WO2021245586A2

WO2021245586A2 - Cd4 binding aptamers and applications thereof

Info

Publication number: WO2021245586A2
Application number: PCT/IB2021/054857
Authority: WO
Inventors: Janice Leigh Limson; Ronen FOGEL; Tamika FELLOWS
Original assignee: RHODES UNIVERSITY
Current assignee: RHODES UNIVERSITY
Priority date: 2020-06-03
Filing date: 2021-06-03
Publication date: 2021-12-09
Anticipated expiration: 2022-12-03
Also published as: WO2021245586A3; ZA202213435B

Abstract

The present invention relates to aptamers having selectivity and specificity for human CD4 protein and/or cells expressing human CD4 protein. Also disclosed is a biosensor device, particularly a point-of-care biosensor device, comprising aptamers having selectivity and specificity for human CD4 protein and/or cells expressing human CD4 protein. The invention further encompasses methods of detecting human CD4 protein and/or cells expressing human CD4 protein in a sample using the aptamers or biosensor disclosed herein, comprising detecting the binding of the aptamers to human CD4 protein and/or cells expressing human CD4 protein.

Description

CD4 BINDING APTAMERS AND APPLICATIONS THEREOF

BACKGROUND OF THE INVENTION

This invention relates to aptamers having selectivity and specificity for human CD4 protein and/or cells expressing human CD4 protein and in particular to a biosensor comprising aptamers having selectivity and specificity for human CD4 protein and/or cells expressing human CD4 protein. The present invention also relates to a method for detecting human CD4 protein and/or cells expressing human CD4 protein in a sample using the aptamers or biosensor of the invention, comprising detecting binding of the aptamers to human CD4 protein and/or cells expressing human CD4 protein.

As of 2013, the World Health Organization (WHO) reported an estimated 35 million people living with HIV/AIDS; 71% of all people living with HIV resided in Sub- Saharan Africa and of these only 37% were receiving adequate treatment coverage. A UNAIDS report (2014) has concluded that a vast majority of people with HIV live in low-income, resource-limited areas which are ill-equipped to combat HIV/AIDS. However, there are still approximately 22 million people living with HIV who do not have routine access to ART in Sub-Saharan Africa, although this number has decreased by 4.7 million since 2010. Access to accurate CD4 tests is one of the major limiting factors of ART penetrance.

HIV targets CD4⁺ T-lymphocytes which act as primary hosts for the virus, thus their decline is linked directly to the spread of the virus and disease progression. Hence, CD4 counts are established indicators of disease progression and survival and remain the most appropriate screening tool to determine ART eligibility in HIV patients. The WHO 2013 Consolidated ART guidelines recommend that patients with fewer than 500 CD4⁺ cells. pi ¹ of whole blood are considered eligible for ART, while patients with fewer than 200 CD4⁺ cells. pi ¹ of whole blood are considered to be immune-deprived and have then contracted AIDS. In a subsequent report released in 2015, WHO guidelines recommended ART be initiated in all patients testing positive for HIV, regardless of their CD4 count: this strategy has been coined “test and treat”. However, CD4 counts remain an important indicator of HIV progression, care and management and its usage in decision-making around ART, especially in initiation and in altering treatment regimens, continues.

There are several socio-economic pitfalls associated with conventional CD4 testing. For example, delayed testing due to high demand, or patients can be lost-to- follow-up (LTFU) procedures. In addition, extensive logistical networks are required to prevent loss and degradation when transporting samples to centralised testing facilities, further increasing the cost of treatment while diminishing its reliability. A 2013 study concluded that point-of-care (POC) CD4 testing at time of HIV diagnosis could reduce the number of patients LTFU, decrease the number of new HIV and TB infections, increase the average life expectancy of HIV patients and result in significant economic savings in health care.

Currently, the most accurate method of measuring the CD4 count is fluorescence-activated cell sorting (FACS), which determines the absolute CD4 count from whole blood through counting of fluorescently-labelled anti-CD4 antibodies binding to cells. However, flow cytometers are generally expensive, laboratory- confined instruments requiring highly-trained personnel to operate and interpret data. As the majority of HIV/AIDS patients live in developing countries and resource-limited areas where centralised equipment is not available, there is a need for effective and portable POC diagnostic tools for CD4+ T-lymphocyte enumeration.

Recent developments in POC devices allow for routine CD4 diagnostics in rural areas, with several existing CD4 counting POC devices based on flow cytometry technology using antibody-based detection methods. The PIMA™ CD4 Analyser (Alere) can conduct CD4 tests rapidly using a specialised disposable cartridge complete with reagents that label CD4⁺ T-lymphocytes and CD3⁺ T-lymphocytes with anti-hCD4 and anti-hCD3 antibodies, respectively. The technology has been tested in Zimbabwe, Thailand, and Mozambique. Although current CD4 counting technologies offer POC facilities in rural areas, these devices do not currently meet the requirements of portability, cost, and accuracy of measurement.

Antibodies have traditionally been the biorecognition agents of choice in biosensors, as evident from the fact that the majority of the CD4 sensors available rely on antibodies to tag CD4-expressing cells. Antibodies are established means of tagging biochemical targets, due to their high target selectivity, affinity, and established immobilisation strategies. However, antibodies are limited by their thermal and chemical stability, their ability to only target antigenic molecules (namely proteins or haptens), and their high synthesis cost due to limited production scale-up. Antibody production is also sensitive to viral and bacterial contamination which leads to a reduction in quality of the final product. Antibodies are further limited by the fact that there is a limited ability to use negative selection pressure to select antibodies against specific cell surface targets unless they are available in a functional recombinant form. Thus, there is a need for alternative sensing molecules that are cost-effective to produce and are capable of sensitive and specific to CD4, to integrate into testing devices to improve access to immunological monitoring strategies in HIV positive patients in POC centres.

The systematic evolution of ligands by exponential enrichment (SELEX) is a process of selection, which permits the isolation of oligonucleotides that have the capability to recognise a wide range of target molecules in a highly specific manner and with high affinity. Further, negative selection pressure in SELEX can be included, to select against cell surface targets. These yield aptamers with very specific binding properties. Thus, aptamers are single-stranded oligonucleotides that have the ability to fold into very specific tertiary structures capable of binding to targets with high affinity and specificity. Aptamers offer several intrinsic advantages as recognition agents relative to antibodies. Aptamers have a long shelf-life due to a very stable phosphodiester backbone with some potential for re-use as denaturation events are reversible. Furthermore, aptamers can also be chemically modified to be resistant to nuclease degradation by incorporating chemical modifications into sugar groups or phosphodiester backbones.

DNA-based biotinylated CD4 aptamers were previously developed through SELEX, which employed the outermost domains of CD4 (2dCD4) as a peptide target for aptamer selection. The resultant aptamer candidate, C27, was found to bind to 2dCD4 proteins with high affinity and specificity when biotinylated. However, this aptamer’s ability to bind to CD4 expressing cells was not validated. An alternative CD4 binding aptamer, F1-62, was identified by Zhao etal. (2014) through Crossover-SELEX which employed CD4-lgG2 recombinant protein and CD4 positive Karpass 299 cells as targets for aptamer selection. In this case, the SELEX approach was customized to select aptamers suitable for therapeutic applications as inhibitors of viral gp-120 binding. The selection factors employed during the SELEX approach significantly influence the characteristics of the resultant aptamer candidates and a Crossover- SELEX approach geared towards the development of CD4 DNA aptamers suitable for diagnostic applications has not yet been described.

SUMMARY OF THE INVENTION

The present invention relates to aptamers having selectivity and specificity for human CD4 protein and/or cells expressing human CD4 protein and a biosensor device, particularly a point-of-care biosensor device, comprising aptamers having selectivity and specificity for human CD4 protein and/or cells expressing human CD4 protein. The invention further relates to a method of detecting human CD4 protein and/or cells expressing human CD4 protein in a sample using the aptamers or biosensor of the invention, comprising detecting the binding of the aptamers to human CD4 protein and/or cells expressing human CD4 protein.

In a first aspect of the invention there is provided for an aptamer comprising or consisting of the nucleotide sequence of any one of SEQ ID NOs:6-32, or a complementary sequence thereof, wherein the aptamer selectively and specifically binds human CD4 protein and/or cells expressing human CD4 protein.

In a preferred embodiment of this aspect of the invention the aptamer comprises the nucleotide sequence of any one of SEQ ID NOs:8, 9, 13-15 and 20, in particular SEQ ID NOs:8, 9, 13 and 20, most particularly SEQ ID NO:13, or a complementary sequence thereof.

In a second embodiment of this aspect of the invention there is provided an aptamer of any one of SEQ ID NOs:6-32, wherein the aptamer is labelled. It will be appreciated that the label may be biotin, a fluorescent label, a luminescent label, a radioactive isotope, amine, aryl azides, thiol, a nanoparticle, an enzymatic label or any other label familiar to those of skill in the art. Preferably, the label is biotin or Cy5.

In a second aspect of the invention there is provided for a biosensor device for detecting human CD4 protein and/or cells expressing human CD4 protein, wherein the biosensor device comprises a labelled aptamer comprising or consisting of the nucleotide sequence of any one of SEQ ID NOs:6-32, or a complementary sequence thereof, wherein the aptamer selectively and specifically binds human CD4 protein and/or cells expressing human CD4 protein.

