aptamers of the kit is modified to increase resistance to RNA nuclease, preferably, the ribose of the pyrimidine of the aptamer or all of the aptamers of the kit carries a fluorine atom at the 2'-position carbon, the aptamer according to claim 1 or the kit according to claim 6.

13. a) Detection of the presence or absence of at least one conformer of F-type α-Syn fibers in a biological sample; b) Determination of the amount of conformers of F-type α-Syn fibers in a biological sample; c) Establishment of the molecular fingerprint of α-Syn fibril conformers, preferably F-type α-Syn fibrils and R-type α-Syn fibrils, in a biological sample; d) Screening for compounds / molecules capable of detecting and / or recognizing the conformer of F-type α-Syn fibrils, preferably screening for compounds / molecules capable of distinguishing the conformer of F-type α-Syn fibrils from the conformer of R-type α-Syn fibrils; or e) Any combination of a) to d), for in vitro use of at least one aptamer according to claim 1, at least one kit according to claim 6, or any combination thereof.

14. An in vitro method for diagnosing synucleinopathy in a subject having at least one symptom of a neurodegenerative disease, comprising: a) contacting a biological sample of the subject with at least one aptamer according to claim 1, at least one kit according to claim 7, or any combination thereof; b) detecting the presence or absence of the conformer of at least one F-type α-Syn fibril, quantifying the conformer of F-type α-Syn fibrils, establishing the molecular fingerprint of α-Syn fibril conformers (preferably F-type and R-type α-Syn fibrils of α-Syn), or any combination thereof, in the biological sample of the subject; and c) diagnosing the presence or absence of synucleinopathy in the subject based on the results of step b). A method comprising the above steps.

15. An in vitro method for stratifying synucleinopathy, pre-diagnosing the prognosis of synucleinopathy, monitoring synucleinopathy, or evaluating the effectiveness of treatment of synucleinopathy in a subject suffering from synucleinopathy, comprising: a) contacting a biological sample of the subject with at least one aptamer according to claim 1, at least one kit according to claim 7, or a combination thereof; b) detecting the presence or absence of at least one conformer of F-type α-Syn fibers, quantifying the conformer of F-type α-Syn fibers, establishing the molecular fingerprint of α-Syn fiber conformers (preferably F-type α-Syn fibers and R-type α-Syn fibers of α-Syn), or a combination thereof in the biological sample of the subject; and c) stratifying synucleinopathy, prognostic diagnosis of synucleinopathy, monitoring of synucleinopathy, evaluating the effectiveness of treatment of synucleinopathy in the subject based on step b), comprising wherein the synucleinopathy is preferably selected from Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), more preferably the synucleinopathy is DLB, a method.

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