Staphylococcal c-s lyase inhibitors

Abstract

The invention relates to the isolation and characterisation of one or more aptamers against staphylococcal C-S lyase. The invention also provides aptamers, aptamer-conjugates, and minimal functional fragments thereof which may be used to inhibit the activity of said lyases and thereby inhibit Staphylococcus associated malodour.

Description

[0001] STAPHYLOCOCCAL C-S LYASE INHIBITORS

[0002] Field of the Invention

[0003] The invention relates to the isolation and characterisation of one or more aptamers against staphylococcal C-S lyase. The invention also provides aptamers, aptamer-conjugates, and minimal functional fragments thereof which may be used to inhibit the activity of said lyases and thereby inhibit Staphylococcus associated malodour.

[0004] Background to the Invention

[0005] It has been known for some time that human sweat itself is generally odourless and that it is the action of certain strains of bacteria present on the human skin which, metabolising compounds in the sweat, produce the malodour. In particular, the generation of thiol based malodorants, namely 3-methyl-3-sulfanylhexan-1-ol (3M3SH), have been attributed to the actions of a small number of staphylococcal species, especially Staphylococcus hominis which is the second most abundant staphylococcal species found in the axillary microbiota.

[0006] Generation of thiol malodorants has been confirmed as being a bacterially mediated process by which axillary S. hominis and closely related species take up cysteinylglycine dipeptide- conjugated percussors derived from the axillary apocrine gland and through the systematic action of a microbial dipeptidase and a specific PLP-dependant cysteine thiol lyase (PatB) release malodours thiols including 3M3SH.

[0007] Malodour development by skin bacteria has been tackled in many ways, most notably by the use of bactericidal deodorants. There have also been some publications describing means by which microbial transformation of malodour precursors in sweat into malodourous substances may be inhibited, to thereby reduce malodour.

[0008] WO1991 / 05541 (Gillette, 1991) discloses amino acid beta-lyase inhibitors as deodorants.

[0009] WO1991 / 11988 (Gillette, 1991) discloses compositions comprising a compound capable of serving as an alternative substrate to a naturally occurring malodour precursor and thereby reducing the conversion of said malodour precursor into a malodourous substance. Certain cysteine and serine analogues were found to be suitable alternative or “competitive” substrates, including O-benzyl serine.

[0010] WO1 995 / 07069 (Gillette, 1995) discloses O-acyl serines as competitive substrate inhibitors of enzymatic creation of body malodour. \N0 2000 / 34232 (Gillette, 2000) discloses particular serine carbonates as precursors for organoleptic compounds, masking agents and antimicrobial agents. They are further disclosed as alternative substrates for malodour producing enzymes.

[0011] EP 1 ,846,436 B1 (Firmenich, 2012) discloses a method of screening compounds capable of reducing malodour comprising the use of a malodour precursor which is a glycine-cysteine- thioalcohol conjugate.

[0012] Recent work in this area Rudden et al. “The molecular basis of thioalcohol production in human body odour” Sci Rep 10, 12500 (2020), has demonstrated, via heterologous expression of the aforementioned cysteine thiol lyase in a non-malodour producing host strain (referred to in this publication as ShPatB) that ShPatB is “both necessary and sufficient” for the generation of thioalcohol-based odour production in the human axilla.

[0013] Given this necessity for an active cysteine thiol lyase for axillary malodour production, sufficient inhibition of this enzyme may offer new approaches for the reduction of axillary malodour. However, there remains a need to develop novel reagents capable of specifically targeting such staphylococcal C-S lyases.

[0014] Aptamers have previously been considered for use in personal care compositions, but not for targeting staphylococcal C-S lyases. WO2020 / 214784 (P&G, 2020) discloses aptamers having a high binding affinity and specificity for malodourous molecules to reduce the intensity of the undesirable smells in personal care compositions.

[0015] It is an aim of some embodiments of the present invention to at least partially mitigate some of the problems identified in the prior art.

[0016] Summary of the Invention

[0017] The present invention relates to the development of one or more aptamers against staphylococcal C-S lyase. Typically, the aptamers are capable of specifically binding to one or more staphylococcal C-S lyases which are pyridoxal phosphate (PLP)-dependent such as ShPatB as further described herein.

[0018] The aptamers described herein are shown to work effectively and provide a simple and quick means of inhibiting staphylococcal C-S lyase activity. For example, the aptamers may be capable of reducing or preventing staphylococcal C-S lyase activity in cleaving Cys-3- methyl-3-sulfanylhexan-1-ol (Cys-3M3SH) to release methyl-3-sulfanylhexan-1-ol (3M3SH). As described herein, 3M3SH is volatile compound released by odour-producing staphylococci. As such, the aptamers of the invention are capable of inhibiting Staphylococcus associated malodour. In certain embodiments, the aptamers are capable of inhibiting staphylococcal C-S lyase activity within bacterial cells. For example, the aptamers of the invention may reduce or prevent binding of the C-S lyase to Cys-3-methyl-3-sulfanylhexan-1-ol (Cys-3M3SH) within staphylococcal cells. Typically, the staphylococcal cells are coagulase negative. Typically, the staphylococcal cells are S. hominis or closely related species as described herein. The staphylococcal cells may be, for example, within body fluids such as saliva or sweat. The staphylococcal cells may be within any fabric contacting such fluids, including items of clothing, seat fabrics, gym equipment, bed linen or the like. Typically, the staphylococcal cells are part of the axial microbiome of human skin, as further described herein.

[0019] In some embodiments, the small size of one or more aptamer(s) of the invention allows them to be internalised within staphylococcal cells, where they can influence metabolic processes of the cell. In some embodiments, the aptamers are conjugated to one or more molecule(s) thereby targeting them to staphylococcal cells (e.g., odour-forming staphylococcal cell) and / or facilitating their entry across the bacterial cell wall. For example, the aptamers may be conjugated to a second aptamer, a peptide and / or a small molecule as further described herein.

[0020] In certain embodiments, the aptamers are capable of specifically binding to staphylococcal C-S lyase comprising a polypeptide sequence as set forth in SEQ ID NO:1, 2, 3, 4, 5 or 6.

[0021] In certain embodiments, the aptamers are capable of specifically binding to staphylococcal C-S lyase comprising a polypeptide sequence as set forth in SEQ ID NO:1, or a protein having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to SEQ ID NO: 1.

[0022] In certain embodiments, the aptamers are capable of specifically binding to a staphylococcal C-S lyase comprising a N-terminal region which includes a hydrophobic pocket selective for branched aliphatic thioalcohol ligands. For example, the N-terminal region may comprise one or more of G21, Y25 and / or E40, wherein the numbering refers to the amino acid residue positions with reference to the polypeptide sequence as set forth in SEQ ID NO: 1. Typically, the N-terminal region comprises a variable region as set forth in any one of SEQ ID NO: 7, 8, 9, 10, or 11. Without being bound to theory, such variable regions may be conserved amongst only staphylococcal C-S lyases capable of metabolising Cys -3M3SH.

[0023] In certain embodiments, the aptamers are capable of specifically binding to a staphylococcal C-S lyase comprising Y25 and / or F274, wherein the numbering refers to the amino acid residue positions with reference to the polypeptide sequence as set forth in SEQ ID NO: 1. Without being bound to theory, such hydrophobic residues may mediate apolar contacts thereby increasing selectivity to aliphatic cysteine-S-conjugates as compared to B. Subtilis Putative Cysteine-S-conjugate p-lyase (PatB) as set forth in SEQ ID NO: 12.

[0024] In certain embodiments, the aptamers of the invention are not capable of binding (or bind with only low affinity) to C-S-lyases from other genus of bacteria. For example, the aptamer of the invention may not be capable of (or bind with only low affinity to) to B. Subtilis PatB as set forth in SEQ ID NO:12.

[0025] In certain embodiments, the invention provides one or more aptamers capable of specifically binding to staphylococcal C-S lyase, wherein the one or more aptamers comprise:

[0026] (a) a nucleic acid sequence selected from any one or more of SEQ ID NOs: 19 to 30;

[0027] (b) a nucleic acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95% or about 99% identity or more with any one or more of the sequences of (a); or

[0028] (c) a nucleic acid sequence having at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more consecutive nucleotides of sequences (a) or (b).

[0029] Advantageously, these aptamers are highly effective in inhibiting staphylococcal C-S lyase as compared to other aptamers screened during the selection process.

[0030] In certain embodiments, the invention provides one or more aptamers capable of specifically binding to staphylococcal C-S lyase, wherein the one or more aptamers comprise:

[0031] (a) a nucleic acid sequence selected from any one or more of SEQ ID NOs: 19, 28 or 30;

[0032] (b) a nucleic acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95% or about 99% identity or more with any one or more of the sequences of (a); or

[0033] (c) a nucleic acid sequence having at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more consecutive nucleotides of sequences (a) or (b).

[0034] Advantageously, these aptamers were the most effective in inhibiting staphylococcal C-S lyase as compared to other aptamers screened during the selection process.

[0035] In certain embodiments, the invention provides one or more aptamers capable of specifically binding to staphylococcal C-S lyase, wherein the one or more aptamers comprise:

[0036] (a) a nucleic acid sequence selected from any one or more of SEQ ID NOs: 19 or 28; (b) a nucleic acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95% or about 99% identity or more with any one or more of the sequences of (a); or

[0037] (c) a nucleic acid sequence having at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more consecutive nucleotides of sequences (a) or (b).

[0038] In certain embodiments, the invention provides one or more minimal effective fragments of any one or more aptamers as described herein. For example, the invention provides one or more minimal fragments of SEQ ID NOs: 19 or 28, as further described herein.

[0039] In certain embodiments, the invention provides one or more aptamers capable of specifically binding to staphylococcal C-S lyase, wherein the one or more aptamers comprise:

[0040] (a) a nucleic acid sequence selected from any one or more of SEQ ID NOs: 31 to 51;

[0041] (b) a nucleic acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95% or about 99% identity or more with any one or more of the sequences of (a); or

[0042] (c) a nucleic acid sequence having at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more consecutive nucleotides of sequences (a) or (b).

[0043] In certain embodiments, the invention provides one or more aptamers capable of specifically binding to staphylococcal C-S lyase, wherein the one or more aptamers comprise:

[0044] (a) a nucleic acid sequence selected from any one or more of SEQ ID NOs: 33, 34, 42, and 43;

[0045] (b) a nucleic acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95% or about 99% identity or more with any one or more of the sequences of (a); or

[0046] (c) a nucleic acid sequence having at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more consecutive nucleotides of sequences (a) or (b).

[0047] In certain embodiments, the invention provides one or more aptamers capable of specifically binding to staphylococcal C-S lyase, wherein the one or more aptamers comprise:

[0048] (b) a nucleic acid sequence selected from any one or more of SEQ ID NOs: 33, 34 and 42;

[0049] (b) a nucleic acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95% or about 99% identity or more with any one or more of the sequences of (a); or

[0050] (c) a nucleic acid sequence having at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more consecutive nucleotides of sequences (a) or (b). Advantageously, these aptamers are highly effective within bacterial cells, as further described herein.

[0051] In certain embodiments, the one or more aptamers are isolated.

[0052] In certain embodiments, the aptamer is an RNA aptamer (e.g., a single stranded RNA aptamer).