In a preferred embodiment of the biosensor device of the invention the aptamer comprises the nucleotide sequence of any one of SEQ ID NOs:8, 9, 13-15 and 20, in particular SEQ ID NOs:8, 9, 13 and 20, most particularly SEQ ID NO:13, or a complementary sequence thereof.

In a second embodiment of the biosensor device of the invention the label is biotin, a fluorescent label, a luminescent label, a radioactive isotope, amine, aryl azides, thiol, a nanoparticle, or an enzymatic label. Preferably, the label is biotin or Cy5.

In a third aspect of the invention there is provided for a method of detecting CD4 in a sample comprising: (a) labelling an aptamer having selectivity and specificity for human CD4 protein and/or cells expressing human CD4 protein with a label, wherein the aptamer comprises or consists of the nucleotide sequence of any one of SEQ ID NOs:6-32, or a complementary sequence thereof; (b) contacting the sample with the labelled aptamer; and (c) detecting binding of the aptamer to human CD4 protein and/or cells expressing human CD4 protein, where binding of the aptamer to human CD4 protein and/or cells expressing human CD4 protein indicates the presence of human CD4 protein and/or cells expressing human CD4 protein in the sample.

In a preferred embodiment of the method of the invention the aptamer comprises the nucleotide sequence of any one of SEQ ID NOs:8, 9, 13-15 and 20, in particular SEQ ID NOs:8, 9, 13 and 20, most particularly SEQ ID NO:13, or a complementary sequence thereof.

In a second embodiment of the method of the invention the label is biotin, a fluorescent label, a luminescent label, a radioactive isotope, amine, aryl azides, thiol, a nanoparticle or an enzymatic label. Preferably, the label is biotin or Cy5.

In a third embodiment of the method of the invention, the step of detecting binding of the aptamer to human CD4 protein and/or cells expressing human CD4 protein is performed using an impedimetric assay, a spectrophotometric assay, a voltammetric assay, a chemiluminescence assay, flow cytometry assay, a radioactive assay, an immunochromatographic assay, a piezoelectric assay, a colourimetric assay, a fluorescence assay, an ELISA assay, an ELONA assay, an acoustic assay, and/or a polymerase chain reaction binding assay. In one embodiment, the assay may be a gold nanoparticle (AuNP) colorimetric assay, wherein the aptamer is conjugated to an AuNP and on binding to CD4 a colour is observed.

BRIEF DESCRIPTION OF THE FIGURES

Non-limiting embodiments of the invention will now be described by way of example only and with reference to the following figures:

Figure 1 : Diagrammatic representation of Crossover SELEX approach to develop aptamers for the enumeration of CD4 expressing cells: Crossover SELEX followed a two-step enrichment process, alternating between CD4-expressing cells (Cell-SELEX) and recombinant human CD4 functionalised paramagnetic beads (Protein-SELEX). Aptamers which did not bind to the CD4 beads were discarded and aptamers which bound were amplified and incubated with the CD4-expressing cell. Aptamers which bound to the CD4⁺ cells were eluted from the cell and amplified, while those that did not bind were discarded. Each SELEX process (either Protein-, or Cell- SELEX) made use of counter-selection, including the absence of binding to a control protein or cell line as a selective pressure to increase the binding specificity of the resultant aptamers. Aptamers that did not bind during positive selection rounds were also discarded.

Figure 2: Analysis of nucleotide residue enrichment following SELEX:

Illustration of frequency of adenosine (A), guanine (G), cytosine (C), and thymidine (T) nucleotides in aptamer, concatemer, and truncated sequences gathered after 5 rounds of Crossover SELEX (m ± SE; n = 30). Student’s t-tests were conducted and p-value > 0.05 are denoted by ^*.

Figure 3: Secondary structure prediction of the CD4-targeting aptamer candidates. The predicted secondary structures were determined by RNAfold analysis for linear DNA at 25 °C using minimum free energy and partition function fold algorithms. Predicted structures are shown at a minimum Gibbs free energy, AG.

Figure 4: Protein-ELONA analysis of ability of selected SELEX aptamer candidates to selectively bind to recombinant human CD4-functionalised, IgG- functionalised, and HSA-functionalised proteins. The rate of TMB radical development was calculated with the linear response of each absorbance versus time graph (n = 3; x ± SD). ANOVA tests were conducted on the variation between each group as indicated by the p-value. Annotated symbols show significant differences between compared groups, as determined by of Fisher LSD post hoc analyses: ^* indicates significantly different assay responses (p-value < 0.05) comparing different aptamer responses to the CD4 protein sample, † indicates significantly higher CD4 assayed response (p-value <0.05) compared to different proteins for a single aptamer.

Figure 5: Fluorescent confocal microscopy analysis of selected Cy5- aptamer controls’ ability to bind to U937 cells. Fluorescent aptamer signal localisation of positive (F1-62 (SEQ ID NO:33)) and negative control aptamers (C27 (SEQ ID NO:34) and MR801 (SEQ ID NO:35)) on CD4-expressing cells was evaluated and presented in this figure.

Figure 6: Fluorescent confocal microscopy analysis of selected aptamer candidates’ ability to bind to CD4-expressing cells. Aptamers derived from SELEX: U4 (SEQ ID NO:8), U14 (SEQ ID NO:20), U20 (SEQ ID NO:9), U26 (SEQ ID NO:13), and U38 (SEQ ID NO:15), were compared to negative control aptamers C27 (SEQ ID NO:34), MR801 (SEQ ID NO:35), and positive control aptamer F1 -62 (SEQ ID NO:33).

Figure 7: Amm-C12 F1 -62 mediated U937 cell capture with Dynabead M-

270 Epoxy: Quantity of aptamer bound to the Dynabead after functionalisation was confirmed by analysing the amount of ssDNA present before functionalisation, and the amount of ssDNA present in the supernatant after functionalisation using the Nanodrop 2000, then compared to a control containing no aptamer.

Figure 8: Amm-C12 F1 -62 mediated U937 cell capture with Dynabead M-

270 Epoxy: Dispersion patterns of the F1 -62 functionalised (F1 -62), and unfunctionalized (no aptamer) beads were visualised at different magnifications (50X, 200X inner, 200X outer) in both the centre of the slide (inner) where Dynabead concentration is highest and along the outskirts (outer).

Figure 9: Amm-C12 F1 -62 mediated U937 cell capture with Dynabead M-

270 Epoxy: Slides were then analysed for the presence of DAPI stained cells and the location of Beads relative to the cells.

Figure 10: Evaluation of selected Amm-C12 modified aptamer candidates’ ability to capture U937 cells capture when immobilised to Dynabead M-270 Epoxy: Beads were assessed for their ability to capture U937 following magnetic separation by preparing microscope slides and counting the number of cells present in 4 different frames at 4 different magnifications. Average number of cells is normalised between different magnifications to achieve the same scale, and compared across different aptamer samples. ANOVA results are indicated by the bar at the top of the graph, while post-hoc Student’s t-tests were conducted between the blank samples (A or G) and their respective aptamer functionalised beads: ^* indicates p-value < 0.05 when blank bead A is compared with other aptamer bead A samples; † indicates p-value < 0.05 when blank bead G is compared with other aptamer bead G samples.

Figure 11 : Visualisation of aptamer mediated cell capture with Dynabead M-270 Epoxy. Beads were assessed for their ability to capture U937 following magnetic separation by preparing microscope slides and visualising them under the Zeiss Axiovert A1 F1-LED epifluorescent microscope at 400 X magnification.

Figure 12: Screening of aptamer binding to hCD4-conjugated magneticbeads, via qPCR. The U4, U14, U20, U26 aptamer and control C27 and F1- 62 sequences at initial concentrations of 1 mM, 0.5 pM, 0.1 pM, 0.05 pM, 0.01 pM, 0.001 pM, 0.0001 pM and 0 pM exposed to 50 pg hCD4-conjugated magnetic-beads. Data points excluded from the modeled kinetic curve are shown as empty circles. The amount of ssDNA retained to hCD4-conjugated beads was determined relative to qPCR calibration curves of each respective aptamer.

Figure 13: Multiple time point study evaluating the rate of colorimetric signal generation by the aptamer gold-nanoparticle conjugates on the LFA screening platforms. A - Initial addition of 50 pL of the various AuNP conjugates; t = 0 min represents the time after the immediate addition of the last U26-AuNP conjugate. B - Colorimetric detection of hCD4 by the various AuNP conjugates; t = 14 min represents the time after the immediate addition of the last U26-AuNP conjugate. C and D - Comparison of the background-corrected quantified signal intensities at the CD4 zones, caused by the accumulation of the SA-AuNPs functionalized with U4, U14, U20, U26 aptamer and control C27 and F1 -62 sequences at a concentration of 0.25 mM.

SEQUENCE LISTING

The nucleic acid and amino acid sequences listed herein and in any accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and the standard three letter abbreviations for amino acids. It will be understood by those of skill in the art that only one strand of each nucleic acid sequence is shown, but that the complementary strand is included by any reference to the displayed strand. In the accompanying sequence listing:

SEQ ID NO:1 - nucleotide sequence of Cy5 conjugated forward SELEX Library Primer. SEQ ID NO:2 - nucleotide sequence of the PC>3-Reverse SELEX Library Primer.

