Theranostic Application of a Novel G-Quadruplex-Forming DNA Aptamer Targeting Malate Synthase of Mycobacterium tuberculosis

Journal Pre-proof Theranostic application of a novel G-Quadruplex forming DNA aptamer targeting glyoxylate cycle enzyme malate synthase of Mycobacterium tuberculosis Abhijeet Dhiman, Chanchal Kumar, Subodh Kumar Mishra, Kriti Sikri, IsharaDatta, Pradeep Sharma, Tej P. Singh, Sagarika Haldar, Neera Sharma, Anjali Bansal, Yusra Ahmad, Amit Kumar, Tarun Kumar Sharma, Jaya SivaswamiTyagi PII:

S2162-2531(19)30274-4

DOI:

https://doi.org/10.1016/j.omtn.2019.09.026

Reference:

OMTN 646

To appear in:

Molecular Therapy: Nucleic Acid

Received Date: 9 July 2019 Accepted Date: 27 September 2019

Please cite this article as: Dhiman A, Kumar C, Mishra SK, Sikri K, IsharaDatta Sharma P, Singh TP, Haldar S, Sharma N, Bansal A, Ahmad Y, Kumar A, Sharma TK, SivaswamiTyagi J, Theranostic application of a novel G-Quadruplex forming DNA aptamer targeting glyoxylate cycle enzyme malate synthase of Mycobacterium tuberculosis, Molecular Therapy: Nucleic Acid (2019), doi: https:// doi.org/10.1016/j.omtn.2019.09.026. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 The Author(s).

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Theranostic application of a novel G-Quadruplex forming DNA aptamer targeting glyoxylate

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cycle enzyme malate synthase of Mycobacterium tuberculosis

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Abhijeet Dhiman1,8, Chanchal Kumar1, Subodh Kumar Mishra2#, Kriti Sikri1#,IsharaDatta1, Pradeep

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Sharma3, Tej P. Singh3,Sagarika Haldar4,5, Neera Sharma6, Anjali Bansal7, Yusra Ahmad8, Amit

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Kumar2, Tarun Kumar Sharma5* and Jaya SivaswamiTyagi1,5*

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1

Department of Biotechnology, All India Institute of Medical Sciences, New Delhi-110029, India.

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2

Discipline of Biosciences and Biomedical Engineering, Indian Institute of Technology Indore, Simrol,

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Indore-453552, India.

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3

Department of Biophysics, All India Institute of Medical Sciences, New Delhi-110029, India.

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4

Department of Experimental Medicine and Biotechnology, PGIMER, Sector12, Chandigarh-160012,

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India.

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5

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Institute, Faridabad, Haryana-121001 India.

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6

Department of Biochemistry, Dr. Ram Manohar Lohia Hospital, New Delhi-110001, India.

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Department of Pediatrics, Dr. Ram Manohar Lohia Hospital, New Delhi-110001, India.

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8

Faculty of Pharmacy, Uttarakhand Technical University, Dehradun-248007, Uttarakhand, India.

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Corresponding Authors

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*Email: Jaya S. Tyagi, [email protected],[email protected], Telefax +91 011-26588491;

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Tarun K. Sharma, [email protected]; Telephone +91 8800931999.

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#

Multidisciplinary Clinical and Translational Research, Translational Health Science and Technology

Contributed equally.

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1

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ABSTRACT

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The successful management of tuberculosis (TB) requires efficient diagnosis and treatment. Further,

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the increasing prevalence of drug resistant TB highlights the urgent need to develop novel inhibitors

26

against both drug susceptible and resistant forms of disease. Malate synthase (MS), an enzyme of the

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glyoxylate pathway, plays a vital role in mycobacterial persistence, and therefore it is considered as an

28

attractive target for novel anti-TB drug development. Recent studies have also ascribed an adhesin

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function to MS and established it as a potent diagnostic biomarker. In this study a panel of

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Mycobacterium tuberculosis (Mtb) MS-specific single-stranded DNA aptamers was identified by

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Systematic Evolution of Ligands by EXponential Enrichment (SELEX). The best-performing G-

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quadruplex-forming 44-mer aptamer, MS10, was optimized post-SELEX to generate an 11-mer

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aptamer, MS10-Trunc. This aptamer was characterized by various biochemical, biophysical and in-

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silico techniques. Its theranostic activity towards Mtb was established using enzyme inhibition, host

35

cell binding and invasion assays.MS10-Trunc aptamer exhibited high affinity for MS (Kd ~19 pM) and

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displayed robust inhibition of MS enzyme activity with IC50 of 251.1nM and Ki of 230 nM. This

37

aptamer blocked mycobacterial entry into host cells by binding to surface-associated MS. In addition,

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we have also demonstrated its application in the detection of tuberculous meningitis (TBM) in patients

39

with sensitivity and specificity each of >97%.