[0053] In certain embodiments, the invention further provides aptamers which compete for binding to the staphylococcal C-S lyase with any aptamer as described herein.

[0054] In certain embodiments, the invention provides an aptamer-conjugate as further described herein.

[0055] In certain embodiments, the aptamers are capable of inhibiting Staphylococcus associated malodour as further described herein.

[0056] Detailed Description

[0057] Brief Description of the Figures

[0058] Certain embodiments of the present invention will be described in more detail below, with reference to the accompanying Figures in which:

[0059] Figure 1 shows the mechanism through which the odourless precursor Cys-Gly-3M3SH is processed in Staphylococcus hominis. Cys-Gly-3M3SH is secreted by axillary apocrine glands in the skin. This is actively taken up by Staphylococcus hominis via the di- / tri-peptide transporter (DtpT). The terminal glycine is cleaved by a dipeptidase (PepA) to release Cys- 3M3SH, which is then further processed by the CS lyase to give the volatile thioalcohol 3- methyl-3-sulfanylhexan-1-ol (3M3SH). This is then excreted from S. hominis and is responsible for body malodour.

[0060] Figure 2 shows the Biolayer Interferometry data used to identify potential aptamer candidates. Individual aptamer candidates were synthesised incorporating a biotin group at the 5’ end. Biotinylated aptamer candidates were immobilised on streptavidin coated biosensor probes and interacted with ShPatB in solution. The sensor data was background corrected to remove any interaction between the ShPatB and an unloaded biosensor. The aptamer candidates are chosen based on their association rate (0-120 sec) and dissociation rate (120-240 sec). The best performing candidates in this screen appear to be 9S_7, 9S_8 and 9S_11 (SEQ ID NOs: 25, 26 and 29 respectively).

[0061] Figure 3 shows the enzyme activity assay used to quantify formation of 3M3SH by ShPatB. Thiol containing 3M3SH was quantified through reduction of Ellman's Reagent, DTNB (5,5- dithio-bis-(2-nitrobenzoic acid), giving a yellow product which is in turn quantified by measuring the absorbance at 405nm. A concentration dependant response is seen when a ShPatB dilution series (1:10 to 1:1,000) is added to the Cys-3M3SH substrate, in either 1x aptamer selection buffer (A) or 1x PBS (B). This confirms that the ShPatB enzyme is active. The same assay can be used to determine the inhibitory effect of the aptamer candidates.

[0062] Figure 4 shows enzyme inhibition assay data used to rank the aptamer candidates based on their inhibition of 3M3SH formation by ShPatB. Aptamer candidates were incubated with ShPatB to allow complex formation, before adding the Cys-3M3SH substrate. Production of 3M3SH was quantified using Ellman's Reagent (DTNB) and absorbance measured at 405nm. This was then plotted for each candidate aptamer, to compare ShPatB activity in the respective sample (A). Each aptamer was tested in independent, duplicate assays (blue and orange bars) and plotted along with the average (grey bar). Buffer alone control (no aptamer) was used as a negative inhibition control. The data was also used to calculate the % reduction in enzymatic activity, compared to the uninhibited ‘Buffer alone control’ (B). The data shows that aptamer candidates 9S_1, 9S_10 and 9S_12 (SEQ ID NOs: 19, 28 and 30 respectively), reproducibly inhibition of ShPatB by -50%, -55% and -30% respectively. Further aptamer candidates 9S_6, 9S_7, 9S_8 and 9S_9 (SEQ ID NOs: 24-27 respectively), show weaker inhibition of ShPatB.

[0063] Figure 5 shows enzyme inhibition assay data used to rank the fragments of aptamer candidates 9S_1 and 9S_10 (SEQ ID NOs: 19 and 28 respectively), based on their inhibition of 3M3SH formation by ShPatB. Aptamer candidates were incubated with ShPatB to allow complex formation, before adding the Cys-3M3SH substrate. Production of 3M3SH was quantified using Ellman's Reagent (DTNB) and absorbance measured at 405nm.

[0064] The data demonstrates that the enzyme is inhibited in the presence of full-length aptamer candidates 9S_1 (A), and 9S_10 (B) (SEQ ID NOs: 19 and 28 respectively) (orange bars), but not the scrambled control. The data also shows that the fragments of each aptamer have a range of activities; select fragments e.g. 9S_1_F12, 9S_1_F14, 9S_1_F15, 9S_1_F16, 9S_1_F18, 9S_1_F19, 9S_10_F1, 9S_10_F2, 9S_10_F3, 9S_10_F4, 9S_10_F6, 9S_10_F8, 9S_10_F9, 9S_10_F14, 9S_10_F15, 9S_10_F18, 9S_10_F19, 9S_10_F20, 9S_10_F21 , 9S_10_F22 and 9S_10_F23 (SEQ ID NOs: 31 to 51 respectively) retain the inhibitory properties.

[0065] Figure 6 shows data from a whole cell assay used to assess the inhibitory properties of aptamer candidates in a whole cell based bacterial assay. S. hominis liquid cultures were treated candidate enzyme inhibitors for 24 hours at 37°C. Samples of the culture supernatant were then tested for thiol production from the Cys-3M3SH substrate, quantified using Ellman's Reagent (DTNB) and absorbance measured at 405nm. A sample containing buffer alone (no aptamer) was used as a negative inhibition control. This data was used to calculate the % inhibition, compared to the uninhibited control.

[0066] Data shows that full-length aptamer candidates 9S_1 or 9S_10 (SEQ ID NOs: 19 and 28 respectively) (yellow bars) inhibited activity -20%, a non-functional scrambled control (red bar) inhibited -15%, where the nominated panel of fragments (blue bars) had a range of activities from non-functional, to -90% inhibition for fragment Optimers 9S_1_F15 (SEQ ID NO: 33), 9S_1_F16 (SEQ ID NO: 34) and 9S_10_F8 (SEQ ID NO: 42) and -70% inhibition for Optimer 9S_10_F9 (SEQ ID NO: 43). These, 9S_1_F15 (SEQ ID NO: 33), 9S_1_F16 (SEQ ID NO: 34), 9S_10_F8 (SEQ ID NO 42) and 9S_10_F9 (SEQ ID NO: 43) are inhibitory in the whole cell assay. This may suggest that these Optimers, as smaller fragments, are taken up by the cultured cells and inhibit the enzyme inside the cells.

[0067] Figure 7 shows data from the enzyme inhibition assay used to assess retention of aptamer function after incubation in axillary derived buffer scrubs. Optimer fragments 9S_1_F15 (blue bars), 9S_1_F16 (orange bars) or 9S_10_F8 (grey bars) (SEQ ID NOs 33, 34, and 42 respectively) were incubated with axilla buffer scrubs at 37°C, for indicated time points, before being assessed for inhibition of ShPatB activity using the purified enzyme assays described herein. Samples were compared to control assays containing enzyme, but no aptamer (green bars).

[0068] Data shows that nominated Optimer fragments 9S_1_F15, 9S_1_F16 and 9S_10_F8 (SEQ ID NOs: 33, 34, and 42 respectively) reduce ShPatB activity to <10% in all samples incubated up to 180 mins (3 hrs). After 1440 mins (24 hrs) ShPatB activity increased to -35- 45%, suggesting some loss of activity, but that all Optimers retain some inhibitory activity after 24 hours at 37°C in axilla buffer scrub medium. Sequence listing

[0069] SEQ ID NO: 1 shows the protein sequence of Staphylococcus hominis PatB (ShPatB) mnynfdeiidrrytnamnvegykgyl f gdadtsdlkdndelirmwvadmdfgtpevvlna irerlnkkilgytnvf gseyyeafvswtkkrygf tfpqehlvf shgivaglielvgyi cd kddkalivtpsygpf kmacdknhistvysplinhhgyyeidfddvrkkveteniklci f a nphnptgrvwseeelatlgqimkendvwlisdeihcdikrsgqshtpf akavpdydkiit tmsqs kafniaglmf sniiiqnesllktwnthhf gtenplsvvatqaayekgedwlqamn hylddnfnyladf lekelphaef kipeatylawvdlsyyikekdidesmakf f iknagvi iegaeqfvhnaeghiriniavprevmkkglqkikaalv

[0070] SEQ ID NO: 2 shows the protein sequence of Staphylococcus hominis Chain A, Aminotransferase (PDB:6QP1_A), which is the crystal structure of the PLP-bound lyase in the external aldimine form when complexed with an inhibitor mgggf avnynfdeiidrrytnamnvegykgyl f gdadtsdlkdndelirmwvadmdf gtp evvlnairerlnkkilgytnvf gseyyeafvswtkkrygf tf sqehlvf shgivagliel vgyicdkddkalivtpsygpf kmacdknhistvysplinhhgyyeidfddvrkkveteni klci f anphnptgrvwseeelatlgqimkendvwlisdeihcdikrsgqshipf akavpd ydki it tmsqs kafniaglmf sniiiqnesllktwnthhf gtenplsvvatqaayekgeg wlqamnhylddnfnyladf lekelphaef kipeatylawvdlsyyikekdidesmakf fi knagviiegaeqfvhnaeghiriniavprevmkkglqkikaalvenlyf qghhhhhhhhh h

[0071] SEQ ID NO: 3 shows the protein sequence of Staphylococcus lugdunensis PatB (SIPatB) mtynfdeiidrrstnamnvegylpyl fgnadvsdlqhtddlirlwiadmdf atpdvvlna irrrldqknlgytqvfhvdyynafvkwtqs rygyhfpqeqlvf shgivaglielvsyicn nedhaliltpsygpf kmacdenqvevhysplineeeyyridfddverqivqhklci f cnp hnpsgrvws reelerf gnimkahdvwlisdeihcdimrkgmqhlpf atvlsdydkvitam sqs kafniaglmf snliirhkgllntwkqqhf gsenplsiaatqaayeqgedwlqamnty Idgnf ewlkqf Iqqelpnakf kipeatylawvdlsdyitqqhisepmakyf ikragviie gqeqfvhnadghirinlavprtvlqqgltkikaalms

[0072] SEQ ID NO: 4 shows the protein sequence of Staphylococcus devriesei PatB (SdPatB) msynfdeiidrrstnamnvegykgyl f gdadvldiedndelirmwvadmdf etpevvlda irdrldkkilgytni f gsdyynafvswterrygytfpqeqlvf shgivaglielvgyica kndkaliltpsygpf kmacdknhistvyspminhngyyeidf edvr qkveteni klci f a nphnptgrvwseeelrqf gqimkdnnvwl is deihcdikrsgqthtpf akavpdydkiit amsqs kafniaglmf sniiipntrllniwkrhhf stenplsivatqaayekgedwleamn dyldgnf qyladf lerelphaef kipeatylawvdlsyyikdndineplakyf iknagvi iegqeqfvhnadghvrinlavprkimqkglqkikdalv