SEQ ID NO:3 - nucleotide sequence of the SELEX Library template DNA.

SEQ ID NO:4 - nucleotide sequence of the PUC M13 forward primer.

SEQ ID NO:5 - nucleotide sequence of the PUC M13 reverse primer.

SEQ ID NO:6 - nucleotide sequence of the UB57 aptamer.

SEQ ID NO:7 - nucleotide sequence of the U1 aptamer.

SEQ ID NO:8 - nucleotide sequence of the U2/4 aptamer.

SEQ ID NO:9 - nucleotide sequence of the U20 aptamer.

SEQ ID NO:10 - nucleotide sequence of the U21 aptamer.

SEQ ID NO:11 - nucleotide sequence of the U22/23 aptamer.

SEQ ID NO:12 - nucleotide sequence of the U24/25 aptamer.

SEQ ID NO:13 - nucleotide sequence of the U26 aptamer.

SEQ ID NO:14 - nucleotide sequence of the U29 aptamer.

SEQ ID NO:15 - nucleotide sequence of the U38 aptamer.

SEQ ID NO:16 - nucleotide sequence of the U42/49 aptamer.

SEQ ID NO:17 - nucleotide sequence of the U45 aptamer.

SEQ ID NO:18 - nucleotide sequence of the U47 aptamer.

SEQ ID NO:19 - nucleotide sequence of the U10/35 aptamer.

SEQ ID NQ:20 - nucleotide sequence of the U14 aptamer. SEQ ID NO:21 - nucleotide sequence of the U30 aptamer.

SEQ ID NO:22 - nucleotide sequence of the U32 aptamer.

SEQ ID NO:23 - nucleotide sequence of the U39 aptamer.

SEQ ID NO:24 - nucleotide sequence of the U46 aptamer.

SEQ ID NO:25 - nucleotide sequence of the U12 aptamer.

SEQ ID NO:26 - nucleotide sequence of the U19 aptamer.

SEQ ID NO:27 - nucleotide sequence of the U16 aptamer.

SEQ ID NO:28 - nucleotide sequence of the U33 aptamer.

SEQ ID NO:29 - nucleotide sequence of the U36 aptamer.

SEQ ID NO:30 - nucleotide sequence of the U37 aptamer.

SEQ ID NO:31 - nucleotide sequence of the U41 aptamer.

SEQ ID NO:32 - nucleotide sequence of the U51 aptamer.

SEQ ID NO:33 - nucleotide sequence of F1 -62 positive control aptamer.

SEQ ID NO:34 - nucleotide sequence of the C27 negative control aptamer.

SEQ ID NO:35 - nucleotide sequence of the MR801 negative control aptamer.

SEQ ID NO:36 - nucleotide sequence of the forward primer for F1 -62 qPCR.

SEQ ID NO:37 - nucleotide sequence of the reverse primer for F1 -62 qPCR.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown.

The invention as described should not be limited to the specific embodiments disclosed and modifications and other embodiments are intended to be included within the scope of the invention. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

As used throughout this specification and in the claims which follow, the singular forms “a”, “an” and “the” include the plural form, unless the context clearly indicates otherwise.

The terminology and phraseology used herein is for the purpose of description and should not be regarded as limiting. The use of the terms “comprising”, “containing”, “having” and “including” and variations thereof used herein, are meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

The inventors of the present invention have employed a Crossover SELEX approach to identify aptamers which could recognise CD4-expressing cells with high affinity and specificity in order to develop a cost-effective POC aptasensor for CD4+ T-cell enumeration. To this end, a modified Crossover SELEX approach was used to enrich a pool of oligonucleotides with aptamers which could bind the purified form of recombinant CD4 protein (Peptide-SELEX) in addition to the endogenously expressed CD4 protein present in the membranes of U937 cells (Cell-SELEX) with high specificity relative to IgG and HSA (Figure 1). Protein-SELEX, and Cell-SELEX were used in alternating rounds to increase the stringency of selection on the ssDNA library. The sequenced pool of aptamers was found to be diverse with a few concatemers, multiple truncated sequences, multiple unsuccessfully ligated sequences, and some aptamer candidates. Despite this, aptamer candidates were successfully identified, and exemplars from each family were chosen for further rounds of analysis.

The inventors of the present invention have evaluated aptamer candidates for their ability to bind to U937 cells and recombinant human CD4 protein, as well as for their specificity for CD4 recombinant proteins when compared to IgG and HSA. Analysis of biotin-aptamer binding ability was assessed with ELONA using protein functionalised paramagnetic beads. Cy5-aptamer binding ability was evaluated using fluorescent confocal microscopy of U937 cells.

Of the aptamer candidates, it was found by the inventors that the U26 aptamer (SEQ ID NO:13) showed good binding affinities for recombinant CD4 protein when conjugated to biotin and U937 cells when conjugated to Cy5. In addition, the U4 aptamer (SEQ ID NO:8) showed high binding specificity for CD4 when functionalised to paramagnetic beads. The previously reported control aptamer, F1-62, was only able to bind to U937 cells when modified with Cy5, while the C27 aptamer control was only able to bind recombinant human CD4 proteins when conjugated to biotin. Compared to the two previously published aptamers, a particularly attractive feature of the U26 aptamer (SEQ ID NO:13) for application in biosensor studies is its ability to retain its affinity for CD4 when bound to either biotin or Cy5. The versatility in modification makes the U26 aptamer (SEQ ID NO:13) attractive for application in existing sensor platforms.

To prove the capability of the identified aptamers to bind endogenous CD4 in complex media, the inventors of the present invention conducted an investigation into the aptamer-mediated magnetic capture of cells revealed that aptamers could be immobilised onto paramagnetic beads coated at epoxy groups through an amino linker modification (Amm C12). These paramagnetic beads also showed capture of U937 cells from solution. Aptamer assessment within this format proved their potential application in CD4-expressing cell numeration. The terms “nucleic acid” or “nucleic acid molecule” encompass both ribonucelotides (RNA) and deoxyribonucleotides (DIMA), including cDNA, genomic DNA, and synthetic DNA. The nucleic acid may be double-stranded or single-stranded. Where the nucleic acid is single-stranded, the nucleic acid may be the sense strand or the antisense strand. A nucleic acid molecule may be any chain of two or more covalently bonded nucleotides, including naturally occurring or non-naturally occurring nucleotides, or nucleotide analogs or derivatives. By “RNA” is meant a sequence of two or more covalently bonded, naturally occurring or modified ribonucleotides. The term “DNA” refers to a sequence of two or more covalently bonded, naturally occurring or modified deoxyribonucleotides. By “cDNA” is meant a complementary or copy DNA produced from an RNA template by the action of RNA-dependent DNA polymerase (reverse transcriptase).

As used herein, the terms “oligonucleotide” and “polynucleotide” both refer to DNA or RNA fragments comprising one or more nucleotides, including naturally occurring or non-naturally occurring nucleotides, or nucleotide analogs or derivatives.

The term “aptamer” refers to a single stranded nucleotide sequence that specifically binds to a particular target molecule. The nucleotide sequence is preferably a DNA sequence, although RNA or other amplifiable nucleic acid based polymers, including naturally occurring or non-naturally occurring nucleotides, or nucleotide analogs or derivatives, can be used. The distinct sequences of the aptamers of the present invention determine the folding of the oligonucleotide molecule into a unique conformational structure. Preferably, an aptamer is a degenerate sequence of about 15-120 nucleotides bases, more preferably of about 30-60 nucleotide bases, in length. The aptamers of the present invention may be flanked by fixed sequences. Those of skill in the art will understand that the sequence of the aptamer may be varied without substantially affecting binding of the target molecule to the aptamer.

The term “sample” refers to a sample isolated or collected from an environmental or biological source and is located ex vivo. Preferably, the sample is a blood or fluid sample.

The term “isolated”, is used herein and means having been removed from its natural environment.

The term “purified”, relates to the isolation of a molecule or compound in a form that is substantially free of contamination or contaminants. Contaminants are normally associated with the molecule or compound in a natural environment, purified thus means having an increase in purity as a result of being separated from the other components of an original composition. The term "purified nucleic acid" describes a nucleic acid sequence that has been separated from other compounds including, but not limited to polypeptides, lipids and carbohydrates which it is ordinarily associated with in its natural state.

The term “complementary” refers to two nucleic acid molecules, e.g., DNA or RNA, which are capable of forming Watson-Crick base pairs to produce a region of double-strandedness between the two nucleic acid molecules. It will be appreciated by those of skill in the art that each nucleotide in a nucleic acid molecule need not form a matched Watson-Crick base pair with a nucleotide in an opposing complementary strand to form a duplex. One nucleic acid molecule is thus “complementary” to a second nucleic acid molecule if it hybridizes, under conditions of high stringency, with the second nucleic acid molecule. A nucleic acid molecule according to the invention includes both complementary molecules.