40 41

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INTRODUCTION

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Active tuberculosis (TB) affected more than 10.4 million people in 2017, while an estimated 1.7

45

billion individuals (~one quarter of the world’s population) are estimated to harbor a latent/ persistent

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infection with Mycobacterium tuberculosis (Mtb).1 The current treatment for TB is prolonged and

47

drug-resistant cases are increasing2, which together pose a significant threat to TB control in the

48

community.3,4 Therefore, there is an urgent need to develop new inhibitory compounds or molecules

49

that have the potential to work against both the drug susceptible and resistant TB. Metabolic pathways

50

of central metabolism have attracted attention in recent times as a source of new targets for novel TB

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drug development.

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The glyoxylate shunt pathway in particular is considered as a prominent target pathway owing to its

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role in mycobacterial survival and persistence, as demonstrated in cell and animal models of Mtb

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infection.5,6 This two-enzyme pathway enables bypass of the decarboxylation steps in the tricarboxylic

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acid cycle and conserves carbon for subsequent gluconeogenesis. Isocitratelyase, the first enzyme of

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this pathway, generates glyoxylate and succinate from isocitrate, and malate synthase (MS), the

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second enzyme, catalyzes the formation of malate using glyoxylate and acetyl-CoA.5 Notably, these

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enzymes appear to be absent in mammals5, a feature which enhances its relevance as an anti-TB drug

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target. In addition to its enzymatic activity, MS was shown to be expressed on the cell wall of Mtb and

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impart host adhering function to bacteria, thus establishing an additional role for it as an adhesin.7

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Furthermore, MS is reported as a potential biomarker for TB infections as it is expressed in the early

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stage of infection and independent of HIV co-infection.8 Thus, the presence of this antigen in

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biological fluid is considered as a direct evidence of infection.

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The crystal structures of both isocitratelyase and MS have been solved

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structures have suggested MS as a more druggable target in comparison to isocitrate lyase.5 A recent

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study demonstrated that knockdown and knock-out of MS leads to Mtb clearance inthe mouse model 3

9,10

and insights from these

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of infection, showing the essentiality of MS for the survival of Mtb during the acute and chronic

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phases of infection.11 Therefore, owing to the attractiveness of MS as a drug target, inhibitors of MS

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have been reported, such as oxalate, phosphoenol pyruvate (PEP), bromopyruvate (BP) and glycolate,

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that exhibit high inhibition constants (Ki) ranging between 60 µM and 900 µM.10 These inhibitors have

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limitations in terms of their use in humans; oxalate is not pharmacologically suitable due to its toxic

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nature, PEP is a key constituent of glycolysis and gluconeogenesis pathways in all organisms

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including humans, while BP exhibits toxicity impairing mitochondrial function.12,13 Recently a series

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of phenyl-diketo-acid (PDKA) molecules having inhibitory activity against Mtb MS enzyme were also

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developed which are under optimization.14,15

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A recent study from our group has established the clinical utility of polyclonal antibody to MS for the

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diagnosis of tuberculous meningitis (TBM), the most lethal form of tuberculosis that affects central

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nervous system.8 However, polyclonal antibody has an inherent problem of batch-to-batch variation,

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thus it is difficult to scale up the assay to a diagnostic test level. To address this limitation associated

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with antibody, it is imperative to develop a diagnostic reagent of uniform quality. Nucleic acid

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aptamers have received a great deal of attention due to their enormous potential to be used as both

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therapeutic and diagnostic agents. By acquiring a complex 2-D and 3-D structure, DNA aptamers

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often recognize their cognate target with unmatched affinity and specificity.16

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Here we report the development of a G-quadruplex (G4) forming aptamer MS10-Trunc against MS of

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Mtb that can serve as a novel theranostic molecule for the management of TB.