[0073] SEQ ID NO: 5 shows the protein sequence of Staphylococcus haemolyticus PatB (ShaPatB) mtynfdeiidrrstnamnvegykgyl f gdadtsdleehdelirmwvadmdf atpevvlda irdrldkkilgytni f gtdyyeaf ms wt err fgy tfpqehlvf shgi vagi i el vs yi cd dddkali f tpsygpf kmacdknni kt vyspminhngyyeidf edvr qkveteni klci f a nphnptgrvwsedelkqlgqimvdndvwiisdeihcdikrdgqthvpf akavpdydkivt amsqs kafniaglmf snliipnrrllktwklhhf s senpl si vatqaayekgkdwlaamn dylddnf kylaqf leqelphaef kipeatylawvdlsyyikakhidepiakyf ikhagvi iegqeqfvhnaeghiriniavpreimikglqkikdalv

[0074] SEQ ID NO: 6 shows the protein sequence of Staphylococcus petrasii PatB mtynfdeiierrytnamnvegykgylf gdadvsdiedndelirmwvadmdf atpevvlda irdrldkkilgytni f gseyyeafvswtkrrf gytfpqeqlvf shgivagiielvsyicd eddkaliltpsygpf kmacdknnvsavysplinnngyyeidykdvrkkveteniklci f a nphnptgrvwsedelkqlgeimqdndvwvisdeihcdikradqthipf akavpdydkiit tmsqs kafniaglmf sniiipnrrllktwklrhf gtenplsiaatqaayekgedwleamn hylddnf kylaef lenelphakf kipeatylawvdlsyyinqkeidesiakyf ikhagvi iegeeqfvhnaeghvriniaiprevmkkglnkikealv

[0075] SEQ ID NO: 7 shows the N-terminal “variable region” of Staphylococcus hominis PatB gykgyl f gdadtsdlkdnde

[0076] SEQ ID NO: 8 shows the N-terminal “variable region” of Staphylococcus lugdunensis PatB gylpyl f gnadvsdlqhtdd

[0077] SEQ ID NO: 9 shows the N-terminal “variable region” of Staphylococcus devriesei PatB gykgyl f gdadvldiednde

[0078] SEQ ID NO: 10 shows the N-terminal “variable region” of Staphylococcus haemolyticus PatB gykgyl f gdadtsdleehde

[0079] SEQ ID NO: 11 shows the N-terminal “variable region” of Staphylococcus petrasii PatB gykgyl f gdadvs di ednde

[0080] SEQ ID NO: 12 shows the protein sequence of Bacillus subtilis PatB mn f dkr ee r 1 gt qsvkwdkt gel f gvtdalpmwvadmdf rapeai teal kerldhgi f gy ttpdqktrdavcgwmqnrhgwkvnpesitf spgvvtalsmavqaf tepgdqvvvqppvyt pf yhmvekngrhilhnpllekdgayamdf edletklsdpsvtl f ilcnphnpsgrswgre dllklgelclehgvtvvsdeihsdlmlyghihtpf aslsddf adisvtf aaps ktfniag Iqasaiiipdrlkrakf saslqrnglgglnaf avtaieaays kggpwldglisyiernmn eaeaf Istelpkvkmmkpdasyliwldf sayglsdaelqqrmlkkgkvilepgtkygpgg egfmrlnagcslatlqdglrrikaals

[0081] SEQ ID NO: 13 shows the N-terminal “variable region” of Bacillus subtilis PatB ktgel fgvtd

[0082] SEQ ID NO: 14 shows a N40B Forward Primer: CCAGTGTAGACTACTCAATGC

[0083] SEQ ID NO: 15 shows a N40B Reverse Primer: GGTTGACCTGTGGATAGTAC

[0084] SEQ ID NO: 16 shows a N40B Reverse Primer binding region:

[0085] GTACTATCCACAGGTCAACC

[0086] SEQ ID NO: 17 shows a N40B_T7_Long Forward Primer: GTGTGTATTGGCTATGTTCCTAATACGACTCACTATAGGGCCAGTGTAGACTAC

[0087] SEQ ID NO: 18 shows a N40B N40B_T7_Short Forward Primer:

[0088] GTGTGTATTGGCTATGTTCC

[0089] SEQ I D NO: 19 shows the full nucleic acid sequence of Aptamer 9S_1 :

[0090] GGGCCAGUGUAGACUACUCAAUGCUACCAGUGUAGACUACUCAAUGCCGUGGACUG

[0091] GUCGGGUUUGGAUUCGGCAGAUGAAUCAGUAGUACUAUCCACAGGUCAACC

[0092] SEQ ID NO: 20 shows the full nucleic acid sequence of Aptamer 9S_2:

[0093] GGGCCAGUGUAGACUACUCAAUGCUACCAGUGUAGACUACUCAAUGCUAGUGUGGU

[0094] GACCUUGACCUCUGGAUAUGGGUGGGAGGGAGGGUACUAUCCACAGGUCAACC

[0095] SEQ ID NO: 21 shows the full nucleic acid sequence of Aptamer 9S_3:

[0096] GGGCCAGUGUAGACUACUCAAUGCUACCAGUGUAGACUACUCAAUGCUCCUAUUCC

[0097] GUAUAGUACGUUAGGUUGGGUAGGUUGGUACGUACUAUCCACAGGUCAACC

[0098] SEQ ID NO: 22 shows the full nucleic acid sequence of Aptamer 9S_4:

[0099] GGGCCAGUGUAGACUACUCAAUGCUACCAGUGUAGACUACUCAAUGCUAGGUUGGU

[0100] UAGGUUUGGUGCAUUCUCUUGUUAUCCUUCUGUACUAUCCACAGGUCAACC

[0101] SEQ ID NO: 23 shows the full nucleic acid sequence of Aptamer 9S_5:

[0102] GGGCCAGUGUAGACUACUCAAUGCUACCAGUGUAGACUACUCAAUGCAGGGUGGGA

[0103] GGGAGGGUAUUGCAUUGCCUAAUCGAGGGUAGUACUAUCCACAGGUCAACC

[0104] SEQ ID NO: 24 shows the full nucleic acid sequence of Aptamer 9S_6:

[0105] GGGCCAGUGUAGACUACUCAAUGCUACCAGUGUAGACUACUCAAUGCUCGACAUUUC

[0106] CGCCCCGACGGCCCUCCUAGUGAUGGGGAGAGUACUAUCCACAGGUCAACC

[0107] SEQ ID NO: 25 shows the full nucleic acid sequence of Aptamer 9S_7:

[0108] GGGCCAGUGUAGACUACUCAAUGCUACCAGUGUAGACUACUCAAUGCAUGGAAGUCA

[0109] UCCUGGUUAACGUUUCUCGUUAACCAAUUUGUACUAUCCACAGGUCAACC

[0110] SEQ ID NO: 26 shows the full nucleic acid sequence of Aptamer 9S_8:

[0111] GGGCCAGUGUAGACUACUCAAUGCUACCAGUGUAGACUACUCAAUGCGCAUGGUUC

[0112] CGGGGACCACUGUCGUGAGCGAAAGACUCAUGGUACUAUCCACAGGUCAACC SEQ ID NO: 27 shows the full nucleic acid sequence of Aptamer 9S_9:

[0113] GGGCCAGUGUAGACUACUCAAUGCUACCAGUGUAGACUACUCAAUGCUGUGUUGAC

[0114] UUGAUCCUGUGGUAUAUGGGUGGGAGGGUUGGGUACUAUCCACAGGUCAACC

[0115] SEQ ID NO: 28 shows the full nucleic acid sequence of Aptamer 9S_10:

[0116] GGGCCAGUGUAGACUACUCAAUGCUACCAGUGUAGACUACUCAAUGCACCCGAGUC

[0117] CGAGUUGCACCUGACCACUAAAUACGAUCCGGUACUAUCCACAGGUCAACC

[0118] SEQ I D NO: 29 shows the full nucleic acid sequence of Aptamer 9S_11 :

[0119] GGGCCAGUGUAGACUACUCAAUGCUACCAGUGUAGACUACUCAAUGCCCUAUCGCAC

[0120] AAGUAGGUUGGCUAGGCUUGGUCUUGUGCAGUACUAUCCACAGGUCAACC

[0121] SEQ ID NO: 30 shows the full nucleic acid sequence of Aptamer 9S_12:

[0122] GGGCCAGUGUAGACUACUCAAUGCUACCAGUGUAGACUACUCAAUGCUCUAGGGGC

[0123] UGCUCGGGAUUGCGGAAGUGGCAUGAAGAGAGUACUAUCCACAGGUCAACC

[0124] SEQ ID NO: 31 shows a minimal fragment (9S_1_F12) of Aptamer 9S_1 :

[0125] GGGCCAGUGUAGACUACUCAAUGCCGUGGACUGGUCGGGUUUGGAUUCGGCAGAU GAAUCAGUA

[0126] SEQ ID NO: 32 shows a minimal fragment (9S_1_F14) of Aptamer 9S_1 :

[0127] GACUGGUCGGGUUUGGAUUCGGCAGAUGAAUCAGUAGUACUAUCCACAGGUCAACC

[0128] SEQ I D NO: 33 shows a minimal fragment (9S_1_F 15) of Aptamer 9S_1 :

[0129] UGGAUUCGGCAGAUGAAUCAGUAGUACUAUCCACAGGUCAACC

[0130] SEQ ID NO: 34 shows a minimal fragment (9S_1_F16) of Aptamer 9S_1 :

[0131] CAGAUGAAUCAGUAGUACUAUCCACAGGUCAACC

[0132] SEQ I D NO: 35 shows a minimal fragment (9S_1_F 18) of Aptamer 9S_1 :

[0133] CUCAAUGCCGUGGACUGGUCGGGUUUGGAUUCG

[0134] SEQ I D NO: 36 shows a minimal fragment (9S_1_F 19) of Aptamer 9S_1 :

[0135] CUCAAUGCCGUGGACUGGUCGGGUUUGGAUUCGGCAGA SEQ ID NO: 37 shows a minimal fragment (9S_10_F1) of Aptamer 9S_10:

[0136] GGGCCAGUGUAGACUACUCAAUGCACCCGAGUCCGAGUUGC

[0137] SEQ ID NO: 38 shows a minimal fragment (9S_10_F2) of Aptamer 9S_10:

[0138] ACCUGACCACUAAAUACGAUCCGGUACUAUCCACAGGUCAACC

[0139] SEQ ID NO: 39 shows a minimal fragment (9S_10_F3) of Aptamer 9S_10:

[0140] GGGCCAGUGUAGACUACUCAAUGCACC

[0141] SEQ ID NO: 40 shows a minimal fragment (9S_10_F4) of Aptamer 9S_10:

[0142] CGAGUCCGAGUUGCACCUGACCACUA

[0143] SEQ ID NO: 41 shows a minimal fragment (9S_10_F6) of Aptamer 9S_10:

[0144] CUCAAUGCACCCGAGU

[0145] SEQ ID NO: 42 shows a minimal fragment (9S_10_F8) of Aptamer 9S_10:

[0146] CACUAAAUACGAUCCG

[0147] SEQ ID NO: 43 shows a minimal fragment (9S_10_F9) of Aptamer 9S_10:

[0148] GGGCCAGUGUAGACUACUCAAUGCACCCGAGUCCGAGUUG

[0149] SEQ ID NO: 44 shows a minimal fragment (9S_10_F14) of Aptamer 9S_10:

[0150] GAGUCCGAGUUGCACCUGACCACUAAAUACGAUCCGGUACUAUCCACAGGUCAACC

[0151] SEQ ID NO: 45 shows a minimal fragment (9S_10_F15) of Aptamer 9S_10:

[0152] ACCUGACCACUAAAUACGAUCCGGUACUAUCCACAGGUCAACC

[0153] SEQ ID NO: 46 shows a minimal fragment (9S_10_F18) of Aptamer 9S_10:

[0154] CUCAAUGCACCCGAGUCCGAGUUGCACCUGACC SEQ ID NO: 47 shows a minimal fragment (9S_10_F19) of Aptamer 9S_10: CUCAAUGCACCCGAGUCCGAGUUGCACCUGACCACUAA

[0155] SEQ ID NO: 48 shows a minimal fragment (9S_10_F20) of Aptamer 9S_10: CGAGUCCGAGUUGCACCUGACCACUAAAUACGAUCCG

[0156] SEQ ID NO: 49 shows a minimal fragment (9S_10_F21) of Aptamer 9S_10:

[0157] CCGAGUUGCACCUGACCACUAAAUACGAUCCG

[0158] SEQ ID NO: 50 shows a minimal fragment (9S_10_F22) of Aptamer 9S_10: CACCUGACCACUAAAUACGAUCCG

[0159] SEQ ID NO: 51 shows a minimal fragment (9S_10_F23) of Aptamer 9S_10: CUCAAUGCACCCGAGUCCGAGUUGCACCUGACCACUAAAUACGAUCCG

[0160] Detailed description

[0161] Further features of certain embodiments of the present invention are described below. The practice of embodiments of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology and recombinant DNA technology, which are within the skill of those working in the art.