As used herein a “substantially identical” or “substantially homologous” sequence is a nucleotide sequence that differs from a reference sequence only by one or more conservative substitutions, or by one or more non-conservative substitutions, deletions, or insertions located at positions of the sequence that do not destroy or substantially reduce the antigenicity of the expressed fusion protein or of the polypeptide encoded by the nucleic acid molecule. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the knowledge of those with skill in the art. These include using, for instance, computer software such as ALIGN, Megalign (DNASTAR), CLUSTALW or BLAST software. Those skilled in the art can readily determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. In one embodiment of the invention there is provided for a polynucleotide sequence that has at least about 80% sequence identity, at least about 90% sequence identity, or even greater sequence identity, such as about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the sequences described herein.

Alternatively, or additionally, two nucleic acid sequences may be “substantially identical” or “substantially homologous” if they hybridize under high stringency conditions. The “stringency" of a hybridisation reaction is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation which depends upon probe length, washing temperature, and salt concentration. In general, longer probes required higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridisation generally depends on the ability of denatured DNA to re anneal when complementary strands are present in an environment below their melting temperature. A typical example of such “stringent” hybridisation conditions would be hybridisation carried out for 18 hours at 65 °C with gentle shaking, a first wash for 12 min at 65 °C in Wash Buffer A (0.5% SDS; 2XSSC), and a second wash for 10 min at 65 °C in Wash Buffer B (0.1% SDS; 0.5% SSC).

The term “SELEX” as used herein refers to any systematic and iterative technique for the selective enrichment of aptamers by exponential amplification and molecular evolution.

As used herein, the term “Crossover SELEX” refers to enrichment of a pool of oligonucleotides with aptamers which could bind the purified form of a peptide, preferably recombinant CD4 protein (Peptide-SELEX), in addition to the endogenously expressed protein in a specific cell type, preferably CD4 protein present in the membranes of U937 cells (Cell-SELEX). The SELEX may inlcude the modification of aptamer candidate sequences with fluorophores (to allow for rapid DNA quantification), and immobilisation of the target onto magnetic beads for ease-of-handling during the SELEX process.

The term “target molecule” or “target” refers to any molecule capable of forming a complex with an oligonucleotide, including, but not limited to, small organic compounds such as drugs, dyes, metabolites, cofactors, transition state analogs, and toxins. Preferably, the target molecule is CD4 protein or CD4+ cells.

The terms “label” and “detectable label” interchangeably refer to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrochemical, chemical, or other physical means. Useful labels include fluorescent dyes (fluorophores), fluorescent quenchers, luminescent agents, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, ³²P and other radioisotopes, gold nanoparticles (AuNPs), haptens, proteins, nucleic acids, or other substances which may be made detectable, e.g., by incorporating a label into an oligonucleotide specifically reactive with a target molecule. The term includes combinations of single labelling agents, e.g., a combination of labels that provides a unique detectable signature.

In some embodiments, the aptamers or aptamer compositions according to the invention may be provided in a kit, together with instructions for use. In other embodiments, the aptamers or aptamer compositions of the present invention may be integrated into an electrochemical impedance spectroscopy biosensing platform for use as a “biosensor” or “aptasensor”.

The following examples are offered by way of illustration and not by way of limitation.

EXAMPLE 1

Preparation for SELEX

Preparation of ssDNA library

Initially, 10 mI (corresponding to ~ 6 c 10¹⁴ molecules of ssDNA library) were dissolved in PBS++ from the original 100 mM stock solution. For all subsequent rounds of SELEX, approximately 1 pg of ssDNA from PCR amplification and lambda exonuclease digestion were dissolved in PBS++. The ssDNA aptamers were encouraged to form secondary structures in PBS++ buffer through heat denaturation and then rapid cooling (Baldrich etal. 2004). At this point, human serum albumin (HSA) (A3782-100MG; Sigma Aldrich) to a final concentration of 150 pM, glucose (0.45% ^w/_v), and MgCh (5 mM) were added, to formulate PBS++, after the heat denaturation in order to avoid degradation or precipitation of products.

PCR Amplification and Conditioning

Amplification of the ssDNA pools generated after the positive selection steps was conducted to increase the copy number of successful target binding sequence. A small-scale cycle optimisation step was set up to determine the correct number of cycles that produce maximum product with no non-specific amplicons. A standard 200 pi KAPA Taq PCR Master mix consisted of 1X Buffer B, 1 mM dNTPs, 0.5 pM Cy5 conjugated truncated forward SELEX Library Primer (-5) having the sequence 5’Cy5- GCCTGTTGTGAGCCTC’3 (SEQ ID NO:1), 0.5 pM 3’ P0₃-Reverse SELEX Library Primer (-3) having the sequence 5’GGGAGACAAGAATAAGC-P03’3 (SEQ ID NO:2), approximately 0.0125 ng.pl^-1 SELEX Library template DNA having the sequence 5’GCCTGTTGTGAGCCTCCTAAC(49N)GCTTATTCTTGTCTCCC’3 (SEQ ID NO:3), and 0.025 U.pl ¹ Taq Polymerase, between 0.0125 - 0.05 ng.pl^-1 of template was added.

PCR amplification was conducted with initial denaturation at 95°C for 5 minutes, denaturation at 95°C for 1 minute, annealing at 59°C, 58°C, 56°C, and 54°C for 1 minutes, extension at 72°C for 1 minute and 30 seconds. Evaluation of the PCR products formed was performed using 10% PAGE. The final PCR products were pooled, and subsequently concentrated and dsDNA column purified. PCR amplification of the selection pool resulted in a dsDNA product which was digested with lambda exonuclease to form an ssDNA population. ssDNA digestion of the phosphorylated anti-sense strand was conducted according to Ardjomandi et al. (2013) with some modifications: approximately 5 pg of dsDNA was added to lambda DNA exonuclease digestion master mix and incubated at 37°C for 2 hours. The reaction was terminated through heat inactivation of the enzyme. The ssDNA was cleaned up and the concentration of DNA was measured using the Nanodrop™ 2000 and stored at -20°C.

Paramagnetic bead functionalisation with recombinant CD4 and IgG for peptide

SELEX

Human recombinant CD4 protein (ab167756; Lot: GR156912) was obtained from Abeam (Cambridge, USA) at a concentration of 1 .36 mg. ml^-1. This protein is fused to the Fc fragment of human lgG1 at the C-terminal and derived from HEK 293 cells. Human immunoglobulin G (IgG), Plasma (401114) was purchased from Calbiochem (Merck Millipore). IgG was used for counter selection, and recombinant CD4 for positive selection steps.

For both proteins, approximately 5 mg of beads were activated through resuspension in 1 ml conjugation buffer, vortexed, and incubated at 4 °C for 10 minutes. Washed beads were resuspended in 0.5 ml of conjugation buffer containing either 0.08 g.L ¹ recombinant CD4 protein, or 0.2 g.L ¹ of IgG protein in 0.15 M HEPES (pH 7.4). An equal volume of ammonium sulphate in conjugation buffer was added, to a final concentration of 0.5 M, and the beads incubated in the protein solutions by slow tilt rotation at 4°C for 24 hours. The supernatant was discarded and the beads subsequently washed four times with conjugation buffer. All unfunctionalised groups were blocked using 0.2 M Tris buffer (adjusted to pH 8.5 using 1 M HCI) for 1 hour at 4°C with slow tilt rotation. The coated beads were washed 4 times with PBS++ and stored at 4 °C for use in peptide SELEX steps. Beads were stored for a maximum of 2 hours before use.

Preparation of mammalian cells for Cell -SELEX

U937 (CRL-1593.2™) hystiocytic lymphoma cells and Ramos (RA1) (CRL- 1596™) Burkitt’s Lymphoma cells were both sourced from ATCC (Middlesex, United Kingdom). Approximately 5 c 10⁶ RA1 or U937 cells with > 95% viability were harvested, washed with PBS++ by centrifugation, and incubated with 500 pmol sheared salmon sperm DNA (D1626-250MG; Sigma Aldrich) for 5 minutes at 25°C with slow tilt rotation. RA1 cells were used as a negative control due to their similarity to U937 cells, and absence of CD4 protein on the cell surface. The number of U937 cells was reduced to 2.5 c 10⁶ in round 2 of Cell-SELEX.

Preparation of competent DH5a E. coli

A single colony of DH5a E. coli (donated by Department of Biochemistry and Microbiology, Rhodes University, South Africa) was inoculated into 25 ml of LB and the culture incubated at 37 ^eC under agitation for 4-6 hours. The cells were placed on ice, collected by centrifugation and resuspended in 10 ml of 0.1 M CaCh. The cell suspension was chilled, after which the cells recollected through centrifugation and resuspended in 5 ml of 0.1 M CaCh supplemented with 15% (7_V) glycerol and dispensed into cold microtubes in 100 pi aliquots. The aliquots were then frozen immediately at -80°C until used. Modified nutrient agar was prepared to screen transformed cells for the presence of plasmid vector containing candidate aptamers. Nutrient agar was prepared as detailed above, once the agar had cooled to 60°C, ampicillin was added to a concentration of 100 pg.ml ¹. Before the plates were inoculated with bacteria, 400 ng of X-gal in _ddhbO and 10 pmoles of IPTG in _ddhbO were added to the surface of each of the agar plates and allowed to dry.