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4

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RESULTS

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In the current study, high-affinity MS-specific single-stranded (ss) DNA aptamers were identified

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from an 80-mer random DNA library (RDL) using SELEX-based screening. The best-performing

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aptamer candidate, MS10, was further optimized post-SELEX to generate MS10-Trunc.The

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theranostic potential of MS10-Trunc was established by its inhibitory activity against MS enzyme,

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interference of Mtb binding and invasion into host cells and ability to detect MS in CSF specimens for

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the diagnosis of TBM.

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Aptamer selection and screening for MS aptamers

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Mtb MS was expressed in E.coli BL21(DE3)pLysS and purified through affinity chromatography (Ni-

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NTA) and used as a target to generate MS-specific aptamers by the process of SELEX (Figure 1). The

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SELEX process included a step of negative selection to exclude nitrocellulose membrane (NCM)

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binders (step a), followed by partitioning of unbound sequences (step b) and a positive selection step

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to obtain binders to MS coated on NCM (step c). The bound DNA was eluted and amplified (step d)

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followed by the generation of ssDNA for the next round of SELEX. After ten iterative rounds of

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SELEX, the archived aptamer populations from rounds 2, 4, 6, 7, 8, 9 and 10 were assessed for

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binding to MS using Aptamer Linked Immobilized Sorbent Assay (ALISA). The enrichment of MS-

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binding aptamers was evident over consecutive rounds of SELEX (Figure 2A). However, the aptamer

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population of rounds 8, 9 and 10 evinced the highest binding over the random DNA library (RDL).

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Interestingly, the round 9 aptamer population showed higher binding to MS compared to round 10

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aptamer pool (Figure 2A). The reason for this could be the improper partitioning between specific and

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non-specific MS binders during the 10th round of selection process or a decrease in the aptamer pool

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complexity or a combination of both.17 The aptamers of rounds R8, R9 and R10 were individually 5

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cloned in the TA cloning vector pTZ57R/T and clones were randomly picked and sequenced (Figure 1,

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step e). Based on the sequencing results, representative aptamer candidates from each round were

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subjected to CLUSTAL-W and BioEdit analysis to identify the primary sequence homology and

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nucleotide richness (Figure S1). Based on the nucleotide richness, all the 22 aptamer candidates were

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categorized into 2 groups G-rich and Non-G rich (Figure S1). Interestingly ~50% aptamer candidates

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were G-rich with high G+C content (Figure S2A and S2B), suggesting the preferential binding of MS

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to G-rich DNA aptamers. These 22 monoclonal aptamer candidates were first screened for their

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binding to MS by ALISA (Figure 2B) and 5 aptamers, namely, MS4, MS5, MS6, MS10 and MS20

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were selected for further study using an arbitrary cut-off of ∆O.D.450nm ≥0.6. Among them, MS4 and

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MS20 were derived from the R8 pool, MS5 and MS6 belonged to the R9 pool while MS10 was

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obtained from the R10 aptamer population.

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In-silico secondary structure prediction of aptamers

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To gain structural insights into aptamers, their secondary structure was predicted by a web-based tool,

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RNA fold, using DNA parameters (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi).

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MS4 and MS20 acquire a typical stem-loop structure while MS5, MS6 and MS10 form only a loop-

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like structure (Figure S3). The selected aptamer candidates belong to the G-rich category that usually

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have propensity to form G4 structures. Analysis of the sequences using an in-silico tool QGRS

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Mapper

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(http://bsbe.iiti.ac.in/bsbe/ipdb/index.php), predicted that all the queried aptamers, namely, MS4, MS5,

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MS6, MS10 and MS20 form G4 structure and show scores comparable or higher than the known G4

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forming aptamer sequences (anti-thrombin, anti-HIV integrase and VEGF-aptamers)18-20 (Table S1).

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Further to validate in-silico the predictions, CD spectroscopy analysis was undertaken which revealed

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that all the aptamers form G4structures. Each aptamer shows a negative peak near 242 nm and a

(http://bioinformatics.ramapo.edu/QGRS/index.php)

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and

G4IPDB

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positive peak close to 265 nm, which typically signify a parallel G4 structure formation by all of these

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aptamers 21-23(Figure 2C).

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Determination of dissociation constant (Kd) of aptamers by SPR

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Purified MS was amine-coupled onto a CM5 chip through EDC-NHS chemistry, followed by injection

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of the individual aptamers, to obtain the dissociation equilibrium constant (Kd) values for each aptamer.