[0162] Most general molecular biology, microbiology recombinant DNA technology can be found in Sambrook et al, Molecular Cloning, A Laboratory Manual (2001) Cold Harbor-Laboratory Press, Cold Spring Harbor, N.Y. or Ausubel et al., Current protocols in molecular biology (1990) John Wiley and Sons, N.Y. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., Academic Press; and the Oxford University Press, provide a person skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0163] Units, prefixes and symbols are denoted in their Systeme International de Unitese (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation and nucleic acid sequences are written left to right in 5' to 3' orientation.

[0164] In the following, the invention will be explained in more detail by means of non-limiting examples of specific embodiments. In the example experiments, standard reagents and buffers free from contamination are used.

[0165] Staphylococcal C-S lyase

[0166] The invention provides one or more aptamers capable of specifically binding to staphylococcal C-S lyase.

[0167] As used herein, “staphylococcus C-S lyase” typically refers to a cysteine-S-conjugate p- lyase (e.g., EC 4.4.1.13) from one or more staphylococci. For example, the staphylococcus C-S lyase may be a Putative Cysteine-S-conjugate p-lyase (PatB) or orthologue thereof.

[0168] In certain embodiments, the staphylococcus C-S lyase is from a coagulase-negative staphylococci. Typically, the staphylococcus C-S lyase is from an odour-producing staphylococcus. For example, the staphylococcus may be capable of taking up and metabolizing (odourless) Cys-Gly-3-methyl-3-sulfanylhexan-1-ol (Cys-Gly-3M3SH)). Typically, the staphylococcus is capable of sequential metabolism of Cys-Gly-3M3SH by a dipeptidase to release glycine and cleavage by the staphylococcus C-S lyase to liberate the volatile (e.g., odorous) 3M3SH.

[0169] In certain embodiments, the aptamer can inhibit (e.g., reduce or prevent) staphylococcal C-S lyase from cleaving Cys-3M3SH thereby inhibiting release of 3M3SH. Typically, the aptamer is capable of inhibiting the activity of Cys-3M3SH within the bacterial cell (e.g., during its metabolism to produce 3M3SH). For example, the aptamer may be capable of being internalised into staphylococcus alone and / or when conjugated to another molecule (e.g., aptamer-conjugate as further described herein).

[0170] In certain embodiments, the staphylococcus cells are within body fluids such as saliva or sweat. The staphylococcus cells may be within any fabric contacting such fluids, including items of clothing, seat fabrics, gym equipment, bed linen or the like. Typically, the staphylococcus is present on human skin. For example, the staphylococcus may be part of the axillary microbiome (e.g., associated with other non-odorous bacteria of the human skin such as S. epidermis which lacks PatB and is not capable of metabolising or releasing 3M3SH). Advantageously, the aptamers of the invention are capable of inhibiting Staphylococcus associated malodour within such fluids or the like.

[0171] In certain embodiments, the staphylococcal C-S lyase is pyridoxal phosphate (PLP)- dependent. For example, the staphylococcal C-S lyase may be from Staphylococcus nepalensis, Staphylococcus cohnii, Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcus devriesei, Staphylococcus haemolyticus, Staphylococcus petrasii, Staphylococcus vitulinis or Staphylococcus sciuri.

[0172] In certain embodiments, the staphylococcal C-S lyase is a cysteine-thiol lyase (C-T lyase).

[0173] In certain embodiments, the staphylococcal C-S lyase is selected from one or more of Staphylococcus hominis (Sh) PatB (SEQ ID NO:1 or 2), Staphylococcus lugdunensis (SI) PatB (SEQ ID NO: 3), Staphylococcus devriesei (Sd) PatB (SEQ ID NO: 4), Staphylococcus haemolyticus (Sha) PatB (SEQ ID NO: 5) or Staphylococcus petrasii (Sp) PatB (SEQ ID NO: 6).

[0174] In certain embodiments, the staphylococcal C-S lyase comprises a N-terminal region comprising a constrained hydrophobic pocket selective for branched aliphatic thioalcohol ligands. For example, the N-terminal region may comprise G21, Y25 and / or E40, wherein the numbering refers to the amino acid positions with reference to the amino acid sequences as set forth in SEQ ID NO: 1. Typically, the staphylococcal C-S lyase comprises a N- terminal variable region as set forth in SEQ ID NO: 7, 8, 9, 10 or 11. Without being bound by theory, these variable regions may be conserved amongst odour-producing staphylococcal C-S lyases, but not present in related C-S lyases from non-odour producing bacteria.

[0175] In certain embodiments, the N-terminal variable region comprises at least one conservative amino acid substitution as compared to SEQ ID NO: 7, 8, 9, 10 or 11. For example, the staphylococcal C-S lyase may contain no more than 2, 3, 4, or 5 conservative amino acid substitutions as compared to SEQ ID NO: 7, 8, 9, 10 or 11. The term “conservative substitution” as used herein refers to substitutions of amino acid residues of a staphylococcal C-S lyase that have no effect on activity or properties of the enzyme. Conservative substitutions may be naturally occurring or non-naturally occurring.

[0176] In certain embodiments, the aptamers are capable of specifically binding to a staphylococcal C-S lyase which comprises (or further comprises) Y25 and / or F274, wherein the numbering refers to the amino acid residue positions with reference to the polypeptide sequence as set forth in SEQ ID NO: 1. Without being bound to theory, such hydrophobic residues may mediate apolar contacts thereby increasing selectivity to aliphatic cysteine-S-conjugates as compared to SEQ ID NO: 12.

[0177] In certain embodiments, the invention provides one or more aptamers capable of specifically binding to Staphylococcus hominis PatB as set forth in SEQ ID NO: 1. In certain embodiments, the aptamers of the invention specifically bind to a protein having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to SEQ ID NO: 1.

[0178] In certain embodiments, the invention provides one or more aptamers capable of specifically binding to the form of Staphylococcus hominis PatB as set forth in SEQ ID NO: 2. In certain embodiments, the aptamers of the invention specifically bind to a protein having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to SEQ ID NO:2.

[0179] In certain embodiments, the invention provides one or more aptamers capable of specifically binding to Staphylococcus hominis PatB as set forth in any one of SEQ ID NO: 3, 4, 5 or 6.

[0180] As used herein, “sequence identity” (in the context of protein sequences) refers to the percentage of amino acids in a candidate sequence that are identical with the amino acids in said sequences after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percentage amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, CLUSTALW or Megalign (DNASTAR) software. For example, % amino acid sequence identity values can be generated using sequence comparison computer programs found on the European Bioinformatics Institute website (http: / / www.ebi.ac.uk).

[0181] An aptamer that binds “specifically” to staphylococcal C-S lyase is an aptamer which binds with preferential or high affinity to one or more staphylococcal C-S lyases as described herein but does not bind (or binds with lower affinity) to C-S lyases from other genus of bacteria. For example, the aptamer may not bind (or binds with lower affinity) to B. Subtilis PatB (SEQ ID NO: 12).

[0182] In certain embodiments, an aptamer that binds with preferential or high affinity to staphylococcal C-S lyase may bind with at least about 2x, 3x, 4x, 5x, 10x, 20x, 50x, 100x or 1000x more affinity as compared to binding to C-S lyase from other genus of bacteria (e.g., B. Subtilis PatB as set forth in SEQ ID NO: 12).

[0183] In certain embodiments, a “strong” binder as described herein is an aptamer that has a target interaction greater than 1nm based on the signal response in a BLI assay. In certain embodiments, a “moderate” binder as described herein is an aptamer that has a target interaction of between 0.5nm to 1nm based on the signal response in a BLI assay. In certain embodiments, a “low” (or no significant) binder as described herein is an aptamer that has a target interaction of less than 0.5nm (e.g., less than 0.3nm) based on the signal response in a BLI assay.

[0184] As used herein, the term “high affinity” is understood to mean, for example, an aptamer that binds to the staphylococcal C-S lyase with a binding dissociation equilibrium constant (KD) of less than about 1 pM, less than about 900nM, less than about 800nM, less than about 700nM, less than about 600nM, less than about 500nM, less than about 400nM, less than about 300nM, less than about 200nM, less than about 100nM, less than about 50nM, less than about 10nM, less than about 9nM, less than about 8nM, less than about 7nM, less than about 6nM, less than about 5nM, less than about 4nM, less than about 3nM, less than about 2nM, less than about 1nM or less.

[0185] In certain embodiments, the aptamers of the invention are capable of specifically binding to staphylococcal C-S lyase with a KD of less than about 1pM or less. For example, the aptamers of the invention preferably have a KD of less than about 900nM, 800nM, 700nM, 600nM, 500nM, 400nM, 300nM, 200nM, 100nM or less.

[0186] The binding affinity of aptamers may be measured by any method known to a person skilled in the art, including, for example, surface plasmon resonance (SPR), Biolayer Interferometry (BLI), ELISA, fluorescence assays such as fluorescence anisotropy or fluorescence polarisation, Microscale Thermophoresis or the like.

[0187] As used herein, the term “low affinity” is understood to mean, for example, an aptamer that binds to a non-staphylococcal C-S lyase (e.g., SEQ ID NO: 12) with a binding dissociation equilibrium constant (KD) of more than about 1 pM, more than about 2 pM, more than about 3 pM, more than about 4 pM, more than about 5 pM, more than about 6 pM, more than about 7 pM, more than about 8 pM, more than about 9 pM, more than about 10 pM or has no detectable response. In certain embodiments, the aptamers of the invention have a rapid association rate and a slow dissociation). Methods of measuring the association rate constant (Ka) and dissociation rate constant (Kd) are also well described in the art.