EXAMPLE 2

Crossover SELEX

Protein SELEX

Protein-SELEX was conducted with fluorescent labelled aptamers selected against control IgG functionalised Dynabead M-270 Epoxy beads, and target recombinant CD4 functionalised beads, as previously described by Stoltenburg et al. (2005) with some modifications.

Counter selection was performed by incubating the ssDNA library with IgG- functionalised beads to select for aptamers which did not bind to IgG or the beads. Glycine and tris were included in the buffer to remove any aptamer sequences specific for these compounds that could be present in the recombinant CD4 protein suspension (in round 1 ). The ssDNA in the supernatant was retained by ssDNA column purification. The pellet was kept for further analysis and the supernatant was used in subsequent positive selection steps.

Positive selection was performed by snap-cooling the ssDNA retained from the counter selection supernatant and adding HSA, glucose, and MgCh (to obtain PBS++), and incubating with recombinant CD4-functionalised beads for 30 minutes (incubation times were decreased to 25 minutes, and then 10 minutes in the subsequent rounds) at 25°C with slow tilt rotation. The beads were washed four times through magnetic separation and the supernatant was analysed and discarded. The bound ssDNA was then eluted from the beads.

Elution of ssDNA from functionalised beads was conducted by incubating the beads with 200 pi of elution buffer (40 mM Tris, 10 mm EDTA, 3.5 M Urea, 0.002% Tween 20, pH 8.8) and heating for 10 minutes at 80°C. The beads were applied to a magnet, the supernatant removed, and the process was repeated. The eluted DNA was recovered from the solution through ethanol precipitation in the presence of sodium acetate and glycogen. The solution was kept at -20°C for 24-48 hours, and then centrifuged for 30 minutes at 4°C, 14000 rpm. The supernatant was removed, ethanol was added and centrifuged. The supernatant was removed and the pellet was dried in a heating block. The resultant pellet was resuspended in 30 mI of _ddH₂0, analysed with the Nanodrop™ 2000, and stored for use in PCR amplification.

The ssDNA retained from the eluate of the CD4 beads was amplified by PCR. The resulting dsDNA was digested to ssDNA with lambda exonuclease. Thereafter, the ssDNA was collected via ssDNA column purification. The prepared ssDNA pool was then subjected to Cell-SELEX phase as described below.

Cell SELEX

Cell-SELEX was conducted as described by Sefah et at. (2010) and Shangguan etai, (2015) with some modifications. Approximately 1 pg of snap cooled ssDNA (the pool attained from Protein SELEX) was incubated with 5 x 10⁶ RA1 cells for 1 hour at 25°C with slow tilt rotation (total volume of 1 ml). Cells were then pelleted through centrifugation. The supernatant was removed, and the cells were washed again and collected via centrifugation. The two fractions of supernatant were then pooled and subjected to Cell-SELEX Positive Selection as described below.

Positive selection was performed by incubating the pooled supernatant with 5 x 10⁶ U937 CD4 expressing cells for 30 minutes (number of cells were decreased to 2.5 x 10⁶ cells and incubation time was decreased to 15 minutes in round two). The cells were incubated with the supernatant at 25°C with slow tilt rotation. Thereafter the bound ssDNA and U937 cells were collected by centrifugation and the supernatant removed for later analysis. The cells were washed 3X through resuspension in 1 ml of PBS++ and then centrifuged. The bound ssDNA library was then eluted from the cells through heat induced DNA denaturation.

The ssDNA sequences were eluted from the collected U937 cells via heat induced denaturation, followed by collection through centrifugation and ssDNA column purified to recover the eluted ssDNA sequences. The ssDNA isolated from the Cell- SELEX positive selection step was then PCR amplified. The resulting dsDNA from PCR was digested to ssDNA with lambda DNA exonuclease for preparation in the subsequent Protein SELEX phase, and the procedure was repeated again from protein SELEX with the enriched ssDNA pool of sequences.

EXAMPLE 3

Candidate Aptamer Characterisation

Bacterial Cloning and Transformation

PCR products from the final selection pool from the last peptide SELEX round were amplified and ligated into the pGEM-T Easy vector for insertion into competent DH5a E. coli cells to allow for the isolation and sequencing of individual aptamer sequences. DH5a E. coli were transformed with the ligated p-GEM-T easy plasmid. The bacterial cells were grown on modified nutrient agar (containing ampicillin, X-gal and IPTG) in discrete colonies for isolation of individual sequences. Untransformed cells were also grown as a negative control. Transformations were plated onto ampicillin-positive plates and ampicillin-negative nutrient agar plates and incubated overnight at 37°C. Transformed bacterial cells were grown on nutrient agar plates containing IPTG and X-Gal for blue/white screening of successfully ligated colonies. At least 50 white and light blue colonies were selected and used to amplify the PUC M13 plasmid containing the insert sequence.

Colony PCR

Colony PCR was conducted according to Alshahni et al. (2009) with some modifications. Bacterial cells were grown on ampicillin positive plates. PCR master mix, including PUC M13 forward primer having the sequence 5’- CCCAGT CACG ACGTT GT AAAACG-3’ (SEQ ID NO:4) and PUC M13 reverse primer having the sequence 5’-AGCGGATAACAATTTCACACAGG-3’ (SEQ ID NO:5), was added to each colony and amplified under the following cycling conditions: initial hot start at 95°C for 5 minutes, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 54°C for 30 seconds, and elongation at 72°C for 30 seconds, followed by an additional elongation step at the end at 72°C for 8 minutes. Each PCR sample was sent for Sanger sequencing to determine the sequence of the insert and potential aptamers.

BigDye Sequencing Protocol The Big Dye® sequencing protocol was performed according to manufacturer’s instructions. Once column purified, the BigDye® PCR samples were for Sanger sequencing by the NRF-SAIAB Molecular Genetics Laboratory (Rhodes University, South Africa).

Sequences were analysed in BioEdit and aligned using Muscle 6.0 Multiple Sequence Alignment tool which was also used to assign phylogeny. The multiple cloning site, flanked by the PUC M13 forward and reverse primers, was removed and the SELEX library primers were identified.

The results from colony PCR, insert PCR and Sanger sequencing are summarised in Table 1. Sanger sequencing confirmed that 34 sequences were positive for insert but found that only 26 of these sequences were of the correct length and contained both primer binding sites while the remaining 8 sequences contained only the reverse primers, or its reverse complement. Sequencing also found that 10 of the successfully inserted sequences were duplicates of one another.

Table 1 : Summary of sequence analysis results.

Table notes: Concatemer: DNA sequence containing multiple repeating copies of primer motifs;

Rp: reverse primer; Fp: forward primer; rRp: reverse primer in opposite direction; rFp: forward primer in opposite direction; ^** indicates sequence » 300 bp; ^*** indicates sequence that is 400 bp.

Bioinformatic Analysis of Sequences

The U-class aptamer pool obtained after five rounds of crossover SELEX with CD4 protein and CD4-expressing cells were cloned into E. coli and sequenced with Sanger sequencing. A summary of all of the sequences obtained from the SELEX process is presented in Table 2.

Table 2: Summary of U-class CD4-specific aptamer sequences gathered after 5 rounds of crossover SELEX. Selection rounds alternated between CD4 and CD4- expressing cells.

U47 (SEQ ID NO:18) : GCCTGTTGTGAGCC : AG ACGTTT AATT AACT CAAGTT G ATCGCTCCT : CATGCTTATTCTTG :

: GTTCACTTCATAATCGT : TCTCCCA

Concatemer Sequ

Sequence : Forward Primer : Variable Region

^* Duplicate sequences were obtained.

Nucleotide enrichment of the selection pool was examined by calculating the average frequency of each of the four nucleotides across all sequences obtained after 5 rounds of SELEX. Figure 2 shows that the frequency of thymidine (T) nucleotides is significantly greater than the frequency of adenosine (A), cytosine (C) and guanine (G) nucleotides in the sequences. The frequency of adenosine residues was also significantly greater than cytosine. However, the frequency of pyrimidines and purines were relatively similar. Thus, thymidine residues are favoured in the selection pool.

Bioinformatic analysis of the sequences was conducted to identify potential aptamer candidates and their secondary structures. Sequences U4, U14, U20 and U26 were selected as representative aptamer candidates from phylogenetic analysis. These sequences show several unique conformational stem and loop secondary structures (Figure 3).

Preparation of target molecules and aptamers for CD4 binding assays

Selected aptamers were ordered from Integrated DNA Technologies (Coralville, USA) and modified at the 5’ end with C12 amine (Amm), biotin, or Cy5 for use in cell capture, fluorescent, and enzymatic assays. The following aptamers obtained from the Crossover SELEX process were assessed for their ability to bind to CD4 expressing U937 cells: U4 (SEQ ID NO:8), U14 (SEQ ID NO:20), U20 (SEQ ID NO:9), U26 (SEQ ID NO:13), and U38 (SEQ ID NO:15). Their binding performance was compared to the following control aptamer sequences:

SEQ ID NO:35 - MR801 : negative control aptamer that binds murine adipocytes:

5’GCCT GTT GT G AGCCT CCT AACT AT AT ATT AT CACGT GG ACAT ACA- TTT ACGG ACACTT AGT CT CGG AT AGCAT GCTT ATT CTT GT CT CCC3’ ;

SEQ ID NO:34 - C27: negative control aptamer previously selected against a 2dCD4 epitope:

5’GCCT GTT GT G ACCCT CCT AACT AGCT CGT AG AAAAAAAAT AT AAAGGG CGT GT GCT GGG ACT GCT CGGG ATT GCGG ACACAT GCTT ATT CTT GT CT CCC3’ ;

SEQ ID NO:33 - F1-62: positive control aptamer developed against CD4 expressing cells:

5ΆT CCAG AGT G ACGCAGCACCACCACCGT ACAATTTTTT CATT ACCT ACT CGGC3’.