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All the aptamers exhibited tight binding with Kd in low micro to nanomolar range; the aptamers were

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ranked in terms of decreasing affinity (Kds) as MS6 (0.28 nM) >MS4 (9.9 nM) >MS10 (12 nM) >

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MS20 (0.91 µM) > MS5 (2.7 µM) (Table 1 and Figure S4).

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Inhibition of MS enzyme activity by aptamers

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Purified MS protein was pre-incubated with selected aptamers (namely MS4, MS5, MS6, MS10 and

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MS20) for 30 min followed by the addition of glyoxylate and acetylCoA to complete the reaction. The

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inhibition of MS enzyme activity by various aptamers was monitored by estimating free CoA as

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described in Methods.10 Based on the extent of inhibition of MS activity, the aptamers were ranked as

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MS10 >MS5 >MS20 >MS4 >MS6. MS10 was determined to be the most potent inhibitor; it evinced

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inhibitory activity of ~91% against purified MS protein (Figure 3A). A control aptamer of the same

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length (C1, Table S2) showed negligible inhibition of MS activity and suggested that MS10 aptamer-

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mediated inhibition was sequence-specific.

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Among all the tested aptamers, MS10 aptamer exhibited maximum inhibitory activity against MS.

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Therefore, MS10 aptamer was taken up for further studies.

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Post-SELEX optimization of MS10 aptamer

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In the literature, numerous G4 forming aptamers are reported as potent inhibitors of biological

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activity.24 MS10 was a G-rich aptamer and to establish its structure-activity relationship (SAR), its G4 7

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forming sequence that was identified through QGRS mapper (Figure S5), was synthesized and

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designated as MS10-Trunc (5’- GGTGGTGGTGG-3’) and assessed for its ability to bind MS using

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ALISA. The 11-mer MS10-Trunc displayed three GGT repeats and exhibited similar binding to MS

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compared to its 44-mer parent aptamer MS10 (Figure 3B), thus representing the smallest functional

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unit of the parent aptamer. To establish the role of each ‘T’ nucleotide present in MS10-Trunc, the ‘T’

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nucleotides were mutated by deleting one ‘T’ nucleotide at a time (Table S3) and the binding of

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individual mutant aptamers (∆T3, ∆T6 and ∆T9) to MS was assessed by ALISA. In terms of aptamer

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binding to MS, the deletion of T3 or T9 was well tolerated, whereas the deletion of T6 led to ~28 %

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reduction in aptamer binding compared to MS10-Trunc (Figure 3B), indicating a crucial role for T6 in

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maintaining the stable G4 aptamer structure. To further evaluate the impact of truncation on aptamer

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binding, the Kd of MS10-Trunc was determined using SPR to be 19 pM (Figure S4), indicating a ~631

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fold improvement in affinity in comparison to the parent aptamer, MS10 (Kd= 12 nM, Table 1).

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Determination of inhibition kinetics of MS10 and its truncated derivative, MS10-Trunc

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The 11-mer MS10-Trunc aptamer exhibited a similar extent of inhibitory activity (~94%) as compared

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to the parent aptamer MS10 (~91%, Figure 3C). A comparative inhibition study was performed for

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MS10 and MS10-Trunc, along with the known MS inhibitors, phosphoenolpyruvate (PEP) and

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Bromopyruvate (BP).10 The IC50 of MS10, MS10-Trunc were determined to be 275.6 nM and 251.1

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nM whereas for BP and PEP it was 114.3 µM and 374.1 µM, respectively (Figure 4A), indicating that

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MS10-Trunc was more potent than its parent aptamer, MS10, as well as the known inhibitors, BP and

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PEP. While the IC50 value of an inhibitor is an indicator of its functional strength (such as inhibiting

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enzyme activity), the Ki value (the equilibrium dissociation constant of the inhibitor) is more reflective

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of its binding affinity for the target. We further determined the Ki value for MS using linear replots of

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Lineweaver-Burk slopes using a range of aptamer and substrate concentrations. MS10 and MS10-

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Trunc showed inhibition constant of 270 nM and 230 nM, respectively (Figure 4B and 4C), which 8

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further supports their robust inhibitory potential. PEP and BP are reported to exhibit Ki ~200 µM and

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~60 µM, respectively.10 The parent aptamer exhibited ~740 and ~220 fold more potency than PEP and

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BP whereas MS10-Trunc displayed ~870 and ~260 fold more potency than PEP and BP.