[0188] Aptamers

[0189] As used herein, the terms “aptamer” or nucleic acid molecule” are used interchangeably to refer to a non-naturally occurring nucleic acid molecule that has a desirable action on, or interaction with a target molecule. For nucleic acid aptamers, for example, a distinction is made between DNA aptamers formed from single-stranded DNA (ssDNA) and RNA aptamers formed from single-stranded RNA (ssRNA), or chemical modifications thereof, including either backbone or base modifications. Typically, the aptamer has high binding affinity to the target molecule compared to an unselected, random or scrambled nucleic acid sequence.

[0190] In certain embodiments, the aptamer is an Optimer™. As such, the invention encompasses both full-length aptamer sequences (e.g., SEQ ID NOs 19 to 30) and / or minimal functional aptamer fragments (e.g., SEQ ID NOs 31 to 51).

[0191] In certain embodiments, the invention provides a single aptamer (e.g., any one of SEQ ID NOs 19 to 51 or variants thereof). However, in other embodiments, the invention provides more than one aptamer, e.g., two, three or more aptamers.

[0192] In certain embodiments, the invention provides two or more aptamers (e.g., an aptamer pair, triplet, or more) that bind to two or more different regions of the staphylococcal C-S lyase. In other words, the two or more aptamers do not compete for binding to the same epitope of the staphylococcal C-S lyase. Thus, in certain embodiments, the invention provides two or more aptamers (e.g., two, three, or more) capable of binding to two or more (e.g., two, three, or more) different regions of the staphylococcal C-S lyase.

[0193] As used herein, the term “Optimer” is understood to mean a fragment (e.g., portion) of the full-length aptamer capable of binding to target molecule with at least the same (or improved) specificity and / or affinity as compared to the full-length aptamer. For example, the Optimer may be about 5% to about 95%, about 10% to about 90% or about 20% to about 80% of the size of the full-length aptamer. Advantageously, the small size of Optimers may provide benefits, including, for example, increased penetration into bacterial cells thereby targeting staphylococcal C-S lyase during bacterial cell metabolism. The small size of an Optimer may also give increased structural stability, leading to improved binding affinity. The small size of an Optimer also gives advantages in reliability and scalability of manufacture and further reductions in batch-to-batch variability.

[0194] In certain embodiments, a minimal effective fragment (e.g., SEQ ID NOs 31 to 51) may compete for binding to the target molecule with the full-length aptamer. By way of example, a panel of fragments representing different regions of the full-length aptamer may be produced by solid phase synthesis (incorporating a 5' biotin group or other appropriate functional group known to those skilled in the art). Each of the individual fragments may then be immobilised onto a separate streptavidin coated Biolayer Interferometry (BLI) sensor probe (or other appropriately functionalised BLI sensor probe), and the interaction with the buffered target molecule monitored using a BLI-based binding assay. A BLI screen may show which fragments retain their binding affinity and which fragments have lost their binding function. These binding and non-binding fragments may then be mapped onto the full-length aptamer sequence to identify the minimal functional fragment (Optimer™).

[0195] Aptamers are characterised by the formation of a specific three-dimensional structure that depends on the nucleic acid sequence. The three-dimensional structure of an aptamer arises due to Watson and Crick intramolecular base pairing, Hoogsteen base pairing (quadruplex), wobble pair formation or other non-canonical base interactions. This structure enables aptamers, analogous to antigen-antibody binding, to bind target structures accurately. A particular nucleic acid sequence of an aptamer may, under defined conditions, have a three-dimensional structure that is specific to a defined target structure.

[0196] The nucleic acid aptamers described herein may comprise natural or non-natural nucleotides and / or or base derivatives (or combinations thereof). In certain embodiments, the nucleic acid molecule comprises one or more modifications such that it comprises a chemical structure other than deoxyribose, ribose, phosphate, adenine (A), guanine (G), cytosine (C), thymine (T), or uracil (U). The nucleic acid molecule may be modified at the nucleobase, at the pentose or at the phosphate backbone.

[0197] In certain embodiments, the nucleic acid molecule comprises one or more modified nucleotides. Exemplary modifications include for example nucleotides comprising an alkylation, arylation or acetylation, alkoxylation, halogenation, amino group, or another functional group. Examples of modified nucleotides include 2'-fluoro ribonucleotides, 2'-NH 2 -, 2'-OCH 3 - and 2'-O-methoxyethyl ribonucleotides, which are used for RNA aptamers. The nucleic acid molecule may be wholly or partly phosphorothioate or DNA, phosphorodithioate or DNA, phosphoroselenoate or DNA, phosphorodiselenoate or DNA, locked nucleic acid (LNA), peptide nucleic acid (PNA), N3'-P5 'phosphoramidate RNA I DNAcyclohexene nucleic acid (CeNA), tricyclo DNA (tcDNA) or spiegelmer, or the phosphoramidate morpholine (PMO) components (see also Chan et al., Clinical and Experimental Pharmacology and Physiology (2006) 33, 533-540).

[0198] In certain embodiments, the aptamer is an RNA aptamer (e.g., ssRNA aptamer). In some embodiments, the RNA aptamer is modified in 2'-OH positions with 2'fluoro ribose, 2'-amino ribose, 2'-O-methyl, or locked nucleic acids. Typically, the RNA aptamer comprises 2'fluoro- pyrmidine RNA.

[0199] In certain embodiments, the aptamer of the invention comprises or consists of a nucleic acid sequence selected from any one of SEQ ID NOs 19 to 51 or variants thereof (e.g., SEQ ID NOs 33, 34, 42, 43 or variants thereof), wherein the aptamer is an RNA aptamer which comprises a 2-fluoro (-F) modification. For example, the aptamer (e.g., SEQ ID NOs 33, 34, 42, 43 or variants thereof) may comprise 2'-Fluoro-Cytidine (2'-F-C) and / or 2'-Fluoro-Uridine (2'-F-U).

[0200] Some of the modifications allow nucleic acid molecules to be stabilized against nucleic acid cleaving enzymes. In the stabilization of the aptamers, a distinction can generally be made between the subsequent modification of the aptamers and the selection with already modified RNA I DNA. The stabilization may not affect the affinity of the modified RNA I DNA aptamers but prevents the rapid decomposition of the aptamers.

[0201] Aptamers to a target molecule may be selected using known processes. For example, an aptamer can be prepared by using the SELEX method or any related in vitro selection approach and an improved method thereof (e.g., Ellington & Szostak, (1990) Nature, 346, 818-822; Tuerk & Gold, (1990) Science, 249, 505-510). In the SELEX method, by setting strict selection conditions by increasing the number of rounds or using a competing substance, an aptamer exhibiting a stronger binding potential for the target molecule is enriched, isolated, and selected. Hence, by adjusting the number of rounds of SELEX and / or changing the competitive condition, aptamers with different binding forces, aptamers with different binding modes, and aptamers with the same binding force or binding mode but different base sequences can be obtained in some cases.

[0202] The in vitro selection method comprises a process of enriching a diverse starting library with target binding sequences through iterative rounds of target binding, recovery, and preferential amplification. The variability of a library is for example in the range of about 1012to 1015different molecules. Starting from the single-stranded DNA or RNA nucleic acid molecule library, the nucleic acid molecules are interacted with the target molecule of interest. Those library members which bind best to the target, are recovered, and amplified by PCR (or RT-PCR for an RNA library). Additional diversity may be introduced into the library during the in vitro selection process by causing a mutation by using error-prone enzymes for amplification, manganese ions and the like in the process. Target binding sequences are enriched cycle by cycle through various selection and amplification steps. Each aptamer selection cycle typically comprises the following sub steps: a) binding of nucleic acid molecule library to target; b) separating target-bound from unbound nucleic acid molecules; c) recovery of target-binding nucleic acid molecules; d) amplification of recovered nucleic acid molecules (e.g. PCR for DNA molecules, reverse transcription PCR for RNA molecules); and e) preparation of relevant single stranded nucleic acids from the amplified product (e.g. ssDNA purification, in vitro RNA transcription).

[0203] After each cycle, the selected and enriched nucleic acid molecule pool is used as the starting material for a next cycle. Typically, 8 to 12 cycles are run through although this number varies depending on the target type, method, and efficiency of selection.

[0204] In certain embodiments, the target used in the interaction with the nucleic acid may comprise one or more forms of the target, including, but not limited to, recombinant proteins or peptides thereof, cells expressing the protein of interest, or bacterial cells or samples of bacteria from the skin known to contain bacterial cells of interest.

[0205] In certain embodiments, the method comprises analysing the nucleic acid sequence of an aptamer which has been identified as binding to the target molecule with high binding affinity. After analysing the sequence, the aptamers (including variants, mutants, fragments, and derivatives thereof) can be prepared by conventional techniques of chemical DNA and RNA synthesis, which are known to the person skilled in the art. Furthermore, the binding properties of individual aptamers to the target molecule can be investigated.

[0206] Aptamers are easily altered through chemical oligonucleotide synthesis methods. For aptamers, by predicting the secondary structure using the MFOLD program, or by predicting the steric structure by X-ray analysis or NMR analysis, it is possible to predict to some extent which nucleotide can be substituted or deleted, where to insert a new nucleotide and the like. A predicted aptamer with the new sequence can easily be chemically synthesized, and it can be determined whether the aptamer retains the activity using an existing assay system.

[0207] Aptamers can be synthesized by methods known per se in the art. One of the synthesis methods is a method using an RNA polymerase. The object RNA can be obtained by chemically synthesizing a DNA having the object sequence and a promoter sequence of RNA polymerase, followed by in vitro transcription using same as a template and according to an already-known method.

[0208] DNA Aptamers can be synthesized using DNA polymerase. DNA having an object sequence is chemically synthesized and, using same as a template, amplification is performed by a known method of polymerase chain reaction (PCR). This is converted to a single strand by an already-known method of denaturing polyacrylamide electrophoresis or enzyme treatment method. When a modified aptamer is synthesized, the efficiency of elongation reaction can be increased by using a polymerase introduced with a mutation into a specific site. The thus-obtained aptamer can be purified easily by a known method.

[0209] RNA Aptamers can be synthesized using RNA polymerase. DNA having an object sequence is chemically synthesized and, using same as a template, multiple RNA copies of the template sequence are prepared by a known method of in vitro transcription. The template DNA is then removed through the use of a DNA specific nuclease, leaving the RNA intact. When a modified aptamer is synthesized, the efficiency of elongation reaction can be increased by using a polymerase introduced with a mutation into a specific site. The thus- obtained aptamer can be purified easily by a known method.

[0210] The DNA or RNA based aptamers can also be synthesized in a large amount by a chemical synthesis method such as amidite method, phosphoramidite method and the like. The synthesis method is a well-known method, and as described in Nucleic Acid (Vol. 2 )1] Synthesis and Analysis of Nucleic Acid (Editor: Yukio Sugiura, Hirokawa Publishing Company) and the like. A synthesizer such as Dr Oligo 96 and the like manufactured by Biolytic, or OligoPilot, OligoProcess and the like manufactured by Cytiva Lifesciences may be used. Purification may be performed using any suitable techniques such as chromatography and the like. In certain embodiments, the aptamers are selected from a nucleic acid molecule library such as a single-stranded DNA or RNA nucleic acid molecule library. Typically, the aptamers are selected from a “universal aptamer selection library” that is designed such that any selected aptamers need little to no adaptation to convert into any of the listed assay formats. In certain embodiments, the “universal aptamer selection library” is as defined in Example 1.

