WO2019035763A1 - Aptamers for detection of zika flaviviral protein - Google Patents

Abstract

Disclosed are one or more aptamers which bind to flaviviral non-structural protein 1 (NS1 ), particularly Zika NS1. Also disclosed are methods for detecting the presence of Zika NS1 in a sample and kit comprising the aptamers.

Description

APTAMERS FOR DETECTION OF ZIKA FLAVIVIRAL PROTEIN

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of priority of Singapore patent application No. 10201706719V, filed 16 August 2017, the contents of it being hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

[002] The present invention generally relates to virology. In particular, the present invention relates to means of detecting flaviviral protein.

BACKGROUND OF THE INVENTION

[003] Diagnosis of a flavivirus infection, for example a Zika virus infection, has received increased attention given the high potential of virus spread from a pregnant woman to her fetus, resulting in microcephaly. Therefore, early detection of a flavivirus infection would be beneficial, giving the possibility to control spread of infection before reaching epidemic proportions, and allowing timely treatment of the infected subjects.

[004] Conventional methods of detection include reverse transcription quantitative polymerase chain reaction (RT-qPCR) or serological methods for detecting flavivirus group antigens or immunoglobulins. However, these methods contain inherent disadvantages, such as false-positive results, or cannot be used for early detection of a flavivirus infection.

[005] In view of the above, there is a need to provide a means of detecting the presence of a flavivirus protein expressed during viral infection. SUMMARY

[006] In one aspect, the present invention refers to an aptamer specifically binding to Zika viral non- structural protein 1 (NS l), wherein the target sequence of the aptamer comprises or consists of SEQ ID NO: 19.

[007] In another aspect, the present invention refers to a method for detecting the presence of flaviviral non-structural protein 1 (NS l) in a sample, the method comprising contacting a sample obtained from a patient with one or more aptamers as disclosed herein; detecting the presence or absence of binding of the one or more aptamers in the sample, wherein the presence of binding of the one or more aptamers in the sample indicates the presence of flaviviral non- structural protein 1 (NSl).

[008] In yet another aspect, the present invention refers to a kit comprising the aptamers as disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[009] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0010] FIG. 1 shows a scatter plot representing data generated using a binding affinity test based on association and dissociation of 7th round of SELEX pool (SE-7) single strand (ss) DNA aptamer pool or random 60 nucleotide ssDNA library to 100 nM Zika NSl protein. The scatter plot shows that the SE-7 ssDNA aptamer pool can bind to 100 nM Zika NSl protein (black) in comparison to the random 60 nucleotide ssDNA library (grey). Binding (nm) refers to changes in optical interference (e.g., shift of the wavelength).

[0011] FIG. 2 shows two scatter plots representing data generated using binding affinity tests based on association and dissociation of aptamers to different concentrations of Zika NS 1 protein. (A) shows the binding affinity test of representative 100 nucleotide aptamer 2 (SEQ ID NO: 15), wherein K_D = 24 pM. (B) shows the binding affinity test of representative 100 nucleotide aptamer 10 (SEQ ID NO: 17), wherein K_D = 134 nM.

[0012] FIG. 3 shows a diagram of four predicted secondary structures and binding affinities of the aptamers. (A) shows full-length ssDNA 100 nucleotide aptamer 2 (SEQ ID NO: 15; K_D = 24 pM). (B) shows the predicted minimum binding domain 41 nucleotide aptamer 2 (SEQ ID NO: 16; K_D = 45 pM). (C) shows the full-length ssDNA 100 nucleotide aptamer 10 (SEQ ID NO: 17; K_D = 134 nM). (D) shows the predicted minimum binding domain 54 nucleotide aptamer 10 (SEQ ID NO: 18; K_D = 240 nM).

[0013] FIG. 4 shows two scatter plots representing data generated using binding affinity tests of the predicted minimum binding domains of aptamers to different concentrations of Zika NS l protein based on association and dissociation. (A) shows the binding affinity test of minimum binding domain 41 nucleotide aptamer 2 (SEQ ID NO: 16), wherein K_D = 45 pM. (B) shows the binding affinity test of minimum binding domain 54 nucleotide aptamer 10 (SEQ ID NO: 18), wherein K_D = 240 nM. [0014] FIG. 5 shows two scatter plots representing data generated using binding specificity tests based on association and dissociation of aptamers to Zika NS l, Dengue NSl serotypes 1 - 4, and the negative control interferon γ (ΓΡΝγ). (A) shows that 41 nucleotide aptamer 2 (SEQ ID NO: 16) can also bind to four Dengue NSl serotypes, but not ΓΕΝγ. (B) shows that 54 nucleotide aptamer 10 (SEQ ID NO: 18) binds only Zika NSl, and does not bind to all Dengue NSl serotypes and ΓΓΝγ. This illustrates that 41 nucleotide aptamer 2 (SEQ ID NO: 16) can bind to flavivirus NSl proteins with some degree of selectivity, but not 54 nucleotide aptamer 10 (SEQ ID NO: 18).

[0015] FIG. 6 shows two scatter plots data representing data generated using sandwich complex formation of aptamer 2-Zika NS l-aptamer 10 complex based on association and dissociation of aptamer 10 to the aptamer 2-Zika NS l complex. (A) shows that aptamer 10 binds to the pre-formed aptamer 2-NS 1 protein complex, compared to the negative control using buffer only. (B) shows the association and dissociation normalized from (A).

[0016] FIG. 7 shows a line graph representing an ELISA assay based on the aptamer 2- Zika NSl-aptamer 10 sandwich complex, wherein the aptamer 2-Zika NSl-aptamer 10 sandwich complex-based ELISA has a detection limit of 100 ng/mL in buffer.

[0017] FIG. 8 shows two scatter plots representing data generated using sandwich complex formation of aptamer 2-Zika NSl-anti-Zika NSl monoclonal antibody complex based on association and dissociation of anti-Zika NSl monoclonal antibody to the aptamer 2-Zika NS 1 complex. (A) shows that anti-Zika NS 1 monoclonal antibody elicits an additional binding to the pre-formed aptamer 2-NS1 protein complex, compared to negative control using buffer only. (B) shows the association and dissociation normalized from (A).

[0018] FIG. 9 shows two line graphs representing data generated using ELISA assays based on the aptamer 2-Zika NSl-anti-Zika NSl monoclonal antibody sandwich complex. (A) shows that the aptamer 2 -Zika NS l-anti-Zika NSl monoclonal antibody sandwich complex-based ELISA has detection limits of 0.1 ng/mL and 10 ng/mL in buffer and 100% human serum respectively. (B) shows that the aptamer 2-Zika NS l-anti-Zika NSl monoclonal antibody sandwich complex-based ELISA has a detection limit of 1 ng/mL in 10% human serum.

[0019] FIG. 10 shows two line graphs representing data generated using ELISA assays based on the aptamer 2-Zika NS l-anti-Zika NSl monoclonal antibody sandwich complex for Dengue NS l serotypes 1, 2, 3 and 4. (A) shows that Dengue NSl serotypes 1, 2, 3 and 4 are not detected. (B) is the zoomed in version of (A).

[0020] FIG. 11 is a photo of a polyacrylamide gel that shows the bands of 5'-biotin- modified 41 nucleotide aptamer 2 (SEQ ID NO: 16) in 98% human serum, treated with urea for 0 minutes, 30 minutes and 60 minutes. The intensity and thickness of the bands at the three time points are similar, illustrating that the amount of aptamers are similar at the time points.

[0021] FIG. 12 shows a line graph representing data generated using the sandwich complex-based ELISA assay for Dengue NS l serotypes 1, 2, 3 and 4, West Nile Virus NS l and Yellow Fever Virus NS l (The Native Antigen Company, UK) using aptamer 2 as the capture agent and anti-Zika NS 1 monoclonal antibody for detection in binding buffer. Anti- rabbit IgG-HRP conjugate was used for the colour development. After colour development using TMB-based substrate and stop solution, absorbance was measured at 450 nm using Cytation3 multi-mode plate reader (BioTek, USA). Background absorbance from the well without Dengue NSl was subtracted. X axis is in loglO scale. Error bars are ±SD.

[0022] FIG. 13 shows a scatter plot representing data generated using a commercial antibody (SQabl610) and an anti-Zika NS l antibody for forming a sandwich complex with Zika NS 1 protein as monitored using BLItz label-free bio-layer interferometry system to test for complementarity.