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We further determined the inhibition mechanism of the MS10 and MS10-Trunc to evaluate the mode

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of aptamer binding to MS that leads to such robust inhibition. There was no significant change in

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inhibition of enzyme activity when the order of addition of aptamer or substrate to MS protein was

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reversed. Both MS10 and MS10-Trunc inhibit MS by mixed inhibition mechanism, indicating that

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they can inhibit catalysis regardless of whether the substrate is bound to the enzyme (Figure 4B and

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4C).

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Inhibition of Mycobacterium tuberculosis invasion in cells

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Since MS is also expressed on the cell surface of Mtb and exhibits adhesin activity that contributes to

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bacterial invasion into host cells7, we assessed the interaction of MS10-Trunc with cell surface-

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associated MS. Flow cytometry analysis (Figure 5A) clearly demonstrated preferential binding of

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MS10-Trunc aptamer with Mtb as compared to the control oligo C2, indicating that the former

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aptamer interacted with cell surface-associated MS. We next assessed the ability of MS10-Trunc to

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modulate invasion/ infection of Mtb H37Rv into human THP-1 monocytic cells. A ~40% decrease in

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Mtb infectivity was observed in the presence of MS10-Trunc (Figure 5B; ***P <0.0001) and no such

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inhibition was observed with 44-mer control oligos (C1 and C2), suggesting that inhibition is aptamer-

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specific and Mtb utilizes a MS-dependent mechanism, at least in part, for host cell entry. Taken

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together these findings demonstrate the potential application of aptamer-based inhibitors as a TB

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therapeutic agent.

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Diagnostic application of MS10-Trunc

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The diagnostic potential of MS10-Trunc aptamer to detect MS in CSF samples was assessed next in 86

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archived CSF specimens belonging to TBM and disease control categories. A detailed description

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about ‘Classification of CSF samples’ is given in Supplementary Methods Section. To determine

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diagnostic accuracy of MS10-Trunc aptamer, a ROC curve was constructed first using ∆OD450nm

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values obtained with CSF from Definite TB (True positive) and NTIM group (True negative) samples

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and a scatter plot was constructed for the two sample categories. The area under the curve was 1.0

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which indicated the robustness of the assay (Figure S6A and S6B). The NTIM category comprised of

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cases of pyogenic bacterial meningitis that included 2 culture confirmed cases of E. coli (n=1), and

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Acinetobacter sp (n=1). The remaining 14 cases were diagnosed on the basis of response to

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appropriate antibiotics, clinical presentation and symptoms. Based on the cut-off derived from the

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ROC curve (0.3444), the performance of MS10-Trunc aptamer ALISA was evaluated (Figure 6). The

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aptamer MS10-Trunc yielded a very high sensitivity and specificity (> 97%) with a highly significant

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discrimination (***p<0.0001) between CSF from TBM and non-TBM subjects was observed. This is a

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highly encouraging finding and requires substantiation in a larger study on geographically distinct

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populations so that this lead can be translated in to a commercially viable diagnostic assay.

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Structural insights into MS10-Trunc

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Secondary structure analysis of MS10-Trunc, the most potent inhibitor among the molecules assessed

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in the study, was predicted to have only a loop configuration, albeit a smaller one, as compared to

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MS10, its parent aptamer, suggesting that the loop is sufficient for aptamer interaction with MS

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(Figure S5). A recent report also suggests that the loop region is sufficient for interaction of aptamer

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to human erythropoietin αwith its cognate target.25 CD spectra analysis revealed that MS10-Trunc

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aptamer, like its parent aptamer (Figure 2C), exhibited a negative peak at 242 nm and a positive peak

223

at 265 nm (Figure 7A), that is typical of parallel-type G4 structure.21,22 This G4 structure was dynamic

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and underwent a structural change in the presence of MS in an aptamer concentration-dependent 10

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manner, as seen by changes in ellipticity (Figure 7A). The formation of G4 structure of MS10-Trunc

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aptamer was confirmed by 1H NMR analysis. The formation of a G4 structure byMS10-Trunc aptamer

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was confirmed by 1H NMR spectrum analysis; the aptamer exhibited a set of merged imino proton

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resonances at 10-12 ppm with broad line widths, clearly demonstrating the formation of a predominant

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G4 fold 26 (Figure 7B). The NMR melting temperature experiment further confirms thermostability of

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the DNA aptamer and showing the existence of imino signals at the higher temperature (upto 65ºC).