[0211] Once selected, the aptamer may be further modified before being used e.g. to remove one or both primer sequences and / or parts of the randomised region not required for target binding.

[0212] Typically, aptamers of the invention comprise a first primer region (e.g. at the 5' end), a second primer region (e.g. at the 3' end), or both. The primer regions may serve as primer binding sites for PCR amplification of the library and selected aptamers.

[0213] The skilled person would understand different primer sequences can be selected depending, for example, on the starting library and / or aptamer selection protocol. For example, aptamers of the invention may comprise SEQ ID NOs: 14 and 16.

[0214] The first primer region and / or second region may comprise a detectable and / or targeting label. For example, in the context of RNA selection processes, the RNA library can be labelled through incorporation of a modified nucleotide (typically UTP) at random positions within the length of the aptamer (body labelling). Examples of modified UTP that can be spiked into an in vitro transcription reaction include fluorescently labelled UTP, biotin labelled UTP etc. In a final aptamer that is prepared by solid phase chemical synthesis, the RNA aptamer may be labelled e.g., through incorporation of a modified phosphoramidite during synthesis. In the context of DNA aptamers, the first and / or second primer region may be fluorescently (e.g. FAM) labelled, biotin labelled or phosphate (PO4) labelled.

[0215] In certain embodiments, the aptamers bind specifically to any staphylococcal C-S lyase as described herein, for example as set forth in any one of SEQ ID NOs 1 to 6 or variants thereof.

[0216] In certain embodiments, the aptamers of the invention comprise or consist of a nucleic acid sequence selected from any one of SEQ ID NOs 19 to 30 or variants thereof. These aptamers are capable of binding specifically to staphylococcal C-S lyase as described herein (e.g., SEQ ID NO: 1 or variants thereof). In certain embodiments, the aptamers of the invention comprise or consist of a nucleic acid sequence selected from SEQ ID NOs 19, 28 or 30 or variants thereof. These aptamers are the best performing full-length aptamers in inhibiting staphylococcal C-S lyase activity.

[0217] In certain embodiments, the aptamers of the invention comprise or consist of a nucleic acid sequence selected from SEQ ID NOs 33, 34, 42, 43 or variants thereof. These aptamers are highly effective within bacterial cells, as further discussed herein.

[0218] In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the nucleotide sequence of SEQ ID NO: 19.

[0219] In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the nucleotide sequence of SEQ ID NO: 28.

[0220] In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the nucleotide sequence of SEQ ID NO: 30.

[0221] In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the nucleotide sequence of SEQ ID NO: 33.

[0222] In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the nucleotide sequence of SEQ ID NO: 34. In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the nucleotide sequence of SEQ ID NO: 42.

[0223] In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the nucleotide sequence of SEQ ID NO: 43.

[0224] As used herein, “sequence identity” (in the context of nucleic acid sequences) refers to the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in said sequences after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, CLUSTALW or Megalign (DNASTAR) software. For example, % nucleic acid sequence identity values can be generated using sequence comparison computer programs found on the European Bioinformatics Institute website (http: / / www.ebi.ac.uk).

[0225] In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence comprising at least about 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides (e.g. up to the total length) of a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity with SEQ ID NO: 19. In this context the term "about" typically means the referenced nucleotide sequence length plus or minus 10% of that referenced length.

[0226] In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence comprising at least about 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides (e.g. up to the total length) of a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity with SEQ ID NO: 28.

[0227] In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence comprising at least about 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides (e.g. up to the total length) of a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity with SEQ ID NO: 30.

[0228] In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence comprising at least about 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides (e.g. up to the total length) of a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity with SEQ ID NO: 33. In this context the term "about" typically means the referenced nucleotide sequence length plus or minus 10% of that referenced length.

[0229] In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence comprising at least about 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides (e.g. up to the total length) of a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity with SEQ ID NO: 34. In this context the term "about" typically means the referenced nucleotide sequence length plus or minus 10% of that referenced length.

[0230] In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence comprising at least about 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides (e.g. up to the total length) of a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity with SEQ ID NO: 42. In this context the term "about" typically means the referenced nucleotide sequence length plus or minus 10% of that referenced length.

[0231] In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence comprising at least about 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides (e.g. up to the total length) of a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity with SEQ ID NO: 43. In this context the term "about" typically means the referenced nucleotide sequence length plus or minus 10% of that referenced length.

[0232] In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence comprising at least about 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides (e.g. up to the total length) of SEQ ID NO: 19.

[0233] In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence comprising at least about 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides (e.g. up to the total length) of SEQ ID NO: 28. In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence comprising at least about 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides (e.g. up to the total length) of SEQ ID NO: 30.

[0234] In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence comprising at least about 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides (e.g. up to the total length) of SEQ ID NO: 33.

[0235] In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence comprising at least about 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides (e.g. up to the total length) of SEQ ID NO: 34.

[0236] In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence comprising at least about 13, 14, 15 or more consecutive nucleotides (e.g. up to the total length) of SEQ ID NO: 42.

[0237] In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence comprising at least about 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides (e.g. up to the total length) of SEQ ID NO: 43.

[0238] Aptamers are characterised by the formation of a specific three-dimensional structure that depends on the nucleic acid sequence. The three-dimensional structure of an aptamer arises due to Watson and Crick intramolecular base pairing, Hoogsteen base pairing (quadruplex), wobble pair formation or other non-canonical base interactions. This structure enables aptamers, analogous to antigen-antibody binding, to bind target structures accurately. A nucleic acid sequence of an aptamer may, under defined conditions, have a three-dimensional structure that is specific to a defined target structure.

[0239] In certain embodiments, the aptamer comprises a secondary structure. The secondary structure analysis of the aptamers may be performed by means of a free-energy minimization algorithm Mfold (M Zuker. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31(13), 3406-3415, 2003). In certain embodiments, the aptamers of the invention may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide variations as compared to any one of SEQ ID NOs 19 to 30 or 31 to 51. Positions where such variations can be introduced can be determined based on, for example, the secondary structures. For example, variations may be introduced at hairpins, stem-loops, G-quadruplexes and / or pseudoknot structures of the aptamer. The variation may be at a bulge region (e.g., an unpaired or non-complementary segment within the secondary structure of the aptamer). Typically, variations are introduced at a stem-loop (e.g., a bulge region of the stem-loop). For example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides may be added or removed from a stem-loop (e.g., a bulge region of the stem-loop) as described herein.

[0240] In certain embodiments, the invention provides an aptamer capable of specifically binding to staphylococcal C-S lyase, wherein the aptamer comprises a nucleic acid sequence of any one of SEQ ID NOs 19 to 30 or 31 to 51, wherein the nucleic acid sequence further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotide additions, substitutions, or deletions at one or more stem-loop secondary structures.

[0241] In certain embodiments, the invention provides an aptamer capable of specifically binding to staphylococcal C-S lyase, wherein the aptamer comprises a nucleic acid sequence of SEQ ID NO: 32, 34, 42 or 43 wherein the nucleic acid sequence further comprises at least 1, 2, 3,

[0242] 4, 5, 6, 7, 8, 9 or 10 nucleotide additions, substitutions, or deletions optionally at one or more stem-loop secondary structures.

[0243] In certain embodiments, the invention provides an aptamer capable of specifically binding to staphylococcal C-S lyase, wherein the aptamer comprises a nucleic acid sequence of SEQ ID NO: 32, 34 or 42 wherein the nucleic acid sequence further comprises at least 1, 2, 3, 4,

[0244] 5, 6, 7, 8, 9 or 10 nucleotide additions, substitutions, or deletions optionally at one or more stem-loop secondary structures.

[0245] The invention also provides aptamers that compete for binding to staphylococcal C-S lyase (e.g., SEQ ID NO: 1) with aptamers as described herein. In certain embodiments, the invention provides aptamers that compete for binding to staphylococcal C-S lyase (e.g., SEQ ID NO: 1) with the aptamers as set forth in any one of SEQ ID NOs 19 to 30 or 31 to 51. In certain embodiments, competition assays or other binding assays may be used to identify an aptamer that bind to staphylococcal C-S lyase (e.g., SEQ ID NO:1). In an exemplary binding assay, immobilised staphylococcal C-S lyase (e.g., SEQ ID NO:1) is incubated in a solution comprising a first labelled aptamer that binds to staphylococcal C-S lyase (e.g., SEQ ID NO: 1) and a second unlabelled aptamer that is being tested for its ability to compete with the first aptamer for binding to staphylococcal C-S lyase (e.g., SEQ ID NO:1). As a control, immobilised staphylococcal C-S lyase (e.g., SEQ ID NO:1) may be incubated in a solution comprising the first labelled aptamer but not the second unlabelled aptamer. After incubation under conditions permissive for binding of the first aptamer to staphylococcal C-S lyase (e.g., SEQ ID NO:1) excess unbound aptamer may be removed, and the amount of label associated with immobilised staphylococcal C-S lyase (e.g., SEQ ID NO:1) measured. If the amount of label associated with immobilised staphylococcal C-S lyase (e.g., SEQ ID NO:1) is substantially reduced in the test sample relative to the control sample, then that indicates that the second aptamer is competing with the first aptamer for binding to staphylococcal C- S lyase (e.g., SEQ ID NO:1).

[0246] In certain embodiments, the aptamer comprises one or more linker sequences and / or is directly labelled.

[0247] In certain embodiments, the aptamer comprises one or more detectable labels. For example, the aptamer may comprise a fluorescent moiety, e.g. a fluorescent / quencher compound. Fluorescent / quencher compounds are known in the art. See, for example, Mary Katherine Johansson, Methods in Molecular Biol. 335: Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols, 2006, Didenko, ed., Humana Press, Totowa, NJ, and Marras et al., 2002, Nucl. Acids Res. 30, el22 (incorporated by reference herein).

[0248] In certain embodiments, the detectable label of the aptamer is FAM. In certain embodiments, the FAM-label is preferably situated at either end of a first or second primer region of the aptamer. The person skilled in the art would understand that the label could also be located at any suitable position within the aptamer. Moieties that result in an increase in detectable signal when in proximity of each other may also be used herein, for example, as a result of fluorescence resonance energy transfer ("FRET"); suitable pairs include but are not limited to fluoroscein and tetramethylrhodamine; rhodamine 6G and malachite green, and FITC and thiosemicarbazole, to name a few.

[0249] In certain embodiments, the detectable label of the aptamer is selected from a fluorophore, a nanoparticle, a quantum dot, an enzyme, a radioactive isotope, a pre-defined sequence portion, a biotin, a desthiobiotin, a thiol group, an amine group, an azide, an aminoallyl group, a digoxigenin, an antibody, a catalyst, a colloidal metallic particle, a colloidal non- metallic particle, an organic polymer, a latex particle, a nanofiber, a nanotube, a dendrimer, a protein, and a liposome.

[0250] In certain embodiments, the detectable label of the aptamer is a fluorescent protein such as Green Fluorescent Protein (GFP) or any other fluorescent protein known to those skilled in the art. In certain embodiments, the detectable label of the aptamer is an enzyme. For example, the enzyme may be selected from horseradish peroxidase, alkaline phosphatase, urease, p- galactosidase, or any other enzyme known to those skilled in the art.