[0023] FIG. 14 shows a line graph representing data generated using sandwich complex- based ELISA assay based on commercial antibody (SQabl610) and an anti-Zika NSl antibody, antibody SQAM610 (Arigo Biolaboratories, Taiwan) was used as the capture agent and anti-Zika NSl antibody was used as the detection agent. Background absorbance from the well without Zika NS 1 was subtracted. X axis is in loglO scale. Error bars are +SD.

DETAILED DESCRIPTION

[0024] Diagnosis of a flavivirus infection, for example, a Zika virus infection, has received increased attention given the high potential of virus spread from a pregnant woman to her fetus, resulting in microcephaly. Therefore, early detection of a flavivirus infection is beneficial to controlling any potential epidemics and allowing timely treatment.

[0025] Zika virus poses great dangers to women at pregnancy as the virus can be passed from pregnant women to their fetus, resulting in microcephaly. It has been also reported that Zika virus infection could be associated with Guillain-Barre syndrome (GBS) and meningoencephalitis in adult. Given a lack of rapid and sensitive diagnostic tools for Zika virus at the present time, the recent outbreak of Zika virus infection in Brazil in 2015 and many others reported in more than 20 countries all over the world with an increase in cases suggest the sensitive diagnostic tools for Zika infection are in urgent demand.

[0026] In clinical setting, early diagnosis of Zika infection would be more effective to control epidemic and for timely treatment. For this purpose, RT-qPCR assay using specific primers to detect viral RNAs is recommended as a preferred diagnostic method. This method however has some inherent disadvantages, including false-negative results from new strains or false-positive results arising from sample contamination. Hence, the RT-qPCR results need to be further confirmed by other types of assays. Another preferred option is to use serological methods for detecting either Zika viral antigens (for example, NSl) or immunoglobulins (for example, IgG and IgM antibodies). Since IgG/IgM detection generally works in later stage of Zika fever (>7 days from symptom onset but variable from case to case), early detection of Zika infection relies more on NS 1 viral antigen (e.g., up to 9 days for Dengue NSl), which is more sensitive than other Zika envelope proteins. A combination of NS l- and IgG/IgM-based detections certainly would be very appropriate and would lead to higher accuracy and reliability of disease diagnostics.

[0027] For the diagnostic purpose, target-binding aptamers selected using SELEX (Systematic Evolution of Ligands by Exponential enrichment) technology offer some advantages over antibodies in terms of high batch-to-batch consistency, lowered cost, better stability, the ease of modification, to name a few. Therefore, in establishing the method disclosed herein for finding diagnostic agents for Zika infection, it was opted to replace antibodies as much as possible with aptamers. The use of aptamers stems further from the fact that no useful aptamers for Zika NSl antigen have been reported so far, while several anti-Zika NSl antibodies for ELISA are presently commercially available in the market.

[0028] As disclosed herein, the SELEX protocol was used to discover a number of single stranded DNA (ssDNA) aptamers, exhibiting pico- to nanomolar binding affinities toward Zika NS l antigen with one aptamer able to highly specifically recognize Zika NSl but not any of four Dengue NS 1 serotypes.

[0029] Moreover, these selected ssDNA aptamers can pair with each other or with an anti- Zika virus antibody, enabling aptamer-mediated highly specific and sensitive detection of Zika NS 1 protein in the sandwich complex -based applications such as ELISA assay. Moreover, the best sensitivity could reach as low as 0.1 ng/mL in binding buffer or 1 ng/mL in 10% human serum.

[0030] Given the prevalence and the well-defined consensus sequence of the non- structural protein 1 (NS1) within the flavivirus family, the Zika NS 1 can be used as an exemplary target as an early diagnosis marker for Zika virus infection. Since the symptoms from Zika virus infections are similar to, if not indistinguishable from, the symptoms of other flavivirus infections mediated by, for example, the Dengue virus, specific discrimination of the infecting agent, as well as the detection of flaviviral infection, is needed and is considered to be beneficial for its medical diagnosis in pregnant women.

[0031] In order to detect the presence or absence of such flaviviral proteins, the present application describes various aptamers. As used herein, the term "aptamer" refers to a type of nucleic acid molecule with antibody-like binding properties toward their targets. Aptamers are known to have advantages over antibodies when used in serological methods such as, for example, batch-to-batch quality, cost, stability and the ease of modification. Therefore, aptamers can be used as an alternative to, for example antibodies, in the diagnosis and as therapeutics.

[0032] Thus, the aptamers disclosed herein bind to flavivirus proteins, and can therefore be used as means to identify, for example, early stages of flavivirus infection, and be incorporated into a diagnostic tool.

[0033] Aptamers can be produced using a method called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). SELEX begins with the step of synthesizing an aptamer library. In one example, the aptamer library comprises sequences that are about 100 nucleotides in length. In another example, the 100 nucleotide long sequences comprise a random region. As used herein, the term "random region" refers to one or more regions within the sequence of a nucleic acid molecule which comprises sequences of undefined random nucleotides (Ideally A:G:C:T = 25%:25%:25:25% ratio in N position). These sequences can be later determined using deep sequencing analysis. In another example, the random region can be, but is not limited to, a length of about 40 to 80 nucleotides, or about 50, about 60, about 70 or about 80 nucleotides in length. In another example, the random region is 60 nucleotides long. In another example, the aptamer library comprises a 100 nucleotide long sequence which comprises a random 60 nucleotide region, as shown, for example, in SEQ ID NO: 1.

[0034] The aptamer library comprises randomly generated nucleotide sequences of fixed length flanked by primers, which are shorter random sequences. The term "primer" as used herein refers to a nucleic acid molecule, for example, an oligonucleotide whether derived from a naturally occurring molecule such as one isolated from a restriction digest or one produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, i.e., in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature and pH. The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. For example, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization. The exact lengths of the primers will depend on many factors, including temperature and source of primer. For example, depending on the complexity of the target sequence, the oligonucleotide primer typically contains at least 15, more preferably 18 nucleotides, which are identical or complementary to the template and optionally a tail of variable length which need not match the template. The length of the tail should not be so long that it interferes with the recognition of the template. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. In one example, the primer can target the 5' end and/or 3' end of the aptamer library. In another example, the primer can be about 15 nucleotide to about 25 nucleotides in length. In another example, the primer can be about 15 nucleotides, about 16 nucleotides, about 17 nucleotides, about 18 nucleotides, about 19 nucleotides, about 20 nucleotides, about 21 nucleotides, about 22 nucleotides, about 23 nucleotides, about 24 nucleotides, or about 25 nucleotides in length. In yet another example, the primer is 20 nucleotides in length.

[0035] In another example, the primer comprises one or more restriction site. The term "restriction site" refers to a specific sequence of nucleotides that are recognized by restriction enzyme. In yet another example, primer comprises one or more restriction site are recognized by restriction enzymes such as, but not limited to, EcoRI, Sac I, Hindlll, Sail, or combinations thereof.

[0036] In another example, the primer comprises one or more tags at the 5' or 3' end. Such tags can be used to, for example, detect or isolate and purify the attached molecules. Thus, a person skilled in the art would know and would be able to use similar tags to attain the same result. These tags can be, but are not limited to, phosphate, histidine FLAG, 3xFLAG, HA, MYC, biotin, streptavidin, and fluorescent tags, such as green fluorescent protein, and multiples or combinations thereof. In yet another example, the primer consists of a phosphate tag at the 5' end. As used herein, these tags are used to recognise the antisense strand, such that the antisense strand can be removed at the end of each SELEX cycle, and the ssDNA library can be used for the next SELEX selection round. In one example, the phosphate tag can be found at the 5' end of the primer, wherein a phosphorylated antisense strand is generated. The phosphorylated antisense strand can be recognised by the enzyme λ- exonuclease and be removed by λ-exonuclease digestion. In a further example, the primer comprises SEQ ID NO: 2 or 3. In yet another example, the primer is SEQ ID NO: 2 or 3.

[0037] SELEX also includes a step wherein the sequences in the library are exposed to a target sequence during the selection step. As used herein, the term "target sequence" refers to the sequence from which the aptamer was derived from during SELEX. In one example, the target sequence of the aptamer comprises a sequence derived from a flavivirus, in other words, a flaviviral protein. In yet another example, the target sequence of the aptamer comprises a sequence of flavivirus NS 1. In yet another example, the target sequence of the aptamer comprises a sequence of Zika NS 1. In yet another example, the target sequence of the aptamer comprises SEQ ID NO: 19. In yet another example, the target sequence of the aptamer is SEQ ID NO: 19.