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These NMR findings are consistent with in-silico prediction of G4 structure by QGRS-Mapper and the

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findings of CD spectral analysis.

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Molecular modeling and in-silico docking studies

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Towards identifying the binding location of MS10-Trunc on MS in accordance to structural insights

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obtained from CD and NMR analysis, we generated a parallel inter-stranded G4 structure model of

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MS10-Trunc aptamer (Figure 8A). This energetically refined model contains 4 chains (O, P, Q, R

237

displayed in different color code) of identical length (11mer) and it was selected as the input ligand for

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docking studies using the crystal structure of Mtb MS (PDB ID: 2GQ3, Figure 8). The analysis of the

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docked structure predicted interactions of MS10-Trunc aptamer with Thr528, Lys484, Thr482, and

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Gln486 residues of MS protein (Figure 9). The G10 nucleotide of R chain forms 2 hydrogen bonds by

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interacting with Thr482 and Gln486 residues of MS, G5 nucleotide of Q chain forms one hydrogen

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bond with Thr528, and G7 of O chain forms one hydrogen bond with Lys484. All these residues map

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in the TIM barrel domain of MS which forms the active site of MS at the interface of TIM barrel

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residues (115-134 and 266-557), residues of domain II (591-727) and residues of a loop region (616-

245

633).10

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247 11

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DISCUSSION

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There is a need for versatile reagents with theranostic activity that can both detect and treat

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tuberculosis. The selection of a unique target that can meet both diagnostic and theranostic criteria

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underlies the successful design of a useful theranostic agent. MS is the second and terminal enzyme of

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the glyoxylate cycle that is essential for sustaining a persistent TB infection 10 of both drug susceptible

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and drug resistant forms of TB. This enzyme is absent in humans which makes MS an attractive TB

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drug target.15,27 Furthermore, in recent years its role as a biomarker for detecting active TB was also

255

established.7,8 Thus, taking together these attributes, MS can be considered as a potent and novel

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theranostic target for the management of TB.

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In recent years, nucleic acid aptamers have gained significant attention from the scientific community

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as promising inhibitors and drug candidates due to their rapid synthesis, high stability, non-toxic

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nature, ease in optimization and functionalization and ability to control pharmacokinetic and

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pharmacodynamic parameters.28-30 We have developed a panel of parallel G4 structure forming

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ssDNA aptamers that showed high-affinity binding and robust inhibition of MS catalytic activity using

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SELEX technology. An 11mer G4 structure forming aptamer, MS10-Trunc, having high performance

263

characteristics, was developed by post-SELEX optimization of its parent aptamer, MS10. Post-SELEX

264

optimization led to ~630-fold improvement in Kd of the 11-mer for MS over its full-length 44-mer

265

counterpart. This finding is consistent with previously published reports where an improvement in

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binding affinity is reported on aptamer truncation.31 Further, MS10-Trunc demonstrated a high

267

inhibitory potential, with low IC50 (230 nM) that was comparable to that of its parent aptamer MS10

268

(270 nM). IC50 data unequivocally demonstrated that the inhibitory activity of MS10-Trunc is

269

considerably superior (~1000 fold) to that of the known MS inhibitors, BP and PEP10 and comparable

270

to that of recently reported PDKA inhibitors which had IC50 values in the range of 0.01 µM to 0.6

271

µM.14 There are reports that G4 forming structures allow aptamers to interact strongly with their 12

18

cognate targets, such as anti-thrombin aptamer

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aptamer.30 More recently, G4 forming inhibitory DNA aptamers against Mtb polyphosphate kinase-2

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were also reported.4 A truncated aptamer with similar length (11-mer) is previously reported by Nadal

275

et al. (2013) for detecting ß-conglutin, an allergen.33 The high affinity binding properties of MS10-

276

Trunc to MS can be ascribed to its ability to form an 11-mer G4 structure (MS10-Trunc) that was

277

confirmed by CD spectroscopy analysis and 1H NMR studies.