[0251] In certain embodiments, the nature of the detection will be dependent on the detectable label used. For example, the label may be detectable by virtue of its colour e.g. gold nanoparticles. A colour can be detected quantitatively by an optical reader or camera e.g. a camera with imaging software.

[0252] In certain embodiments, the detectable label of the aptamer is a fluorescent label e.g. a quantum dot. In such embodiments, the detection means may comprise a fluorescent plate reader, strip reader or similar which is configured to record fluorescence intensity.

[0253] In embodiments in which the detectable label of the aptamer is an enzyme label, the detection means may, for example, be colorimetric, chemiluminescence and / or electrochemical (for example, using an electrochemical detector). Typically, electrochemical sensing is through conjugation of a redox reporter (e.g. methylene blue or ferrocene) to one end of the aptamer and a sensor surface to the other end. Typically, a change in aptamer conformation upon target binding changes the distance between the reporter and sensor to provide a readout.

[0254] In certain embodiments, the detectable label of the aptamer may further comprise enzymes such as horseradish peroxidase (HRP), Alkaline phosphatase (APP) or similar, to catalytically turnover a substrate to give an amplified signal.

[0255] Aptamer-conjugates

[0256] In certain embodiments, the aptamer of the invention is conjugated to one or more molecule(s). An “aptamer-conjugate” as used herein is a complex formed by conjugating an aptamer or target binding nucleic acid molecule with one or more molecule(s). For example, the aptamer may be conjugated to another aptamer (“aptamer-aptamer”), a peptide (“aptamer-peptide”) or small molecule (“aptamer-small molecule). Techniques of conjugating an aptamer to one or more molecule(s) are further described herein.

[0257] In some embodiments, the aptamer region of the conjugate is capable of specifically binding to staphylococcal C-S lyase. The one or more molecule(s) of the conjugate may be capable of targeting the aptamer to a desired bacterial cell as described herein. The one or more molecule(s) of the conjugate may be capable of otherwise modulating or improving the performance of the aptamer or imparting other beneficial properties to the aptamer.

[0258] As used herein, “capable of specifically binding” refers to the ability of the aptamer to selectively attach itself to the target molecule. Selective binding means that the interaction between the molecule is highly specific, typically the result of molecular recognition where the aptamer has a binding site that matches the shape, charge, or chemical properties of the target molecule. This specific interaction allows the aptamer to recognise and attach to the target molecule while having minimal binding or affinity for other molecules in the sample.

[0259] An aptamer may be conjugated to one or more molecule(s) using any chemical modification technique which allows for the attachment of the modified aptamer to one or more compatibly modified molecule(s), while preserving the binding affinity and specificity of both components.

[0260] To facilitate conjugation, specific and compatible functional groups may be introduced to the aptamer and / or one or more molecule(s). Common function groups used for conjugation will be known to those skilled in the art. Aptly common function groups used for conjugation include, for example, primary amines (NH2), sulfhydryl groups (SH), ‘Click chemistry’ groups such as an azide or alkyne and carboxyl groups (COOH). This may be introduced during the solid phase synthesis of the aptamer using phosphoramidites carrying the functional group; or may be achieved through chemical modification of the aptamer post synthesis, or one or more molecule(s) having existing functional groups. The functional groups may be activated using any suitable techniques known to those skilled in the art, e.g., using cross-linking reagents or chemical reactions that create reactive sites for conjugation. Common reagents include N-hydroxysuccinimide (NHS) and maleimide.

[0261] In certain embodiments, the aptamer region of the aptamer-conjugate comprises one or more primary amines (NH2), sulfhydryl groups (SH), azide, alkyne and / or carboxyl groups (COOH).

[0262] In certain embodiments, the one or more molecule(s) comprises one or more primary amines (NH2), sulfhydryl groups (SH), azide, alkyne and / or carboxyl groups (COOH).

[0263] The appropriately modified aptamer and one or more molecule(s) may then be combined under any suitable condition which allows specific conjugation, whilst minimising any side reactions. The specific reaction conditions (e.g., pH, temperature, time) may vary depending on the conjugation chemistry and method chosen. Typically, the aptamer-conjugate is then purified to remove any unreacted aptamers and / or molecules and reaction by-products. Typical purification methods may include, for example, size exclusion chromatography, affinity chromatography, dialysis, or the like. The aptamer-conjugate may then be further characterised and / or stored under any suitable conditions.

[0264] EXAMPLES

[0265] In the following, the invention will be explained in more detail by means of non-limiting examples of specific embodiments. In the example experiments, standard reagents and buffers free from contamination are used.

[0266] EXAMPLE 1 - Aptamer selection and preliminary characterisation

[0267] Recombinant His tagged ShPatB protein (SEQ ID NO: 2) was supplied by Unilever, human Microbiome Department. The protein was characterised by UV spectroscopy and SDS- PAGE analysis for quality control purposes. The protein was immobilised onto His-Tag Isolation and Pulldown magnetic Dynabeads™ (ThermoFisher Scientific, UK), according to manufacturer’s protocols. The protein loading density was determined spectrophotometrically.

[0268] The aptamer selection process was carried out starting from synthetic ssDNA oligonucleotide sequences of an aptamer library (manufactured by IDT, Belgium). The nucleotide sequences of the aptamer library have the following structure (in a 5’ to 3’ direction): T7S - T7 - P1 - R - P2, wherein T7 is a promoter region which is recognised and bound by the T7 RNA polymerase, T7S is a primer region, P1 is a first primer region, R is a randomized region (40 nucleotides in length) and P2 is a further primer region wherein R or a portion thereof are involved in target molecule binding.

[0269] The following modified primers were used in the amplification of the oligomers by means of Reverse Transcription and PCR: a forward primer (P1) with the sequence: 5’ - CCAGTGTAGACTACTCAATGC - 3’ (SEQ ID NO: 14), a reverse primer (P2) with the sequence: 5’ - GGTTGACCTGTGGATAGTAC - 3’ (SEQ ID NO: 15), a long forward primer that contains the external T7S sequence (T7S), the promoter sequence for T7 RNA polymerase (T7) and the forward primer sequence (P1) with the sequence: 5’ - GTGTGTATTGGCTATGTTCCTAATACGACTCACTATAGGGCCAGTGTAGACTAC - 3’ (SEQ ID NO: 17), and a short T7S forward primer (T7S) with the sequence: 5’ - GTGTGTATTGGCTATGTTCC - 3’ (SEQ ID NO: 18). The selection process consisted of iterative selection rounds with increasingly stringent selection conditions. In Cycle 1, 166pmol of naive RNA aptamer library was incubated with the target immobilised beads in 1x aptamer selection buffer (50 mM MES pH 6.2, 10 mM MgCI2, 2 mM CaCI2, 60 mM NaCI, 4.5 mM KCI, 0.01% Tween, 0.01% (w / v) BSA), using binding conditions established in preliminary binding studies. The beads were washed to remove loosely bound aptamers, and the remaining aptamers were eluted in water. The recovered RNA aptamer population was reverse transcribed using the reverse primer (SEQ ID NO: 15), and FIREScript Reverse Transcriptase (Solis BioDyne OU, Estonia) according to manufacturers’ protocols. The resulting ‘complimentary DNA’ (cDNA) copy was then annealed to a complimentary long primer containing the T7 promoter (SEQ ID NO: 17) and extended in an extension reaction using HOT FIREPol® DNA Polymerase (Solis BioDyne OU, Estonia) according to manufacturers’ protocols. The resulting dsDNA was then amplified by PCR using both T7S forward and reverse primers (SEQ ID NOs: 18 and 15 respectively) and HOT FIREPol® DNA Polymerase (Solis BioDyne OU, Estonia) according to manufacturers’ protocols.

[0270] An enriched RNA library is prepared for the subsequent cycle of selection using a 500pl in vitro transcription reaction containing 1x transcription buffer (40mM Tris HOI pH8, 6mM MgCI2, 2mM Spermidine HOI, 10mM NaCI), 5mM each nucleotide (ATP, GTP, 2’F UTP, 2’F CTP), 0.134pM template dsDNA, 0.05x reaction volume T7 RNA polymerase, all prepared using nuclease free water. If labelled RNA is required (e.g. biotin labelled RNA for Biolayer Interferometry studies), the reaction is also spiked with 0.05mM labelled UTP. After incubation at 37°C for 6 hours, template dsDNA is removed by treatment with DNase 1 (ThermoFisher Scientific, UK) following manufacturers protocols.

[0271] The nascent RNA aptamer library is purified using AxyPrep Mag PCR Clean-up Kit (Axygen Biosciences, USA) according to manufacturers’ protocol. In Cycle 2 (and all subsequent rounds), the same process is followed but aptamer-target incubations are carried out with increasingly stringent conditions. Counter selection was carried out against blank His-Tag Isolation and Pulldown magnetic Dynabeads™, to remove bead binding sequences.

[0272] After aptamer selection, the refined aptamer populations were assessed for the ability to bind to ShPatB using a Biolayer interferometry assay. The experiments described here were conducted using an Octet RED384 instrument (Sartorius Corporation, USA) based on manufacturers defined protocols. The aptamer population was prepared using the in vitro transcription protocol described above. The reaction mix was spiked with biotin-16-UTP (Jena Bioscience GmbH, Germany)) to produce a partially biotinylated RNA aptamer population. Biotinylated ssRNA was then immobilised onto the surface of streptavidin coated biosensor probes (Streptavidin-SA Dip & Read Biosensors, Sartorius Corporation, USA) following manufacturer protocols. The aptamer populations were prepared at 50nM in 1x aptamer selection buffer (50 mM MES pH 6.2, 10 mM MgCI2, 2 mM CaCI2, 60 mM NaCI, 4.5 mM KCI, 0.01% Tween, 0.01% (w / v) BSA). Target protein stocks were also prepared in the 1x aptamer selection buffer. All buffer I blank I baseline interactions were carried out in 1x aptamer selection buffer with no added ShPatB. The interaction between the immobilised aptamer population and ShPatB was monitored. All data was reference corrected using a blank sensor probe (no immobilised aptamer) to allow correction of buffer effects.

[0273] Aptamer populations which have improved target binding relative to the naive library, were analysed by Next Generation Sequencing (NGS) to identify potential candidate aptamer sequences. The population analysis was carried out using an Ion PGM Ion Torrent Next Generation Sequencing system. Libraries were prepared using Ion Torrent™ Ion Plus Fragment Library kit, according to manufacturer protocols (ThermoFisher Scientific, UK), and barcoded using Ion Xpress™ Barcode Adapters 1-96 Kit following manufacturer protocols (ThermoFisher Scientific, UK). Barcoded populations were then templated onto Ion Sphere Particles (ISP) using the Ion OneTouch™ 2 System, following manufacturer protocols (ThermoFisher Scientific, UK). After successful templating, the prepared templated ISPs were processed and loaded onto an Ion 318™ Chip Kit v2 BC following manufacturer protocols and the samples run using the Ion PGM Ion Torrent Next Generation Sequencing system. Sequence reads were then analysed in Linux to identify enriched candidate sequences within each population. In-house Python scripts were used to process the sequencing data and trim each sequence so that only the variable region is present in the data. Datasets were then clustered using FastAptameR2.0 (https: / / fastaptamer2.missouri.edu / )) to group similar sequences into families or ‘clusters’. The highest read sequence from each cluster is then taken forward as the candidate sequence for synthesis and subsequent screening by Biolayer Interferometry (BLI) on the Octet RED384 instrument (Sartorius Corporation, USA). The obtained sequence data is set forth in SEQ ID NOs: 19 to 30 as described herein.