[0038] The selection step in SELEX can be repeated, resulting in the enrichment of aptamers, wherein more rounds of selection indicate a higher stringency in terms of target aptamer binding. In one example, the aptamer can be enriched by SELEX. In yet another example, the aptamer can be enriched after 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 rounds of SELEX. In yet another example, the aptamer can be enriched after 7 rounds of SELEX. This is shown, for example, in FIG. 1 , wherein the aptamer pool enriched after 7 rounds of SELEX bound to Zika NS 1. [0039] The enriched aptamer pool comprises aptamer clones, wherein the aptamer clones are sequences that comprise a deviation of n in the sequence in comparison to the original sequence In other words, the aptamer library utilised contained fully randomized 60 nucleotide region, as described in previous section, and the aptamers can be selected and enriched depending on the target binding affinity and specificity during SELEX rounds. The identity of the sequences present in the initial library were not known, as the library was fully randomized. In one example, the aptamer clone can be sequences of between about 90 to about 110 nucleotides in length. In yet another example, the aptamer clone is about 100 nucleotides in length. In yet another example, the aptamer clone comprises SEQ ID NO: 4. In another example, the aptamer clone comprises or consists of the whole or part of one or more consensus sequences as disclosed herein. Further examples of the sequences on the random 60 nucleotide region and consensus sequences are shown in Table 1 below. For example, clone 3 has the sequence CACAGACTCC ATCTTGGATT GCAAAGGtCT GCTGTGTGGT AGTCTGTGGA GGCCATGTCT on the random 60 nucleotide region (5' to 3'), wherein the underlined section "GGtCTGCT" is the consensus sequence, wherein t is a mutation on the consensus sequence.

SEQ ID NO of

SEQ ID NO of sequence on

Clone Sequences on random 60 nucleotide consensus random 60

region (5' to 3') sequence, if nucleotide

present region (5' to 3')

ATACCGGTGC CATATTCCAC

1 (42.9%) AAGGGGGACT GCTCGGGATT 5 20

GCGGATTTGT GGAATTGTTG

ACTAGGTTGC AGGGGACTGC

2 (7.4%) TCGGGATTGC GGATCAACCT 6 21

AGTTGCTTCT CTCGTATGAT

CACAGACTCC ATCTTGGATT

3 (6.5%) GCAAAGGtCT GCTGTGTGGT 7 22

AGTCTGTGGA GGCCATGTCT

ACTCCGCGAT AGACGGTTCT

4 (5.0%) GCATGCACGT TCCTCCGACG 8 n.a.

TCCCGCCTCT GGTTGCTATC

ACTATGGAGT AGATCAAACA

5 (4.3%) TCGGTAGATC ATGCTTGTCG 9 n.a.

GGGGATTGCC ATTCCGGTCT

6 (3.8%) GGTATGGTAT TCAACCAGGC 10 n.a. TCTAGGTACC AGCTTGTCCT

GGCGTGAAGA GGGTTTGGCT

CCTCTTGTAG ACCTGAAGCT

7 (3.7%) GACAGAGGGG gCTGCTCGGG 11 23

ATTGCGaATA TCTGGTGGGT

CCCCCAGTTA GGACAGATCT

8 (3.2%) TaGGGtCTGC TCGGGATTGC 12 24

GGAeGATCTG TTCCACTCGC

ATTCAGTTGA CGTCGGCCTT

9 (2.0%) GACCAAGCTC ATAtaGGACT 13 25

GCTtaGGATT GCGaAgTTGA

GGCTGTTGTT GTTACCTATT

10(1.5%) GCGTGGCGAT CGGACTTTCG 14 n.a.

ATTCCGATTA ACGCCGGAGG

Table 1. Sequences of the N60 random region of the selected aptamers with >1.5% occurrences. Number in parenthesis refers to the percentage of occurrence of the clone with identical sequences in all sequenced clones (from a total of 20,313 clones. Only sequences occurring for more than 1% of the total number of clones were shown.) Underlined sequences are the consensus sequence among sequenced clones. Small letters indicate a mutation on the consensus sequences.

[0040] In another example, the aptamer comprises or consists of a sequence that can be, but is not limited to, SEQ ID NO: 6, 14, 15, 16, 17, 18, 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12 and 13, or combinations thereof. In another example, the aptamer comprises or consists of a consensus sequence. In yet another example, the aptamer comprises or consists of a consensus sequence that can be, but is not limited to, SEQ ID NO: 20, 21, 22, 23, 24 and 25, or combinations thereof. In another example, the aptamer comprises or consists of a sequence, wherein aptamer comprises or consists of a consensus sequence. In yet another example, the aptamer comprises or consists of a sequence the can be, but is not limited to, SEQ ID NO: 6, 15, 16, 5, 7, 11, 12, 13, or combinations thereof, wherein the aptamer comprises or consists of a consensus sequence that can be, but is not limited to, SEQ ID NO: 20, 21, 22, 23, 24 and 25, or combinations thereof. In another example, the aptamer comprises a sequence of, but not limited to, SEQ ID NO: 5, wherein the consensus sequence is SEQ ID NO: 20. In another example, the aptamer comprises a sequence of, but not limited to, SEQ ID NO: 6, 15, 16, 5, or combinations thereof, wherein the consensus sequence is SEQ ID NO: 21. In another example, the aptamer is a sequence of SEQ ID NO: 6, 15, 16 or 5, wherein the consensus sequence is SEQ ID NO: 21. In another example, the aptamer comprises a sequence of, but not limited to, SEQ ID NO: 7, wherein the consensus sequence is SEQ ID NO: 22. In another example, the aptamer comprises a sequence of, but not limited to, SEQ ID NO: 11, wherein the consensus sequence is SEQ ID NO: 23. In another example, the aptamer comprises a sequence of, but not limited to, SEQ ID NO: 12, wherein the consensus sequence is SEQ ID NO: 24. In another example, the aptamer comprises a sequence of, but not limited to, SEQ ID NO: 13, wherein the consensus sequence is SEQ ID NO: 25. As referred to in this paragraph, the term "combinations thereof also includes the term chimeric structure. As used herein, the term "chimeric structure" refers to a structure which is composed of at least two different SEQ ID NOs. The term "combinations thereof also includes sequences with deviations in the consensus sequences. For example, based on the consensus sequences, a partially randomize library can be designed and synthesized, from which an aptamer can be re- selected, resulting similar ap tamers with slightly different sequence context.

[0041] In another example, the aptamer disclosed herein comprise chimera structures or tandem repeat structures. In one example, the chimera structure comprises at least two sequences which can be any two of SEQ ID NOs: 20, 21, 22, 23, 24 and 25. In yet another example, the chimera structure comprises at least two sequences which can be any two of SEQ ID NO: 6, 15, 16, 5, 7, 11, 12, and 13. In a further example, the chimera structure comprises the sequences SEQ ID NO: 15, SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18. Such chimeric structures can comprise appropriate linker sequences, which can increase the binding affinity of the chimeric structure or which can allow multiple binding to the target or binding of the chimeric structures to multiple targets.

[0042] The aptamer as disclosed herein consist of nucleic acids. Such nucleic acids can be either naturally occurring nucleic acids or synthetic nucleic acids. As used herein, the term "nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymer in either single or double stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. In another example, the aptamer is a single-strand nucleic acid or double-strand nucleic acid. As used herein, the term "single-strand" refers to one strand of nucleic acid, and the term "double-strand" refers to two strands of nucleic acid, which are bound together by base pairing. In yet another example, the aptamer is DNA or RNA. In a further example, the aptamer is single-strand DNA, double-strand DNA, single-strand RNA or double-strand RNA. In another example, the aptamer is single-strand DNA.

[0043] In another example, the aptamer comprises or consists of one or more secondary structures, wherein examples of the secondary structure are, but are not limited to, helix, stem loop and pseudoknot, or any combinations thereof. In one example, the aptamer comprises a stem or stem-loop region. In another example, the stem or stem loop region is between 5 to 15 base pairs in length. In yet another example, the stem or stem loop region is about 6 base pairs, about 7 base pairs, about 8 base pairs, about 9 base pairs, about 10 base pairs, about 11 base pairs, about 12 base pairs, about 13 base pairs, or about 14 base pairs long. As a person skilled in the art would appreciate, the stem or stem-loop region of an aptamer may be shortened, or the sequence truncated, so long as the resulting structure is stable and the binding affinity is still present. Using methods in the art, a person skilled in the art would be able to determine the stability and the binding affinity of resulting truncated/shortened structures.