All aptamers were conjugated to Cy5 (Cyanine 5 fluorescent dye) for fluorescent detection, biotin for detection of aptamer with ELONA, or Amm-C12 for immobilization of aptamer onto Dynabead M-270 Epoxy for magnetic bead capture.

EXAMPLE 4

Biotin labelled aptamer evaluation through Peptide ELONA

In order to determine aptamer affinity for both CD4-expressing cell and the recombinant CD4 proteins, biotin-conjugated aptamers were used in a colorimetric ELONA assay. Protein and cell ELONAs were conducted in order to assess the affinity of the chosen SELEX aptamers for the recombinant protein, CD4 and CD4-expressing U937 cells, respectively, while also providing some indication of specificity of the aptamer by comparing the extent of binding to the control proteins human IgG, and human serum albumin (HSA). This also provided an indication of the potential applicability of the aptamer in a colorimetric sensor format.

Dynabead M-270 Epoxy were either left unlabelled, or functionalised with CD4, HSA or IgG. After functionalisation, the beads were incubated with 1 c 10⁹ moles of snap-cooled, biotinylated aptamer, followed by strep-HRP. The beads were then washed four times and resuspended in 50 pi of PBS++. 1-Step Ultra TMB was added and absorbance was ws measured in the SpectraMax at 450 nm.

The average rate of TMB radical development of unfunctionalised (no protein) beads appeared fairly consistent across the tested aptamers in Figure 4. This is supported by the ANOVA which indicates that there is no significant difference between the unfunctionalised beads lacking aptamers and those incubated with aptamers (p-value = 0.600; F = 0.778 < F crit = 2.848). This suggests that all aptamers trialled in this investigation do not bind non-specifically to the unfunctionalised beads.

The ANOVA (p-value = 0.134; F = 2.488 < F crit = 4.066) conducted on the different no aptamer beads indicates that there was no specific response that could be observed between the unfunctionalised beads and the functionalised beads, with specific reference to CD4 indicating that any variation in response on CD4 beads occurs as a result of aptamer- mediated interactions.

Prior research indicated that biotin-C27 would have a higher affinity for CD4 proteins than for IgG, HSA, or unlabelled beads. Figure 4 shows that the rate of TMB radical development in the presence of biotin-C27 is higher in CD4 functionalized beads than HSA, and significantly higher than IgG, or unfunctionalized beads, this is supported by Student’s t-test where p is equal to 0.031 and 0.038, respectively. This also indicates that C27 (SEQ ID NO:34) can bind to the recombinant and CD4 protein when biotinylated, with high affinity and high specificity, although partial binding to HSA is observed.

Zhao et al. (2014) identified the aptamer F1 -62 (SEQ ID NO:33) which bound to CD4 expressing cells and recombinant CD4 proteins with high affinity and specificity when tagged with Cy5. It was also expected that biotin-F1 -62 bind to CD4 proteins with higher affinity than for IgG, HSA, or unlabelled beads. The ANOVA (p-value = 0.14; F = 2.404 < F crit = 4.066) conducted in Figure 4 shows that no significant differences were observed in the rate of TMB radical development between different beads labelled with F1 -62. This indicates that biotin F1 -62 does not bind to the recombinant form of the CD4 protein, or that it does not bind to CD4 when biotinylated. Post-hoc Student’s t-test reveal that the aptamers biotin-U26 and biotin-U4, in Figure 4, show significantly greater binding to CD4 beads relative to unfunctionalised, IgG and HSA. This preliminary screening study indicates that these aptamers bind to CD4 recombinant proteins with high affinity.

Furthermore, inter-aptamer Student’s t-test conducted on CD4 bead samples indicate the rate of TMB development of the CD4 beads labelled with biotin-U26 and biotin-U4 is significantly greater than the observed response for C27 (p-value = 0.002 and 0.05, respectively) and all other aptamers which did not show positive binding. As the same concentration of aptamer and protein was used in each sample, this response indicates that biotin-U26 and biotin-U4 bind with higher affinity to CD4 recombinant protein than biotin-C27.

EXAMPLE 5

Cy5 fluorescently labelled aptamer evaluation through confocal microscopy

The ability of Cy5 labelled aptamer candidates to bind to U937 cells was assessed using fluorescent spectroscopy and confocal microscopy. Approximately 9 x 10⁵U937 cells were harvested and washed with PBS++ and resuspended in different solutions of prepared aptamer binding buffers. The cells were incubated with Cy5 conjugated prepared aptamers, washed and resuspended in PBS++ and 10 pi of the solution was transferred to a PLL coated coverslip for assessment through confocal microscopy. The cells were fixed and stained with 1 pg.ml ¹ DAPI. Dried slides were mounted with DAKO mounting medium and visualised using the Zeiss LSM 780 Confocal Scanning Microscope using three illuminating lasers (UV Laser 355 nm for DAPI excitation, Argon multiline 458/488/514 for Cy3 excitation and, Argon Laser 633 nm for Cy5 excitation). Images were analysed using Zen 2 software.

Unlabelled U937 cells were compared to cells labelled with Cy5-C27, and Cy5- F1 -62 as negative and positive controls, respectively. The fluorescent staining pattern is shown in Figure 5. Unlabelled cells exposed to both the UV and Argon lasers showed only a fluorescent blue nucleus stained with DAPI. The control aptamer Cy5-C27 selected in an alternative SELEX process only showed a fluorescent blue nucleus with no presence of the Cy5 fluorophore on the cell membrane. The lack of binding to CD4- expressing cells could be attributed to two factors: either this aptamer cannot recognise the endogenous form of CD4 protein on the cell membrane; or, the conformation of the aptamer at the active site could be altered with the addition of an alternative conjugation molecule, Cy5. The positive control aptamer, F1 -62 (SEQ ID NO:33), showed positive aptamer binding, demarcated as an increase in the Cy5 fluorescent intensity located at the peri nuclear cell membrane. This staining pattern is consistent with that previously observed wherein CD4 is located in small clusters evenly distributed around the cell membrane. The small clustering effect is likely due to the enrichment of CD4 in lipid rafts which plays a role in enhanced immune response.

The candidate aptamers identified from the SELEX process were screened for their affinity for U937 cells in Figure 6. This figure shows that there is a high fluorescent signal emitted in the range of Cy5 by the aptamer Cy5-U4, and Cy5-U14. Flowever, the fluorescent profile of these aptamers indicates that the signal occurs within the cell in the nuclear region and not near the cell membrane. This indicates that the increased fluorescent signal is due to cellular uptake of the aptamer by compromised cell membranes. Separately, the aptamer Cy5-U20 and Cy5-U38 both show very little fluorescent signal in the Cy5 region.

The only aptamer which indicates a distinct positive signal for specific aptamer binding to the cells’ extracellular membrane, similar to Cy5 F1 -62, is the aptamer U26 (SEQ ID NO:13). This staining pattern is identical to that exhibited by the aptamer F1 - 62 which also shows clusters of red fluorescent signal around the cell membrane and an increased in the fluorescent intensity in the Cy5 range in the extranuclear region of the cell as demarcated by DAP I.

EXAMPLE 6

Evaluation of Amm-C12 aptamer mediated magnetic bead cell capture

As part of the development of a point-of-care CD4 aptasensor, it was also important to validate the application of different aptamers in various sensor formats. The CD4 aptasensor of the present invention aims to make use of aptamers as both capture and detection molecular recognition elements. As a capture agent, the aptamer will be able to concentrate CD4 cells from a complex matrix while a secondary aptamer can quantitatively label the CD4 cells, ideally in a colorimetric format.

Aptamer mediated magnetic bead capture of U937 cells

Amm-C12 F1-62 aptamer labelled Dynabead M-270-Epoxy were assessed for their ability to capture U937 cells from a sample. Amm-C12 is an an amine group which allows for aptamer binding to the epoxy residue on the surface of the beads. Dynabead M-270 Epoxy functionalisation was conducted using 0.3 mg of beads (activated in 0.15 M HEPES pH 7.4 at room temperature for 10 minutes) each incubated with 9 c 10 ¹⁰ moles of Amm-C12 modified aptamer (No aptamer, C27, F1 -62, U4, U14, U20, U26, U38) in either 3 M ammonium sulphate (in 0.15 M HEPES, pH 7.4) or 0.05 M glutamate (in 0.15 M HEPES, pH 7.4) for 24 hours at 37°C with rotation. The supernatant was removed, and the beads were washed twice through magnetic separation with PBS++ and transferred to a microscope slide. The dispersion properties of the unlabelled versus labelled beads were assessed using the Zeis Axiovert A1-F1-LED epifluorescent microscope.