278

In-silico modeling and docking studies have provided useful insights into the molecular interactions

279

between MS10-Trunc aptamer and its cognate target, MS that may contribute to its theranostic activity.

280

In-silico generated parallel G4 structure of MS10-Trunc was predicted to have 4-stranded structure

281

containing R, O, Q and P chains. This observation is in agreement in the previous reports where a 6-

282

mer anti-HIV DNA molecule forms a similar parallel G4 structure involving four chains.34 G-

283

nucleotides mapped to the R, O and Q chains of G4 structure of MS10-Trunc were predicted to have

284

H-bonding interactions with 4 amino acid residues (Thr482, Thr528, Lys484 and Gln486) that are

285

located in the TIM barrel domain of MS. Since the TIM barrel domain forms the active site interface

286

by interacting with the residues of domain II and loop region (616-663), therefore binding of MS10-

287

Trunc aptamer to residues of the TIM barrel domain may possibly lead to a conformational change

288

in/near the active site of MS that adversely influences catalytic function of MS and results in an

289

inhibition of enzymatic activity of MS in presence of MS10-Trunc. It is also established that MS acts

290

as an adhesin and is known to play a vital role in bacterial invasion into host cells.7 Our findings

291

clearly demonstrate that MS10-Trunc aptamer blocks bacterial invasion by ~40%. In addition to MS,

292

Mtb is also known to use other adhesins, including but not limited to HupB, PstS-1 and LpqH (19-kDa

293

antigen)35, during host cell invasion. A previous study has also reported aptamer-mediated inhibition

294

of Mtb entry into murine macrophages although the identity of the aptamer target was not known.36

13

, nucleolin-targeting aptamer

32

272

and anti-VEGF

295

Therefore, to achieve more robust inhibition of bacterial invasion, a combinatorial approach (a cocktail

296

of aptamers targeting various adhesins of Mtb) may possibly be used.

297

This study offers proof of concept for a TB theranostic reagent that is directed against MS. Our

298

findings indicate that MS10-Trunc aptamer has the potential to target Mtb infection. However, the

299

stability and delivery of aptamers into mycobacteria poses a challenge for their therapeutic application.

300

Although G4 based aptamers are relatively more stable in comparison to their non-G4 counterparts,

301

their stability may further be improved by chemical modification strategies.37,38 Efforts are ongoing in

302

our laboratory to suitably modify the aptamer using various functionalization and conjugation

303

strategies to achieve high stability and efficient delivery of MS10-Trunc aptamer into Mtb.

304

The future challenges relating to aptamer delivery in animal model or humans can possibly be

305

addressed by administration of aptamer in the form of an inhaled aerosol mist or by injecting the

306

aptamer into the blood. The delivery of aptamer through the inhalation route might protect the

307

truncated aptamer from the exonucleases in serum while delivery of aptamer as injectables would

308

require suitable modification such as blockage of the 3' end with PEG, modification of the backbone

309

and introduction of modified bases etc. to avoid enzymatic breakdown and rapid clearance by the

310

kidneys and liver.

311

The performance of aptamer-based TBM diagnostic test developed in the current study is superior to

312

various previously reported diagnostic modalities (Table 2). MS10-Trunc aptamer has shown a very

313

high diagnostic accuracy (>97% sensitivity and specificity) with an ability to efficiently discriminate

314

between TBM and non-TBM subjects (p-value of ***p < 0.0001). This aptamer being a synthetic

315

DNA molecule, will evince minimal/no batch-to-batch variation and can easily be adapted on to

316

various biosensing platforms for developing a rapid point of care test for TBM and possibly other

317

forms of TB. 14

318

In conclusion, we have identified a panel of G4 forming ssDNAaptamers that represents a novel class

319

of theranostic molecules against Mtb MS. The theranostic potential of the best-performing and

320

optimized G4 forming aptamer, MS10-Trunc was established through binding studies using ALISA

321

and SPR, enzyme inhibition assay, binding and inhibition of Mtb invasion into host cells and

322

demonstrating its diagnostic utility in TBM. We anticipate that MS10-Trunc holds promise for the

323

development of novel MS-targeted theranostics approach against Mtb and may complement the

324

existing diagnostic and therapeutic modalities in the near future.