[0044] In another example, the aptamer can be between about 20 to about 110 nucleotides in length. In yet another example, the aptamer can be between about 20 to about 26, about 25 to about 31, about 30 to about 36, about 35 to about 41, about 40 to about 46, about 45 to about 51, about 50 to about 56, about 55 to about 61, about 60 to about 66, about 65 to about 71, about 70 to about 76, about 75 to about 81, about 80 to about 86, about 85 to about 91, about 95 to about 101, about 100 to about 106 or about 106 to about 110 nucleotide in length. In yet another example, the aptamer can be about 30 nucleotides, about 31 nucleotides, about 32 nucleotides, about 33 nucleotides, about 34 nucleotides, about 35 nucleotides, about 36 nucleotides, about 37 nucleotides, about 38 nucleotides, about 39 nucleotides, about 40 nucleotides, about 41 nucleotides, about 42 nucleotides about 43 nucleotides, about 44 nucleotides, about 45 nucleotides, about 50 nucleotides, about 51 nucleotides, about 52 nucleotides, about 53 nucleotides, about 54 nucleotides, about 55 nucleotides, about 56 nucleotides, about 57 nucleotides, about 58 nucleotides, about 95 nucleotides, about 96 nucleotides, about 97 nucleotides, about 98 nucleotides, about 99 nucleotides, about 100 nucleotides, about 101 nucleotides, about 102 nucleotides, about 103 nucleotides, about 104 nucleotides, or about 105 nucleotides in length. In yet another example, the aptamer is about 41, about 54 or about 100 nucleotides in length. Exemplary aptamers can be found, for example, in FIG. 3(A) to (D).

[0045] In another example, the aptamer comprises one or more mutations. In another example, the aptamer comprises of one or more mutations in the 60 nucleotide random region of the aptamer. In another example, the aptamer comprises of up to 10% mutation. In yet another example,, the aptamer comprises of 1 to 4%, 3 to 6%, 4 to 7%, 5 to 8%, 6 to 9%, 7 to 10%. In another example, the aptamer comprises of 1 to 2%, 2 to 3%, 3 to 4%, 4 to 5%, 5 to 6%, 6 to 7%, 7 to 8%, 8 to 9% and 9 to 10% mutation. In another example, the aptamer comprises of 1.7% mutation (corresponding to mutation in one nucleotide), 3.3% mutation (which corresponds to mutations in two nucleotides), 5% mutations (which corresponds to mutations in three nucleotides), 6.7% mutations (which corresponds to mutations in four nucleotides), 8.3% mutations (which corresponds to mutations in five mutations) and 10% mutations (which corresponds to mutations in six nucleotides). In another example, the mutation is present between nucleotide number 23 to 82 (inclusive), as numbered in SEQ ID NO: 4, or combinations thereof provided here. In yet another example, the mutation is present between nucleotide number 23 to 28, 27 to 32, 31 to 36, 35 to 41, 40 to 45, 44 to 50, 49 to 54, 53 to 58, 57 to 62, 61 to 66, 65 to 71, 70 to 75, 74 to 80 or 79 to 82. In yet another example, the mutation is present on one or more of nucleotide numbers 44, 48, 50, 53, 56, 57, 66, 67, 69, 76, and/or 78 , or combinations thereof. In yet another example, the mutation is present on nucleotide number 50 in sequence of SEQ ID NO: 7. In yet another example, the mutation is present on nucleotide numbers 53 and 69 in sequence of SEQ ID NO: 11. In yet another example, the mutation is present on nucleotide numbers 44, 48 and 66 in sequence of SEQ ID NO: 12. In yet another example, the mutation is present on nucleotide numbers 56, 57, 66, 67, 76 and 78 in sequence of SEQ ID NO: 13.

[0046] In one example, the aptamer can comprise 5', or 3 ' end modifications, or combinations thereof. In yet another example, the aptamer comprises 5' end modifications. In yet another example, the aptamer comprises 5' end modifications that can be, but not limited to, biotin, biotin-TEG, amino C₃, amino C₆, amino C₁₂, phosphate, thiol, digoxigenin, dinitrophenol, spacers, crosslinking, dihydro bases, hydroxylated bases, oxo bases or fluorescent tags.

[0047] In yet another example, the aptamer is stable, wherein the term "stable" used herein refers to the aptamer is not cleaved into 2 or more parts under specific conditions. Such conditions can be, but are not limited to highly acidic conditions, highly basic conditions, or high temperature conditions. In yet another example, the aptamer is stable when tested for cleavage using urea treatment. In yet another example, the aptamer comprising 5' end modifications which are stable when tested for cleavage using urea treatment. In yet another example, the aptamer of SEQ ID NO: 16, comprising a 5'-biotin modification, is stable when tested for cleavage using urea treatment. Examples of such stability can be found in FIG. 11.

[0048] In another example, the target protein of the aptamer is a flaviviral protein. As used herein, the term "bind" refers to being secured together by a chemical bond, wherein the chemical bond can be, but not limited to, covalent bond, electrostatic force, ionic bond, and hydrogen bond. In another example, the aptamer binds to a flavivirus protein. Such a flavivirus protein can be, but is not limited to, a protein from any one or more of the following viruses: Zika virus, Dengue virus, Japanese encephalitis virus, St. Louis encephalitis virus, West Nile virus, tick-bourne encephalitis virus or yellow fever virus. In one example, the flaviviral protein is a Zika virus protein. In another example, the flavivirus protein is a Dengue virus protein.

[0049] Flavivirus non- structural protein is a protein encoded by the flavivirus, wherein the protein is found during flavivirus infection. In yet another example, the aptamer binds to the flavivirus non-structural protein. In yet another example, the aptamer binds to the flavivirus non-structural 1 (NS l) protein. In yet another example, the aptamer binds to a Zika NSl protein. In yet another example, the aptamer binds to a Dengue NSl protein, wherein the Dengue NS l protein can be of Dengue NSl serotype 1, 2, 3, 4, 5 or combinations thereof. In yet another example, the aptamer can bind to Zika NS l and/or Dengue NSl, as shown for example in FIG. 5 A. In yet another example, the aptamer can specifically bind to Zika NSl, as shown for example in FIG. 5B. In another example, the aptamer capable of binding to Dengue NSl comprises the sequence of any one of SEQ ID NO: 6, 15, or 16. In another example, the aptamer capable of binding to Dengue NS 1 comprises the sequence of SEQ ID NO: 16.

[0050] Biological molecules can be characterised by their binding affinities to specific partners. For example, antibodies are often characterised using the binding affinities generated from binding assays with specific antigens. These binding affinities ("KD") are commonly referred to as dissociation constant ("K_d"), and have an inverse relationship with association constant ("K_A"). As used herein, the term "K_D" refers to binding affinity, which is the strength of the binding interaction between two binding partners, for example, between an aptamer and its flaviviral protein target, whereby it can be said that the smaller the K_D value, the stronger the binding between the two partners. As used herein, the term refers to the dissociation constant, which is the point wherein the Zika NS1 binds to the aptamer, or vice versa. It can be generally said that the smaller the value for K_d, the stronger the binding between the binding partners. As used herein, the term "K_a" refers to the association constant, which is the point wherein the Zika NS 1 detaches from the aptamer, or vice versa. It can be generally said that the larger the value for K_a, the stronger the binding between the binding partners. The aptamers disclosed herein have been tested for their binding affinities to their respective target sequences, as shown for example in Table 2 below. Therefore, in one example aptamer 2 binds Zika NS1 with a K_D of 45 pM, as shown for example in FIG. 3B and 4A. In yet another example, the aptamer binds to a Zika NS1 with a K_D of 240 pM, as shown for example in FIG. 3D and 4B.

Table 2. Binding affinities (K_D) of selected single-strand DNA aptamers.

[0051] Also disclosed herein are methods for using the described aptamers in detecting flaviviral proteins. Such methods can be, but are not limited to enzyme-linked immunosorbant assay (ELISA), sandwich ELISA, competitive ELISA, immunoprecipitation or western blot. The underlying concept between all of the methods listed above is that the aptamer, as disclosed herein, forms a complex with the one or more flaviviral proteins which may or may not be present in a sample. In one example of the method, in case more than one aptamer is used in a sample, these at least two aptamers can bind to two different sites on the flaviviral protein, thereby forming a sandwich complex between the two aptamers and the target flaviviral protein. In one example, a complex or sandwich complex is formed by the binding of the aptamer to one or more flavivirus proteins. In another example, a complex or sandwich complex is formed by the binding of the aptamer to one or more flavivirus NSl proteins. In yet another example, a complex or sandwich complex is formed by the binding of one or more aptamers as disclosed herein to one or more Zika NSl and/or Dengue NSl proteins. In yet another example, a complex or sandwich complex is formed by the binding of one or more aptamers to Zika NSl. In yet another example, a complex or sandwich complex is formed by the binding of one or more aptamers to Dengue NS 1. In yet another example, the complex or sandwich complex is formed by the binding of aptamer of SEQ ID NO: 15 or 16 to Zika NS l. In yet another example, the sandwich complex is formed by the binding of aptamers of SEQ ID NO: 15 or 16 and Zika NS 1, followed by the binding of the second aptamer of SEQ ID NO: 17 or 18, as shown for example in FIG. 6A and 6B.