Indirect aptamer quantification was used to determine if the aptamer had bound to the beads successfully. This process compared the difference in ssDNA concentration between the supernatant after functionalisation and the initial solution. Figure 7 shows that there is a decrease in ssDNA concentration in the supernatant when compared with the initial ssDNA concentration before incubation indicating that the Dynabead was successfully functionalised with ~ 15. 353 pg if it is assumed that no aptamers were eluted from the beads during additional wash steps.

Figure 8 shows that the dispersion properties of the unlabelled and aptamer labelled beads are very different: unlabelled beads tend to stack together more tightly, forming network-like shapes which can be seen under lower magnifications. The aptamer labelled beads exhibit a more uniform distribution, clustering only around U937 cells. This is a further indication that the aptamer functionalisation process was successful. Further, only the aptamer labelled beads were able to capture cells. Figure 9 shows that cells are only present in the F1 -62 aptamer labelled bead samples. The beads are also tightly clustered around the cells at multiple sites suggesting that the aptamer binds at multiple protein sites which allows for the cell to be well anchored to the magnet during separation.

Assessment of Amm-C 12 modified aptamers as U937 cell capture agents

Approximately 30 c 10⁵ U937 cells were harvested, ~ 1 c 10⁵ U937 cells were incubated with the aptamer labelled beads for 30 minutes at room temperature and then washed twice through magnetic separation with PBS++. The beads were then resuspended in 100 pi of PBS++ and 10 pi were placed onto a PLL coated coverslip. The cells were allowed to settle onto the coverslip for 10 minutes after which they were fixed with 4% (7_V) paraformaldehyde in PBS for 30 minutes. Excess paraformaldehyde was removed and the cells were stained with 1 :1000 DAPI for 1 minute in the dark. The coverslips were washed with _ddH₂0 and mounted with DAKO mounting medium. The cells were visualised under the Zeiss Axiovert A1 -F1 LED Epifluorescent microscope to obtain cell counts. Figure 10 shows that A C27, G U4 and G U14 have the greatest affinity for the U937 cells relative to the unlabelled ‘blank’ beads. Both U4 (SEQ ID NO:8) and C27 (SEQ ID NO:34) can bind to U937 cells or CD4 proteins when biotinylated, this investigation indicates that they can also bind to U937 cells when immobilised to a bead through an Amm-C12 epoxy group. U26 (SEQ ID NO:13), U38 (SEQ ID NO:15), and F1-62 (SEQ ID NO:33) also all seem to show greater affinity for U937 cells than the unfunctionalised beads in the presence of glutamate and ammomium sulphate. Figure 11 shows that the cells are heavily coated by the functionalised and unfunctionalised beads indicating that there is a degree of non-specific cell capture taking place.

EXAMPLE 7

Binding affinity characterization via magnetic-bead-based qPCR

Before qPCR kinetic analysis, the 5'-biotin-labeled ssDNA aptamers were heat- denatured at 95 °C for 5 min in 1 x PBS pH 7.4, thereafter rapidly cooled and maintained at 4 °C for 30 min. For each aptamer, separate 50 pg hCD4-conjugated bead amounts were incubated with aptamer concentrations of 1 pM, 0.5 pM, 0.1 pM, 0.05 pM, 0.01 pM, 0.001 pM, 0.0001 pM in 100 pL 1 PBS pH 7.4 at 25 °C for a period of 2 h. Following incubation, the supernatant (unbound aptamer fraction) was discarded from the hCD4-conjugated beads collected by magnetic force. The magnetic-beads were washed twice by 200 pL 1 x PBS pH 7.4 containing 0.001% Tween-20, once with 200 pL 1 PBS pH 7.4, and resuspended to a final volume of 50 pL in 1 x PBS pH 7.4. Aptamer sequences were amplified directly off the hCD4- conjugated beads using a reaction mixture utilizing a SYBR Green PCR master mix (qMax Green No Rox qPCR Mix, Accuris, USA). A single qPCR reaction included 1 pg magnetic-bead ssDNA template, 0.5 pM SELEX library forward primer (SEQ ID NO:1 ), 0.5 pM 5'-phosphorylated SELEX library reverse primer (SEQ ID NO:2) and 1 master mix (containing reaction buffer, nucleotides, fluorogenic dye and Hot Start DNA polymerase) made up to 10 pL with ddH20. The PCR cycle parameters were as follows: initial hold at 30 °C for 30 s followed by 95 °C for 2 min; 40 cycles of denaturation at 95 °C for 5 s, annealing at 54 °C for 20 s and extension at 72 °C for 10 s.

Minor modifications of the qPCR procedure were necessary for the F1-62 aptamer, utilizing forward and reverse primers of 5'-ATCCAGAGTGACGCAGCA-3' (SEQ ID NO:36) and 5'-GCCAG AGT AGGT AAT G AA-3' (SEQ ID NO:37). All qPCR amplification was performed in triplicate. The derived C_q values of the hCD4-conjugate bead-bound ssDNA were normalized to those obtained from 5 fmol, 1 fmol, 0.5 fmol, 0.1 fmol, 0.01 fmol, 0.001 fmol, 0.0001 fmol standard additions of each corresponding aptamer amplified by qPCR as described above. For affinity analysis, normalized C_q values were modeled to the Langmuir isotherm, using the least-squares regression algorithm in Statistica 13, by the following equation:

where A_beadbound is the amount of aptamer bound to the hCD4-conjugated beads (nmol) quantified by qPCR, [A] is the concentration of aptamer initially incubated with the hCD4-conjugated beads (mM), A_max is the modeled maximum bound (nmol) and ¼ is the modeled dissociation constant of the aptamer-bead complex (pM).

Antibody magnetic-bead based ELISA validated both bead immobilization of hCD4 and exposure of the CD4 extracellular domain to solvent-accessible areas. As hCD4 was randomly orientated onto the magnetic-beads, evident exposure of the hCD4 extracellular domain proved varied epitope presentation to aptamer candidates during the qPCR-based binding assay. From Figure 12, the successive increase in sequence retention to hCD4-conjugate beads was indicative of candidate aptamer binding affinity to hCD4 in a concentration-dependent manner. Among the aptamer sequences generated, U26 showed the highest affinity for hCD4 with a K_d value of 2.93 ± 1.03 nM. The dissociation constants of the sequences U20, U14, U4, C27 and F1 - 62 were evaluated as 457.61 ± 126.67 nM, 118.26 ± 46.19 nM, 19.04 ± 9.18 nM, 287.61 ± 72.48 nM and 221 .07 ± 24.49 nM respectively. Variation in the apparent maximal binding capacities of the aptamer candidates to 50 pg hCD4-conjugated beads (A_max) was noted to two orders of magnitude, similar to the variation in K_d. Variation in A_max was not expected to arise from sequence-specific variation in amplification efficiencies of the aptamers (controlled by comparing bead-bound responses of each aptamer through standards generated using each respective sequence, nor from differences in the available protein sites between each aptamer (as bead masses were maintained between samples). Rather, observed differences in A_max between candidates may relate to differences in the rate constants by which the complexes assemble and dissociate. The evaluated binding affinity of F1-62 was lower than the previously reported K_d of 1 .59 nM, determined through flow cytometry analysis of Cy5-labeled F1-62 binding to the CD4+ T-cell lymphoma cells, Karpas-299. This may be attributed to differences in the ligand-analyte pair and interaction environment between cell and magnetic-bead based affinity assays. Association of the C27 control provides evidence of nonspecific affinity to the hCD4-conjugated bead matrix by the selected aptamer candidates in the nM range. Despite potential nonspecific interactions, comparison of binding affinities tended to favour hCD4 binding in order of the sequences U26, U4, U14, F1-62 and U20.

EXAMPLE 8

Screening of the Apt-AuNP conjugates for suitability in binding hCD4 in a Lateral Flow Assay (LFA) format

An LFA device comprising 3 primary components was used: sample pad, nitrocellulose strip and wicking pad. Protein solutions (0.5 mg mL-1 ) of the target hCD4 (test protein) and control IgG proteins were separately drop-dried in 2 ^c 0.25 pL aliquots onto the nitrocellulose strip at their designated zones, using a 37 °C drying time of 10 min between each aliquot addition. To remove nonspecific binding sites on the material, the entire nitrocellulose strip and wicking pad were blocked for 20 min by immersion in 1 c PBS containing 5% (w/v) milk powder. The blocked strip and pad were subsequently dried at 37 °C for 2 h and stored in an airtight container at 4 °C, for a maximum of 7 days until use. The wicking pad, prepared nitrocellulose strip and sample pad were assembled on the surface of adhesive cardboard and mounted into a plastic cassette. The LFA was stored at room temperature until use.

Colloidal nanosphere gold particles (AuNP) were synthesized using conventional citrate reduction with minor modifications: a solution of 50 mL of water containing 400 pM dissolved HAuCI4-3H20 was heated to boiling under reflux and 2 mL of 1% (w/v) trisodium citrate was added slowly, in increments. After 30 min, the solution turned characteristically deep red in color and was subsequently cooled to 4 °C in the dark for storage and later use.