[0274] Individual aptamer candidate sequences were synthesised using standard solid phase phosphoramidite oligonucleotide synthesis methods. RNA phosphoramidites (Glen Research, USA) were used to prepare the candidate Optimers using a Dr Oligo 96 High throughput oligonucleotide synthesiser, following manufacturer’s instructions (Biolytic Inc. USA). Synthesised oligonucleotides were cleaved from solid support and deprotected using AMA (1:1 v / v solution of 28% ammonium hydroxide and 40% methylamine). 2' protecting groups were removed by treatment with triethylammonium trihydrofluoride. Optimers were then purified using Glen-Pak DNA purification cartridges following the manufacturer’s instructions (Glen Research, USA). Optimers were analysed by polyacrylamide gel electrophoresis before being used in screening assays.

[0275] Each individual aptamer was then analysed for binding to their respective target; using the BLI assay described above.

[0276] EXAMPLE 2 - Assessment of aptamer inhibition of ShPatB enzyme activity

[0277] A ShPatB activity assay was established for use in candidate aptamer screening.

[0278] 10mM stock of CS lyase substrate, Cys-Gly-3M3SH (Cys-Gly- 3-methyl-3-sulfanylhexan-1- ol) was prepared in DSMO. This was then diluted to 100pM in 5ml of 1x PBS (2.5ml of 2x PBS, 2.45ml water, 50pl substrate stock).

[0279] 2mM DTNB (5,5-dithio-bis-(2-nitrobenzoic acid) stock was prepared fresh for each assay by dissolving 20 mg DTNB (ThermoFisher Scientific, UK) in 25ml 1x PBS. This DTNB stock was then diluted by combining 1 ml of the 2mM DTNB stock, with 2ml 100mM Tris HCI pH7.4 and 15ml 18.2 MQ water.

[0280] A dilution series (1 :10, 20, 50, 100, 200, 500, 1000) of ShPatB enzyme (Unilever, human Microbiome Department) was prepared in 1x PBS.

[0281] 1 ,056pl 10mM substrate stock was combined with 2,640pl of 2x aptamer selection buffer (100 mM MES pH 6.2, 20 mM MgCI2, 4 mM CaCI2, 120 mM NaCI, 9 mM KCI, 0.02% Tween) and 1 ,531 ,2pl nuclease-free water. This was mixed, then 198pl was aliquoted into each well in 3 columns of a 96-well microtiter plate. 2pl of the enzyme dilution series was added to each column (including a no enzyme control), mixed, then incubated at 37°C for 4 hours, with gentle shaking (140 to 180 rpm). Thiol detection was performed in a separate microtiter plate by combining 100pl enzyme / substrate reaction mixture, with 100pl diluted DTNB. This was mixed, then incubated at room temp for 10 mins before reading in a plate reader at 405nm. Data is plotted as an average of triplicate absorbance readings vs enzyme dilution and compared to the ‘No enzyme’ control.

[0282] Data in Figure 3 demonstrates enzyme concentration dependent increase in thiol production, demonstrating that ShPatB is active and converts Cys-Gly-3M3SH substrate into 3M3SH.

[0283] Aptamer candidates were screened using the same ShPatB activity assay. In this case, ShPatB was diluted 1 :50 in 1x aptamer selection buffer, then combined with an equal volume of each aptamer candidate (also in 1x aptamer selection buffer) to give 2pM final aptamer concentration and 1:100 diluted enzyme. This was incubated for 10 minutes at room temperature before adding 2pl of aptamer-enzyme mix to 198pl of diluted substrate, and incubating at 37°C for 4 hours, with gentle shaking (140 to 180 rpm). Thiol production was quantified using DTNB as described above.

[0284] Data in Figure 4 demonstrates that ShPatB is inhibited in the presence of aptamer candidates 9S_1 , 9S_10 and 9S_12 (SEQ ID NOs: 19, 28, 30), as less Cys-Gly-3M3SH substrate is converted into 3M3SH.

[0285] EXAMPLE 3 - Assessment of aptamer fragment inhibition of ShPatB enzyme activity

[0286] After nominating preferred aptamer candidates 9S_1 and 9S_10 (SEQ ID NOs: 19, 28), the minimal functional fragment Optimer is identified. A panel of fragments representing different lengths and regions of the full-length aptamer were synthesised using standard solid phase phosphoramidite oligonucleotide synthesis methods as described above. The aptamer fragments were screened using the ShPatB inhibition assay described above.

[0287] Data in Figure 5A and 5B show ‘% inhibition of ShPatB’ from the full-length aptamer candidates 9S_1 and 9S_10 (SEQ ID NOs: 19, 28) respectively, and Optimer fragments thereof. The data demonstrates that the enzyme is again inhibited in the presence of full- length aptamer candidates 9S_1 and 9S_10 (SEQ ID NOs: 19, 28) (orange bars), but not the scrambled control. The data also shows that the fragments of each aptamer have a range of activities; select fragments e.g. SEQ ID NOs: 31 to 51 , retain the inhibitory properties.

[0288] EXAMPLE 4 - Assessment of aptamer fragment inhibition of ShPatB activity in whole cell assays

[0289] Aptamer candidates and Optimer fragments shown to inhibit ShPatB activity in purified enzyme assays above, were also assessed for function in whole cell assays.

[0290] S. hominis cultures were grown overnight at 37°C on SS agar (10 g L"1tryptone, 5 g L"1Lemco powder, 3 g L"1yeast extract, 13 g L"1agar no. 1, 10 g L"1sodium pyruvate, 0.5 g L-1glycine, 22.5 g L"1potassium thiocyanate, 1.2 g L"1disodium hydrogen orthophosphate dihydrate, 0.67 g L"1sodium dihydrogen phosphate, 2 g L"1lithium chloride, 10 mL L-1glycerol, pH 7.2, with 0.002% (w / v) sodium azide and 30 g L'1egg white both added after autoclaving). A sterile loop was used to inoculate 25ml of TSB (prepared as per manufacturer instructions (Merck)), before incubation overnight at 37°C, shaking at 250rpm. Overnight liquid cultures were harvested by centrifugation (13000rpm, 10 mins) and cell pellets suspended and diluted in 1x PBS to ODe20nm ~5.

[0291] Whole cell odour biotransformation reactions were carried out in triplicate as follows; 20pl of each full-length aptamer or Optimer fragment (from 100pM stock) was added to 160pl of resuspended cells and 20pl of Cys-Gly-3M3SH substrate (from 20mM stock). Cell, aptamer / Optimer mixes were incubated at 37°C for 24 hours, before removing cells by centrifugation (13000rpm, 10 mins). 100pl of supernatant was taken from each sample and thiol production quantified using DTNB as described above.

[0292] Data in Figure 6 shows that after 24 hours, Optimer fragments 9S_1_F15, 9S_1_F16, 9S_10_F8 and 9S_10_F9 (SEQ ID NOs: 33, 34, 42 and 43 respectively) have significantly reduced thiol production, indicating that these Optimer fragments are capable of inhibiting ShPatB activity in whole cell cultures.

[0293] EXAMPLE 5 - Assessment of aptamer fragment stability in axilla wash samples

[0294] To be useful in a cosmetic application, nominated aptamers or Optimers must retain their function on skin. To assess the Optimer stability and retention of function in this media, axilla buffer scrubs were collected using an established collection method (Williamson, P.; Kligman, A. M. A New Method for the Quantitative Investigation of Cutaneous Bacteria. The Journal of Investigative Dermatology 1965, 45 (6), 498-503, herein incorporated by reference). Collected buffer scrub wash was split into 6x 2ml aliquots. 150pl of nominated Optimer fragments 9S_1_F15, 9S_1_F16 or 9S_10_F8 (SEQ ID NOs: 33, 34 or 42 respectively) were each added to two separate buffer scrub wash aliquots (final concentration ~6.66pM), mixed, then incubated at 37°C. 100pl samples were taken from each mix after incubation for 0, 30, 60, 120, 180 and 1440 mins (24 hrs). Samples were then assessed for inhibition of ShPatB activity using the purified enzyme assays described above. Samples were compared to control assays containing enzyme alone (no aptamer).

[0295] Data in Figure 7 shows thiol production as a measure of ShPatB activity (relative to enzyme alone control), in samples treated with each Optimer after incubation at 37°C for various times, in axilla buffer scrub medium. Data shows that nominated Optimer fragments 9S_1_F15, 9S_1_F16 and 9S_10_F8 (SEQ ID NOs: 33, 34 or 42 respectively) reduce ShPatB activity to <10% in all samples incubated up to 180 mins (3 hrs). After 1440 mins (24 hrs) ShPatB activity increased to -35-45%, suggesting some loss of activity, but that all aptamers retain some inhibitory activity after 24 hours at 37°C in axilla buffer scrub medium.

Claims

Claims1. One or more aptamer(s) capable of specifically binding to staphylococcal C-S lyase.

2. The aptamer of claim 1 , wherein the aptamer is capable of inhibiting staphylococcal C-S lyase activity.

3. The aptamer of claim 1 or 2, wherein the staphylococcal C-S lyase is an S. hominis C-S lyase.

4. The aptamer of any one of claim 1 to 3, wherein the aptamer comprises:(a) a nucleic acid sequence selected from any one or more of SEQ ID NOs: 19 to 30 or 31 to 51;(b) a nucleic acid having at least about 50% identity with any one or more of the nucleic acid sequences of (a); and / or(c) a nucleic acid sequence having at least about 20 consecutive nucleotides of any one or more of the nucleic acid sequences of (a) to (b).

5. The aptamer of claim 4, wherein the one or more aptamer(s) comprise:(a) a nucleic acid sequence selected from any one or more of SEQ ID NOs 33, 34, 42 or 43;(b) a nucleic acid sequence selected from any one or more of SEQ ID NOs 33, 34, or 42;(c) a nucleic acid having at least about 50% identity with any one or more of the nucleic acid sequences of (a) or (b); and / or(d) a nucleic acid sequence having at least about 20 consecutive nucleotides of any one or more of the nucleic acid sequences of (a) to (c).

6. The aptamer of claim 4 or 5, wherein the one or more aptamer(s) comprise:(a) a nucleic acid sequence selected from any one or more of SEQ ID NOs:19, 28 or 30;(b) a nucleic acid sequence selected from any one or more of SEQ ID NOs:19 or 28;(c) a nucleic acid having at least about 50% identity with a nucleic acid sequence of (a) or (b); and / or(d) a nucleic acid sequence having at least about 20 consecutive nucleotides of any one or more of the nucleic acid sequences of (a) to (c).

7. The aptamer(s) of any one of the preceding claims, wherein the aptamer is an RNA aptamer.

8. An aptamer that competes for binding to staphylococcal C-S lyase with the aptamer of any one of claims 1 to 7.

9. An aptamer-conjugate, wherein the aptamer region of the conjugate is as according to any one of claims 1 to 7, and the aptamer region is conjugated to one or more molecule(s).

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