[0052] In another example of a sandwich complex method, in case one aptamer is used for detecting flaviviral proteins in a sample, a further binding partner must be present and bind to a different site on the flaviviral protein other than the aptamer binding site. This forms a sandwich complex between the aptamer, the target flaviviral protein, and the further binding partner. In one example, the capture agent (that is the agent immobilised on the surface of the immunoassay vessel, for example) is the aptamer. In such a case, a further detection agent would be used to detect binding of the flavivirus protein to the immobilised aptamer. In another example, the capture agent is an antibody. In this situation, a further detection agent would be used to detect binding of the flavivirus protein to the immobilised antibody. As the flaviviral NS l is known to form hexameric structures, multiple binding of aptamer and antibody is also envisioned. The further binding partner can be, but not limited to, an antibody, a conjugated antibody, an enzyme-linked antibody or a fluorescent-tagged antibody. In one example, a sandwich complex is formed by the binding of the aptamer to one or more flavivirus proteins, for example flaviviral NS l, followed by binding to an antibody. In another example, a sandwich complex is formed by the binding of the aptamer to one or more flavivirus NS 1 proteins (for example, any flavivirus NS 1 proteins present in the sample), followed by binding to an anti-flavivirus NS l antibody, resulting in, for example, the specific detection of flaviviral NS l using specific NSl antibody. In yet another example, a sandwich complex is formed by the binding of one or more aptamers as disclosed herein to one or more Dengue NSl proteins, followed by binding to an anti -Dengue NSl antibody. In one example, the formation of hexameric structures between the flaviviral NS 1 protein and the aptamers (through multiple binding of aptamer and antibody is also envisioned. In yet another example, a complex or sandwich complex is formed by the binding of one or more aptamers to Zika NS1, followed by binding to an anti-Zika NS 1 antibody. In yet another example, the sandwich complex is formed by the binding of aptamers of SEQ ID NO: 15 or 16, the Zika non- structural protein 1, and an anti-Zika NS1 antibody, as shown in FIG 8A and 8B.

[0053] In another example, the complex or sandwich complex can be free-floating or immobilized on a coated solid surface. Where a coated surface is used, such surfaces can be coated with substrates such as, but not limited to, streptavidin, Protein A, Protein G, glutathione, antibodies or metal-chelate. In one example, the surface can be coated with one or more of the aptamers as disclosed herein.

[0054] Thus, as disclosed herein, a method, as disclosed herein, can be used to detect the presence or absence of one or more flaviviral proteins. In one example, the method is used to detect the presence or absence of flaviviral NS1. In another example, the method is used to detect the presence or absence of one or more proteins, which are, but are not limited to, Zika NS 1, Dengue serotype 1 NS1, Dengue serotype 2 NS 1, Dengue serotype 3 NS 1, Dengue serotype 4 NS 1 or Dengue serotype 5 NS1. In another example, the method is used to detect the presence or absence of a Zika NS 1 protein. In a further example, the method as disclosed herein is used to detect the presence or absence of a Dengue NS1 protein.

[0055] The method as disclosed herein can be used to for detecting the presence of flaviviral NS 1 in a sample. The sample can be, but is not limited to, sputum, blood, blood plasma, urine, feces, semen, tissues, organs, fractions, cell extracts or cells.

[0056] The sample as disclosed herein can be obtained from a healthy or a diseased subject. In one example, the subject is a mammal. In another example, the subject is, but is not limited to, human, bovine, equine, porcine, feline, murine, simian, or canine. In one example, the subject is human.

[0057] In one example, the method to detect the presence or absence of flaviviral NS1 comprises contacting a sample obtained from a subject with one or more aptamers. The term "capture aptamer" can be used to describe the one or more aptamers, as disclosed herein, that contacts the flaviviral NS 1 in a sample obtained from a subject. In a further example, the capture aptamer can be used to detect the presence or absence of Zika NS1 and/or Dengue NS 1. In another example, the capture aptamers can be the same, or different aptamers. In case different aptamers are used as capture aptamers, these capture aptamers can be used in combination of two or more. In other words, a combination of two, three, four or more aptamers can be used as capture aptamers. In yet another example, the capture aptamer can comprise or consist of SEQ ID NOs: 6, 15, or 16 This is exemplified by FIG. 7, wherein Zika NS 1 was detected by forming a complex with aptamer 2.

[0058] In another example, the method to detect the presence or absence of flaviviral NSl can further comprise the detection of the presence or absence of flaviviral NSl with one or more aptamers. The term "detection aptamer" can be used to describe the one or more aptamers, as disclosed herein, used for the detection of the presence or absence of flaviviral NS 1. In another example, the detection aptamer can be used to detect the presence or absence of Zika NS l. In another example, the detection aptamers can be the same, or different aptamers. In case different aptamers are used as detection aptamers, these detection aptamers can be used in combination of two or more. In other words, a combination of two, three, four or more aptamers can be used as detection aptamers. In another example, the detection aptamer that can be used to detect the presence or absence of Zika NS 1 comprises of SEQ ID NO: 14, 17 or 18, or combinations thereof This is exemplified by FIG. 7, wherein aptamer 10 was used to detect Zika NS 1.

[0059] In another example, the method to detect the presence or absence of flaviviral NSl can also comprise the detection of the presence or absence of flaviviral NS l with an anti- flaviviral NSl antibody. In another example, the flaviviral NS l antibody used can be an anti- Dengue NSl antibody or an anti-Zika NSl antibody. In another example, the flaviviral NSl antibody used is an anti-Zika NSl antibody, as shown for example in FIG. 9, wherein Zika NS l was first bound to the capture aptamer, followed by the use of an anti-Zika NSl antibody to detect the presence of Zika NS 1.

[0060] In another example, the method to detect the presence or absence of Zika NSl further comprises the use of a further detection agent to detect the presence or absence of the detection aptamer. As used herein, the term "further detection agent" can refer to, but not limited to, an enzyme-conjugated antibody, enzyme, or antibody that can produce and/or intensify a reaction. In another example, the enzyme horseradish peroxidase (HRP) can be used to detect the detection, as shown for example FIG. 7, wherein streptavidin-HRP conjugate was used to detect the detection aptamer and produce the signals that correspond to the data points. In another example, a HRP conjugated antibody can be used to detect the detection, shown for example FIG. 9A and 9B, wherein anti-rabbit IgG-HRP conjugated antibody were used respectively to detect the detection aptamer and produce the signals that correspond to the data points.

[0061] In another example, the method as disclosed herein is, but not limited to, enzyme - linked immunosorbent assay (ELISA) or sandwich ELISA. In one example, the method can be a sandwich ELISA comprising 5'-biotinylated aptamer 2 as a Zika NSl capture agent, and aptamer 10 as a detection agent . In one example, truncated versions of the aptamers disclosed herein are used. For example, FIG. 7 shows that the aptamer 2-Zika NSl-aptamer 10 sandwich complex-based ELISA has a detection limit of 100 ng/mL in buffer. In yet another example, the method can be a sandwich ELISA comprising 5'-biotinylated aptamer 2 as a Zika NS l capture agent, and an anti-Zika NSl antibody as a detection agent. For example, FIG. 9A shows that the aptamer 2 -Zika NS 1 -anti-Zika NS l monoclonal antibody sandwich complex-based ELISA has detection limits of 0.1 ng/mL and 10 ng/mL in buffer and 100 % human serum respectively, whilst FIG. 9B shows that the aptamer 2-Zika NS1- anti-Zika NSl monoclonal antibody sandwich complex-based ELISA has a detection limit of 1 ng/mL in 10 % human serum. However, it is of note that such detection limits, in other words the sensitivity of the disclosed method, can be improved by, for example, increasing the affinity of the secondary antibody or the cognate antibody used. It is also of note that compared to commercially available kits, the presently disclosed method and the disclosed aptamer- antibody hybrid system are more economical in terms of cost, as well as application and time required for detection.

[0062] Using the aptamer and method as disclosed herein, a kit can be made. In one example, the kit can be comprised of, but not limited to, the requisite buffers, one or more primary and secondary detection agents, reaction vessels, substrate for coating the reaction vessel, the aptamers disclosed herein, and negative/positive controls.