Citrate-stabilized AuNPs were used as colorimetric reporter molecules in the aptamer-based LFA. For aptamer functionalization, 1 pg streptavidin (1 mg mL^-1 in PBS) was first incubated in 1 mL citrate stabilized AuNP solution for 2 h at room temperature under slow tilt-rotation. For each test, 200 pL of the AuNP solution was collected by centrifugation at 7000g for 5 min and the supernatant was removed. To immobilize the aptamer sequences, streptavidin-coated nanoparticles (SA-AuNP) were incubated with 250 nM heat-treated aptamer in 100 pL 1 * PBS for 1 h at room temperature under slow tilt-rotation. The aptamer-functionalized AuNP conjugates (Apt-AuNP) were collected at 7000g for 5 min and the supernatant was removed. The Apt-AuNP were resuspended in 50 mI_ 1^c PBS and directly added to the sample well of the prepared LFA. To serve as a positive control, two rabbit (1 :1000 dilution) monoclonal antibodies targeting the extracellular domain of CD4 were each separately incubated in citrate stabilized AuNPs for 2 h at room temperature under slow tilt- rotation. For each test, 200 pl_ of the antibody-coated AuNPs were collected by centrifugation at 7000g for 5 min, resuspended in 50 mI_ 1^c PBS and directly added to the LFA sample well.

Image and video footage were captured for 15 min upon initial addition of the Apt-AuNPs to the LFA using a Samsung Galaxy Note 10+ primary camera (12MP sensor with 1.4 pm pixels, 26 mm (equivalent) variable-aperture f/1 .5-2.4 lens, dualpixel AF, OIS). Still images captured every minute of the LFA run were evaluated for colorimetric signal intensity using the colour histogram function of the Fiji image- processing package. Here, the sum of square differences in individual RGB colour components of a 30 c 30 pixel sample between the target protein and blank regions of the LFA was returned as a measure of colorimetric signal intensity in the following equation:

AI = J(R_S - R_oy + (C_s - Go)² + (B_s - B₀)²

Where the colorimetric signal intensity is represented by D/, the RGB colour of the target protein /¾, G_s and B_s and corresponding R₀, Go and So as the RGB of the LFA blank region.

Figure 13 presents the results of the multiple time point study of the various AuNP conjugates towards 250 ng of hCD4. As the time-dependent responses across the same sensors were examined, a repeated measures variant of ANOVA was used to assign significant differences in the sensor responses, compared to the measured signal intensities at t = 0. Upon addition to the Apt-AuNP screening platforms, the conjugates began migrating towards the test and control zones of the nitrocellulose via passive capillary action. Notably, the U4 Apt-AuNP conjugate was not recoverable following centrifugation potentially due to factors leading the conjugate to an aggregated state. The average migration rate of the various conjugates was consistent and rapid. For all stable Apt-AuNP conjugates, slight fluctuations were observed in D/ for the initial 1-3 min due to discoloration of the nitrocellulose strip by the Apt-AuNP solution when compared to the dry nitrocellulose membrane. The D/ of each sensor achieved a consistent baseline response following saturation of the nitrocellulose strip with the Apt-AuNP solution upon 4 min. F1 -62 produced a statistically significant (p = 0.000) increase in D/ compared to the baseline response 7 min (p = 0.000) after the addition of the Apt-AuNP conjugate. Similarly, U26 produced a statistically significant response after 9 min (p = 0.000) and the U20 Apt-AuNP conjugate at 9 min (p = 0.029).

From the response time, sensitivity and specificity of hCD4 detection, the LFA screening platform identified two novel aptamers disclosed herein (U20 and U26) for potential application in rapid, low-cost and selective aptamer-based CD4 lateral-flow sensors.

EXAMPLE 9

Summary of aptamer binding assays to CD4

The results of all the different aptamer binding assays are summarised in Table 3. While some aptamers may have a preference for the endogenous form of the protein in the whole cell, all aptamers here, including F1 -62 (SEQ ID NO:33) were selected through a crossover SELEX approach using recombinant CD4 proteins and CD4- expressing cells as targets. It was found that F1 -62 (SEQ ID NO:33), the control aptamer, only bound to either form of CD4 when tagged with Cy5. This disruption in binding activity could be due to disruptions in the secondary structure of the aptamer by the biotin molecule as this sequence is only 53 bp long which is just over half the length of the SELEX library (90 bp). Zhao etal. (2014) did not measure the amount of aptamer biotin-F1 -62 bound to CD4 expressing Karpass 299 cells directly, but they did notice that when the aptamer was biotinylated and bound to streptavidin in tetrameric forms they were able to reduce gp-120 binding.

Table 3: Influence of reporter molecule or aptamer configuration on aptamer candidates’ ability to bind to target: Different aptamer candidates were assessed for their ability to bind to CD4 proteins or U937 cells when labelled with biotin or Cy5, or immobilised onto a paramagnetic bead through an Amm C12 linker.

Table Notes: FCM: Fluorescent confocal microscopy; AMC: Aptamer mediated cell capture;

Cy5: Cyanine 5; ELONA: Enzyme linked oligonucleotide assay; Amm C12: Amino group attached to a 12-carbon molecule. Asterisk (*) indicates that the ‘Yes’ is based on results of preliminary investigation.

In contrast, U26 (SEQ ID NO:13) bound to recombinant CD4 proteins when tagged with biotin and U937 cells when tagged with Cy5. Surprisingly, the aptamer U4 (SEQ ID NO:8) also showed good binding ability to CD4 proteins when biotinylated while exhibiting no binding ability to U937 cells when tagged with Cy5. When aptamers were immobilised onto paramagnetic beads and used to capture cells from a solution, all aptamer functionalised beads except U20 (SEQ ID NO:9) were able to capture significantly more beads than the unfunctionalised aptamers. This gives a good preliminary indication of potential sensor applications as detailed herein. The LFA screening platform identified U20 (SEQ ID NO:9) and U26 (SEQ ID NO:13) for potential application in rapid, low-cost and selective aptamer-based CD4 lateral-flow sensors.

Claims

1 . An aptamer comprising the nucleotide sequence of any one of SEQ ID NOs:8, 9, 13 and 20, or a complementary sequence thereof, wherein the aptamer selectively and specifically binds human CD4 protein and/or cells expressing human CD4 protein.

2. The aptamer of claim 1 , wherein the aptamer comprises the nucleotide sequence of SEQ ID NO:13, or a complementary sequence thereof.

3. The aptamer of claim 1 , wherein the aptamer is labelled.

4. The aptamer of claim 3, wherein the label is biotin, a fluorescent label a luminescent label, a radioactive isotope, amine, aryl azides, thiol, a nanoparticle, or an enzymatic label.

5. The aptamer of claim 4, wherein the label is biotin, Cy5 or a gold nanoparticle.

6. A biosensor device for detecting human CD4 protein and/or cells expressing human CD4 protein, comprising a labelled aptamer comprising the nucleotide sequence of any one of SEQ ID NOs:8, 9, 13 and 20, or a complementary sequence thereof, wherein the aptamer selectively and specifically binds human CD4 protein and/or cells expressing human CD4 protein.

7. The biosensor of claim 6, wherein the aptamer comprises the nucleotide sequence of SEQ ID NO:13, or a complementary sequence thereof.

8. The biosensor of claim 6, wherein the label is biotin, a fluorescent label, a luminescent label, a radioactive isotope, amine, aryl azides, thiol, a nanoparticle, or an enzymatic label.

9. The biosensor of claim 8, wherein the label is biotin, Cy5 or a gold nanoparticle.

10. A method of detecting human CD4 protein and/or cells expressing human CD4 protein in a sample, the method comprising: a) labelling an aptamer having selectivity and specificity for human CD4 protein and/or cells expressing human CD4 protein with a label, wherein the aptamer comprises the nucleotide sequence of any one of SEQ ID NOs:8, 9, 13 and 20, or a complementary sequence thereof; b) contacting the sample with the labelled aptamer; and c) detecting binding of the aptamer to human CD4 protein and/or cells expressing human CD4 protein, where binding of the aptamer to human CD4 protein and/or cells expressing human CD4 protein indicates the presence of human CD4 protein and/or cells expressing human CD4 protein in the sample.

11. The method of claim 10, wherein the aptamer comprises the nucleotide sequence of SEQ ID NO:13, or a complementary sequence thereof.

12. The method of claim 10, wherein the label is biotin, a fluorescent label, a luminescent label, a radioactive isotope, amine, aryl azides, thiol, a nanoparticle, or an enzymatic label.

13. The method of claim 12, wherein the label is biotin, Cy5 or a gold nanoparticle.

14. The method of claim 10, wherein detecting binding of the aptamer to human CD4 protein and/or cells expressing human CD4 protein is performed using an impedimetric assay, a spectrophotometric assay, a voltammetric assay, a chemiluminescence assay, a flow cytometry assay, a radioactive assay, an immunochromatographic assay, a piezoelectric assay, a colourimetric assay, a fluorescence assay, an ELISA assay, an ELONA assay, an acoustic assay, and/or a polymerase chain reaction binding assay.