[0063] By way of the following example, the method and concept disclosed herein is explained. SELEX was carried out to identify Zika NS l -binding ssDNA aptamers using a random 60-nt ssDNA aptamer library consisting of —1016 sequences. Negative selection against 6x histidine tag peptides and magnetic Ni-NTA beads was carried out to remove nonspecific bead-bound binders for every SELEX cycle, and the enrichment of the Zika NS1- binding aptamer pool was monitored using BLItz label-free biolayer interferometry system (FIG. l). After 7 rounds of selection with increasingly reduced amounts of the target Zika NS 1 protein (500 nM for first cycle, 100 nM for second to fifth cycle, 10 nM for sixth cycle and 1 nM for seventh cycle), the enriched aptamer pool binds to 100 nM of Zika NS1 protein more significantly than the initial random ssDNA library, suggesting that Zika NS 1 -binding aptamers have been successfully enriched after 7 rounds of SELEX selection.

[0064] Therefore, the enriched aptamer pool was sequenced using an Ion Torrent deep sequencing system to identify the specific binders for Zika NS 1. A total of 20313 clones were sequenced and sequences with >1.5 occurrences were further characterized (Table 1). Among sequenced clones, it was seen that clones 1, 2, 3, 6, 8, and 9 share the whole or part of the consensus sequence (Table 1). The results show that the specific sequences, which are thought to have a specific structure preferred by the target, were enriched after 7 rounds of SELEX cycles.

[0065] Binding affinities and specificities for the selected aptamers were determined using BLItz label-free biolayer interferometry system. After preliminary screening for the affinity and the specificity for Zika NS 1 protein (data not shown), aptamers 2 (100-nt in length including a 60-nt random region and two 20-nt fixed sequences, FIG. 3A and 3C), which has the best binding affinity (KD = 24 pM, FIG. 2A), and aptamer 10, which has the best specificity but relatively weaker in binding (K_D = 134 nM, FIG. 2B), were chosen for structure optimization and further analysis. K_D values of selected clones were also summarized in Table 2.

[0066] To determine the minimum binding domain responsible for the observed binding affinity, several truncated versions of aptamers 2 and 10 were designed based on their secondary structures predicted using M-fold web server. Binding affinities determined using biolayer interferometry show that aptamers 2 and 10 can be truncated from 100-nt (FIG. 3A and 3C, and Table 2) to 41 and 54-nt (FIG. 3B and 3D), respectively, with comparable binding affinities (24 and 45 pM for 100-nt long and 41-nt long aptamer 2, and 134 and 240 nM for 100-nt long and 54-nt long aptamer 10, respectively). Therefore, these two truncated versions containing minimum binding sequences were used for further analysis. It might be worth pointing out that the truncated aptamer 10 of 54-nt in length contains the 3 ' fixed sequence, suggesting that the fixed sequence plays an important role in target binding (FIG. 3D).

[0067] Next, the specificities of the truncated aptamers were assessed since full-length aptamer 2 binds to Dengue NS 1 protein while aptamer 10 does not (data not shown). As shown in FIG. 5A, although the truncated aptamer 2 does not bind to unrelated proteins such as interferon-γ (IFN-γ) and bovine serum albumin (BSA, ~ 15 μΜ in binding buffer), it does bind to all four Dengue NS1 serotypes, ranked in the order of Zika NS1 > Dengue NS1 serotype 4 > serotype 1 > serotype 2 > serotype 3. On the other hand, the truncated 10 does not bind to any of Dengue NS 1 serotypes as well as IFN-γ and BSA, confirming that aptamer 10-mediated recognition of Zika NS 1 proceeds in a highly specific fashion (FIG. 5B).

[0068] Despite advances in molecular diagnostics, the false-negative or -positive results, which could arise from a high degree of sequence conservation among related viruses, sequence variations from new strains, etc., are still common issues in terms of sensitivity for Zika infection. For these reasons, serological assays are still generally considered as important diagnostic tool. For example, ELISA is the gold standard assay with high sensitivity while the paper-based assay is rapid and of lower costs. For these methods, finding a pair of ligands with complementary binding sites able to sandwich the target protein is the prerequisite.

[0069] To identify the aptamer or the antibody that could pair with aptamer 2 for forming the sandwich complex for detecting Zika NS 1, a number of Zika NS 1 -binding aptamers, commercial antibodies or in-house rabbit anti-Zika NS1 monoclonal antibodies were screened in label-free interferometry system. Briefly, 5'-biotinylated aptamer 2 was immobilized on streptavidin (SA) sensor, and Zika NS1 protein from Brazil strain was added and the solution was incubated to pre-form the aptamer 2-Zika NS 1 complex.

[0070] Next, aptamers or antibodies to be screened were added, and the association and dissociation of the preformed aptamer 2-Zika NS1 complex were monitored. From the screening, aptamer 10 (FIG. 6 A and 6B) and an in-house anti-Zika NS1 monoclonal antibody (FIG. 8 A and 8B) were found to be capable of pairing with aptamer 2 to form a Zika NS1- sandwiched complex. Accordingly, aptamer 2/10 or 2/anti-Zika NS 1 antibody were chosen as the pair in the ELISA assay using a 96-well plate (FIG. 7 and 9A). For aptamer 2/10 pair, ELISA was carried out in the binding buffer (FIG. 7) with 2 immobilized onto the plate as the capture agent, 5 '-biotin-modified 10 as the detection agent and streptavidin-HRP conjugate for signal amplification. The data shows that Zika NS1 can be reliably detected at a concentration ranging from 100 ng/mL to 1 μg mL. For aptamer 2/anti-Zika NS1 antibody pair, ELISA assay was carried out in both binding buffer and human serum (FIG. 9A) with aptamer 2 as the capture agent and the antibody as the detection. In binding buffer, this a tamer- antibody pair excitedly lowers the detection sensitivity to 0.1-1 ng/mL, which is comparable with those reported using immunoassays (e.g., 0.2 ng/mL and 3 ng/mL in PBS buffer using impedimetry and capacitance detection modes, respectively). Even in 100% human serum containing many other types of proteins, protease, small molecules and ions, Zika NS l of as low as >10 ng/mL can be detected. This is significant because nucleic acid aptamers often are not stable or easily deform in structure in human serum. In the present case, after incubation in human serum for 30 minutes at room temperature, aptamer 2 still could function properly and bind to Zika NSl protein. In fact, no significant degradation of aptamer 2 could be observed in gel after 1 hour (FIG. 11). Upon diluting 100% human serum 10-fold using binding buffer (FIG. 9B), the detection limit could be increased 10-fold to ~1 ng/mL, which is comparable to the detection limit of 0.5-30 ng/mL obtained using capacitive or impedimetric immunoassays and of 0.2 ng/mL for commercially available antibody-based Zika NSl ELISA kits. For comparison, under the identical conditions, this aptamer- antibody pair does not detect any of four Dengue NSl serotypes, West Nile Virus NS 1 and Yellow Fever Virus NS 1 (FIG. 12).

[0071] Lastly, although aptamer 2/antibody-mediated detection of Zika NSl protein was tested in human serum or diluted 10% human serum with high selectivity and no cross- reactivity from other unrelated proteins (IFN-γ and BSA), further testing on various clinical specimens including plasma, blood or urine samples is required before the performance of this assay on true clinical samples can be validated. Even though the detection limit using aptamer 2/10 pair is not as optimal as the aptamer 2/antibody pair, aptamer 2/10 pair constitutes one of very rare aptamer-aptamer pairs with successful applications in the sandwich ELISA format and/or other sandwich assays. Further research and optimization using such as doped library is needed to discover aptamers that are complementary to aptamer 2 in binding site and that come with higher binding affinity, selectivity and slower dissociation rate.

[0072] The presently disclosed aptamer/antibody approach has been compared with the other two alternative approaches (for example, antibody/aptamer and antibody/antibody) where antibody was used as the capture agent. In the reversed antibody/aptamer approach, some technical problems were encountered, such as low efficiency of antibody immobilization and lack of defined orientation for antibody, which are general issues for antibody immobilized as a capture agent. Without being bound by theory, and likely because of these problems, significantly lowered signals were observed for Zika NSl (data not shown). In addition to overcoming this sensitivity issue by using aptamer 2 as the capture agent, other added values of using aptamer, when compared to antibody, include the relatively lower cost in aptamer production and high batch-to-batch consistency in product quality.

[0073] As for antibody/antibody approach, screening some commercial antibodies identified antibody SQAbl610 (Arigo Biolaboratories, Taiwan) as the complementary antibody for pairing with an in-house antibody to form a sandwich complex with Zika NSl protein (FIG. 13). Using this antibody/antibody pair and the same ELSIA assay protocol, a detection limit of 10 ng/mL was obtained in binding buffer (FIG. 14), which is 100-fold less sensitive than the hybrid aptamer/antibody approach with a detection limit of 0.1 ng/mL (FIG. 9A).

[0074] In summary, several consensus sequence- sharing Zika NS l-binding ssDNA aptamers have been found, and developed ELISA-based assay for highly specific and sensitive detection of Zika NSl antigen in buffer using a pure aptamer-aptamer pair (for example, aptamer 2/10) and in human serum using a hybrid aptamer/antibody pair (for example, aptamer 2/antibody). With the use of aptamer 2/antibody pair, detection limits could reach 0.1-1, 1-10 and >10 ng/mL in buffer, 10% human serum and 100% human serum, respectively. In particular, aptamer 2 (of 41 nucleotides in length) exhibits exceedingly high binding affinity of 45 pM toward Zika NSl antigen. The aptamer 2/10 pair is one of limitedly available aptamer-aptamer pairs with proven applications for antigen detection with a detection limit of >100 ng/mL in buffer. Thus, hybrid 2/antibody pair is understood to be clinically relevant to applications in medical diagnosis of Zika virus infection.

[0075] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred examples and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0076] As used in this application, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a genetic marker" includes a plurality of genetic markers, including mixtures and combinations thereof.

[0077] As used herein, the term "about", in the context of concentrations of components of the formulations, typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value.

[0078] Throughout this disclosure, certain examples may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0079] Certain examples may also be described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the examples with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0080] The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0081] Other examples are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

EXPERIMENTAL SECTION

Material and Methods

Selection Conditions and Procedures for Systematic Evolution of Ligands by Exponential enrichment (SELEX)

[0082] Oligonucleotides including random aptamer library and primers were purchased from Integrated DNA Technologies (USA) or Sangon Biotech (China). All procedures were carried out at room temperature. For aptamer selection, a random 60 nucleotide aptamer library (SEQ ID NO: 1) (5' -GAT A GAATTC GAGCTC GGGC (N: 60) GCGG GTCGAC AAGCTT TAAT-3') is heated at 95°C for 5 minutes and cooled to room temperature to allow refolding. 6x histidine peptide and magnetic Ni -NT A beads were incubated with the random aptamer library in SELEX buffer (20 mM Tris pH 7.5 with 150 mM NaCl, 2.7 mM KC1, 1 mM MgCl₂ and 0.005% NP-40) to remove non-specific binders. After quick centrifugation, the supernatant containing the aptamer library was incubated with Zika NS1 protein containing a His tag at C-terminus (Aero Biosystems; accession no. ALU33341 in GenBank database; SEQ ID NO: 19). After washing three times with binding buffer, the bound sequences were eluted using a solution containing 1 M NaCl and 10 mM NaOH, and then further extracted using phenol extraction method. The eluted sequences were purified using G-25 micro-spin columns and the sequences were amplified by PCR using a taq polymerase system for the next cycle of selection. The following forward and reverse primers were used for PCR [95 °C for 5 minutes, 10 to 20 cycles (95 °C for 30 seconds, 55 °C for 30 seconds and 72 °C for 30 seconds) and 72 °C for 3 minutes]:

Forward primer (SEQ ID NO: 2)

5' -GAT A GAATTC GAGCTC GGGC-3 '

EcoRI Sad

Reverse primer (SEQ ID NO: 3)

5 '-Phosphate- ATT A AAGCTT GTCGAC CCGC-3'

Hindlll Sail

For the next cycle of selection, ssDNA aptamers were recovered by lambda exonuclease reaction which eliminates complementary sequences containing a 5' phosphate group. After further purification by phenol extraction and ethanol precipitation, the recovered aptamer sequences were used for the next cycle of SELEX. After 7 rounds of SELEX with reduced amounts of the target (500 nM to 1 nM), binding affinity of enriched DNAs was tested against NS1 proteins.

Deep Sequencing

[0083] Deep sequencing was carried out for the 7^th round of aptamer pool using an Ion Torrent sequencing system and sequencing kits (Life Technologies) according to the manufacture's protocol.

Determination of Minimum Binding Domain and Secondary Structure Prediction

[0084] To determine the minimum binding domain responsible for the observed binding affinity, several truncated versions of aptamers 2 and 10 were designed based on their secondary structures predicted using M-fold web server.

Determination of Binding Affinity (KD) using BLItz Label-Free Biosensor System

[0085] Single- stranded DNA (ssDNA) aptamers were purchased from Integrated DNA technologies. BLItz label-free biosensor system for biolayer interferometry assay (ForteBio, USA) was used for the binding test and the determination of binding affinity (KD). Briefly, 5'- biotinylated (5'-biotin-TEG) ssDNA aptamer (100 nM) was loaded onto streptavidin (SA) sensor (10 to 20 seconds) for immobilization. Various concentrations of Zika NS1, which mainly exist in the form of dimer or hexamer in the membrane -bound or solution state, respectively, were incubated with the aptamer-immobilized SA sensor or control SA sensor in binding buffer (30 mM Tris at pH 7.5 with 150 mM NaCl, 1 mM MgCl₂ and 0.1% bovine serum albumin (BSA)). Both association and dissociation events were recorded for 300 seconds for the kinetic analysis after a waiting time of 60 seconds for baseline to stabilize. Binding kinetics data were analysed using BLItz Pro 1.2 software.

[0086] For binding specificity test, a solution containing Zika NS1, Interferon-gamma (IFN-γ, Sino Biological) or any of four Dengue NS1 serotypes (10 nM for aptamer 2 or 500 nM for aptamer 10, BioRad) was loaded onto a SA sensor immobilized with the aptamer. After a waiting time of 60 s for baseline to stabilize, both association and dissociation events were recorded for 300 s for the kinetic analysis.

[0087] For screening of aptamer-aptamer pair or aptamer-antibody pair for sandwich ELISA assay, biotinylated aptamer 2 (100 nM) was loaded onto SA (Streptavidin) sensor (150 or 20 seconds) for immobilization after a waiting time of 30 seconds for baseline to stabilize. 100 nM of Zika NS 1 protein was then incubated with the aptamer-immobilized S A sensor for 300 seconds in binding buffer. After 120 seconds (or 60 seconds for antibody screening) for baseline to stabilize, 1 μΜ of identified aptamer or in-house anti-Zika NS1 monoclonal antibody (rabbit IgG) was loaded to check if these aptamers or antibodies show competitive binding or additional binding to the preformed aptamer 2-Zika NS 1 complex. Both association and dissociation events were monitored for 300 seconds, respectively. Data were analysed using BLItz Pro 1.2 software. Truncated versions of the aptamers disclosed herein were used in these experiments.

Sandwich ELISA for Detecting Zika NS1 Antigen

[0088] Sandwich complex -based ELISA assays for Zika NS 1 were carried out using aptamer 2 (capture agent) and aptamer 10 (detection agent). Briefly, 60 pmole of 5'-amino C6-containing aptamer 2 was immobilized (Maximum binding capacity is > 125 pmole/well in 100 pL) on amine-binding, maleic anhydride activated plates (Pierce) using manufacturer's protocol. Then, non-specific binding sites were blocked using SuperBlock solution (Thermo Fisher Scientific). After incubation with Zika NS 1 protein at various concentrations in binding buffer (100 pL) on a 96-well plate, the well was washed with a binding buffer containing 0.1% Tween 20 (200 μΕ). 100 nM of 5'-biotinylated aptamer 10 was incubated (100 pL) for 30 minutes. After washing, streptavidin-HRP conjugate (Jackson ImmunoResearch, 1 μg/mL) was incubated for 15 minutes, and the well was washed three- times with binding buffer containing 0.1% Tween 20. Following colour development using TMB-based substrate and stop solution using manufacturer's protocol (KPL, USA), absorbance was measured at 450 nm using Cytation3 multi-mode plate reader (BioTek, USA).

[0089] In a similar experiment, sandwich comple -based ELISA assays for Zika NS1 or Dengue NS 1 serotypes 1, 2, 3 and 4 were carried out using aptamer 2 (capture agent) and in- house rabbit anti-Zika NS1 monoclonal antibody (detection agent). Briefly, 60 pmole of 5'- biotinylated aptamer 2 (20 pmole in 200 pL) was immobilized (Maximum binding capacity is >15 pmole/well in 200 pL) on streptavidin-coated plates (Kaivogen) using manufacturer's protocol. Then, non-specific binding sites were blocked using SuperBlock solution (Thermo Fisher Scientific). After incubation with Zika NS 1 protein at various concentrations (100 pL) in binding buffer or in 100 % or 10% human serum (prepared by diluting 10 pL 100% human serum in 90 pL buffer) or Dengue NS1 serotypes 1, 2, 3 and 4 at various concentrations in binding buffer on a 96-well plate, the well was washed with binding buffer containing 0.1% Tween 20 (200 μ¾. 10 nM of rabbit anti-Zika NS 1 antibody (100 pL) was added and incubated for 30 minutes. After washing, anti-rabbit IgG-HRP conjugate (Promega, 0.5 μg/mL) was incubated for 15 minutes, and the well was washed three-times with binding buffer containing 0.1% Tween 20. Following color development using TMB -based substrate and stop solution using manufacturer's protocol (KPL, USA), absorbance was measured at 450 nm using Cytation3 multi-mode plate reader (BioTek, USA).

Determining Aptamer Stability using Polyacrylamide Gel Electrophoresis

[0090] 2 μL· of aptamer (100 μΜ) was mixed with 98 μΐ. of human serum (100%) to prepare aptamer-containing 98% human serum. 10 μΐ_^ of such solutions mixed with 110 μΐ_^ of 10 M Urea in IX TBE for 0, 30 and 60 minutes, respectively, were gel electrophoresed in 20% polyacrylamide gel containing 7 M Urea and visualized using SYBR Gold dye (Invitrogen). The band intensity was analysed using Image J software.

Description of SEQ ID

[0091] Table 3 below details the SEQ ID NOs referenced herein and their corresponding sequences. A brief description of the sequences is also provided. Underlined sequences are the consensus sequence among sequenced clones. Small letters indicate a mutation on the consensus sequences.

SEQ ID Sequence Description

NO (Underlined refers to consensus

sequence; small letters refer to

mutations with regard to nucleotide

sequences)

1 GATAGAATTC GAGCTCGGGC Sequence of aptamer library with random

NNNNNNNNNN NNNNNNNNNN 60 nucleotide region

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN GCGGGTCGAC AAGCTTTAAT

2 GATAGAATTC GAGCTCGGGC Forward primer used during SELEX

3 ATTAAAGCTT GTCGACCCGC Reverse primer used during SELEX

4 GGGAUAGAAU UCGAGCUCGG Sequence of aptamer clone that includes

GCNNNNNNNN NNNNNNNNNN a N60 random region

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNGCGGGUCG ACAAGCUUUA AU ATACCGGTGC CATATTCCAC Sequence of the N60 random region of AAGGGGGACT GCTCGGGATT the selected aptamer 1, wherein the first GCGGATTTGT GGAATTGTTG nucleotide corresponds to nucleotide number 23.

ACTAGGTTGC AGGGGACTGC Sequence of the N60 random region of TCGGGATTGC GGATCAACCT the selected aptamer 2, wherein the first AGTTGCTTCT CTCGTATGAT nucleotide corresponds to nucleotide number 23.

CACAGACTCC ATCTTGGATT Sequence of the N60 random region of GCAAAGGtCT GCTGTGTGGT the selected aptamer 3, wherein the first AGTCTGTGGA GGCCATGTCT nucleotide corresponds to nucleotide number 23.

ACTCCGCGAT AGACGGTTCT Sequence of the N60 random region of GCATGCACGT TCCTCCGACG the selected aptamer 4, wherein the first TCCCGCCTCT GGTTGCTATC nucleotide corresponds to nucleotide number 23.

ACTATGGAGT AGATCAAACA Sequence of the N60 random region of TCGGTAGATC ATGCTTGTCG the selected aptamer 5, wherein the first GGGGATTGCC ATTCCGGTCT nucleotide corresponds to nucleotide number 23.

GGTATGGTAT TCAACCAGGC Sequence of the N60 random region of TCTAGGTACC AGCTTGTCCT the selected aptamer 6, wherein the first GGCGTGAAGA GGGTTTGGCT nucleotide corresponds to nucleotide number 23.

CCTCTTGTAG ACCTGAAGCT Sequence of the N60 random region of GACAGAGGG GsCTGCTCGG the selected aptamer 7, wherein the first GATTGCGaAT ATCTGGTGGGT nucleotide corresponds to nucleotide number 23.

CCCCCAGTTA GGACAGATCT Sequence of the N60 random region of TaGGGtCTGC TCGGGATTGC the selected aptamer 8, wherein the first

GGAgGATCTG TTCCACTCGC nucleotide corresponds to nucleotide number 23.

ATTCAGTTGA CGTCGGCCTT Sequence of the N60 random region of GACCAAGCTC ATAtaGGACT the selected aptamer 9, wherein the first GCTtaGGATT GCGaAsTTGA nucleotide corresponds to nucleotide number 23.

GGCTGTTGTT GTTACCTATT Sequence of the N60 random region of GCGTGGCGAT CGGACTTTCG the selected aptamer 10, wherein the ATTCCGATTA ACGCCGGAGG first nucleotide corresponds to nucleotide number 23.

GATAGAATTC GAGCTCGGGC Aptamer 2 (100 nucleotide) sequence ACTAGGTTGC AGGGGACTGC TCGGGATTGC GGATCAACCT AGTTGCTTCT CTCGTATGAT GCGGGTCGAC AAGCTTTAAT

CTAGGTTGCA GGGGACTGCT Aptamer 2 (41 nucleotide) sequence CGGGATTGCG GATCAACCTA G

Claims

Claims

1. An aptamer specifically binding to Zika viral non-structural protein 1 (NS 1), wherein the target sequence of the aptamer comprises or consists of SEQ ID NO: 19.

2. The aptamer according to claim 1, wherein the aptamer is single-strand DNA or single-strand RNA.

3. The aptamer according to any of the preceding claims, wherein the aptamer comprises a consensus sequence selected from the group consisting of SEQ ID

NO: 20 , 21, 22, 23, 24, 25.

4. The aptamer of claim 3, wherein the consensus sequence is SEQ ID NO: 21.

5. The aptamer according to any of the preceding claims, wherein the aptamer is between 30 nucleotides to 100 nucleotides in length.

6. The aptamer according to claim 1 or 2, wherein the aptamer comprises a sequence selected from the group consisting of SEQ ID NO: 6, 14, 15, 16, 17, 18, 1, 2, 3, 4, 5, 7 to 13; wherein the aptamer comprises up to 10% of mutations, wherein the mutations are present between nucleotide number 23 to nucleotide number 82 (inclusive).

7. The aptamer according to claim 3, wherein the aptamer comprises a sequence selected from the group consisting of SEQ ID NO: 6, 15, 16, 5, 7, 11, 12, 13.

8. The aptamer according to claim 2, wherein the aptamer comprises a sequence selected from the group consisting of SEQ ID NO: 14, 17, 18, 1, 2, 3, 4, 8, 9, 10.

9. The aptamer according to any of the preceding claims, wherein the aptamer is capable of binding to both Zika and Dengue non- structural protein 1.

The aptamer of claim 9, wherein the aptamer comprises or consists of the sequence selected from the group consisting of SEQ ID NO: 6, 15, and 16.

A method for detecting the presence of flaviviral non- structural protein 1 (NS1) in a sample, the method comprising

a) contacting a sample obtained from a subject with one or more aptamers as described in any one of claims 1 to 10;

b) detecting the presence or absence of binding of the one or more aptamers in the sample, wherein the presence of binding of the one or more aptamers in the sample indicates the presence of flaviviral non-structural protein 1 (NS 1).

The method of claim 11, wherein the flaviviral non-structural protein 1 (NS1) is selected from the group consisting of Zika, Dengue serotype 1, Dengue serotype 2, Dengue serotype 3, Dengue serotype 4, and Dengue serotype 5.

The method of any one of claims 11 to 12, wherein a) comprises a further detection agent.

The method of claim 13, wherein the one or more aptamers are the same or different from each other.

The method of claim 13, wherein, if two aptamers are used, the two aptamers are selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 14.

The method of any one of claims 13 to 15, wherein the further detection agent is an antibody or a conjugated antibody.

The method of any one of claims 11 to 16, wherein the method is selected from the group consisting of enzyme -linked immunosorbent assay (ELISA) or sandwich ELISA.

18. A kit comprising the aptamers according to any one of claims 1 to 10. The kit according to claim 18 for performing enzyme-linked immunosorbent assay (ELISA) or sandwich ELISA.