325 326

MATERIAL AND METHODS

327

Reagents and Samples

328

All oligonucleotides and other primers utilized in the study were purchased from Integrated DNA

329

Technologies (IDT, USA). Other Routine reagents and chemicals were purchased from Sigma Aldrich,

330

USA. Bacterial expression plasmids of MS (GlcB), MPT51, MPT64, CFP-10 and purified proteins

331

Ag85C, HspX, GroES, LAM, ESAT-6 and culture filtrate proteins (CFPs) were provided by BEI

332

Resources, NIAID, NIH, USA, (please refer to acknowledgement for details).

333

Purification of MS

334

Plasmid pMRLB.8 encoding C-terminal hexa-histidine fusion of Mtb MS (Rv1837c) was used to

335

overexpress recombinant MS protein in E.coli BL21(DE3)pLysS. Bacterial cultures were grown in

336

2×YT medium at 37°C to an O.D595 of ~0.5 followed by an induction with 1 mMisopropyl-1-thio-β-D-

337

galactopyranoside (IPTG) for 5 hours at 25°C. MS was purified from the induced culture by Ni-NTA

338

affinity chromatography as described.8

339

Selection of DNA Aptamer against Mtb MS antigen using SELEX

15

340

A synthetic 80-mer single-stranded (ss) random DNA library (RDL) containing a random region of 44

341

nucleotides flanked by fixed primer binding sequences of 18-mer each that was used in our previous

342

study39 was employed to screen MS specific aptamers. The fixed primer binding sequences allow

343

DNA amplification by PCR using DRF (Forward- 5’ GTC TTG ACT AGT TAC GCC-3’) and DRR

344

(Reverse -5’ GAG GCG CCA ACT GAA TGA-3’) primers. The subtractive nitrocellulose membrane

345

(NCM)-based SELEX approach was used as described. For Round 1, 10 µg of MS was blotted onto

346

the NCM and allowed to dry at room temperature (RT). Mean while the RDL (1500 pmole) was

347

heated at 92°C followed by snap chilling on ice and finally brought to RT. The denatured library was

348

first incubated with NCM in a step of negative selection. The unbound sequence pool was then

349

incubated with pre-dried NCM blotted with MS in SELEX screening buffer [10 mM Tris pH 7.5

350

supplemented with 10 mM MgCl2, 50 mM KCl, 25 mM NaCl, 0.05% Tween-20 (v/v), 0.1 µg/µL yeast

351

tRNA and 0.1µg/µL BSA]. The unbound ssDNA was washed away with selection buffer (SB, 10 mM

352

Tris pH 7.5, 10 mM MgCl2, 50 mM KCl, 25 mM NaCl) supplemented with 0.5% Tween-20. The

353

bound DNA was eluted using nuclease-free water, followed by heating at 92°C for 5 min. The eluted

354

DNA binder population was used as a template for PCR amplification with the forward and reverse

355

primers for an optimized number of cycles of thermal cycling; 94°C for 30 sec, 55°C for 30 sec and

356

72°C for 30 sec with a final extension at 72°C for 5 min. In order to avoid formation of any undesired

357

high molecular weight amplicon, PCR cycle numbers were optimized. Subsequently, ssDNA was

358

prepared using alkaline treatment as described previously39,40 and used in the next round of SELEX.

359

The rigorousness of selection was intensified in every successive round of SELEX by increasing the

360

strength of Tween-20 during washing and by decreasing the interaction time with the target during

361

selection. In order to minimize cross-reactivity, counter-selection was employed by incubating the

362

binder population with other Mtb proteins, namely, purifiedHspX,CFP-10, ESAT-6, Ag85C,LAM,

363

MPT51, GroES and culture filtrate proteins (CFPs). After 10 rounds of selection, the high-affinity 16

364

pools were cloned in pTZ57R/T vector system (Thermo ScientificTMInsTAClone PCR cloning Kit)

365

and transformed in E.coli DH5α. The obtained colonies were randomly picked and subjected to DNA

366

sequencing.

367

Monitoring the rounds of SELEX

368

After ten iterative rounds of SELEX, the archived aptamer populations from rounds R2, R4, R6, R7,

369

R8, R9 andR10 were amplified through PCR using 5’ biotinylated forward primer and rA-containing

370

reverse primer, followed by strand separation using denaturing Urea-PAGE as described previously.39

371

These ssDNA pools of different rounds were checked for their binding to MS by Aptamer Linked

372

Immobilized Sorbent Assay (ALISA). For this, 500 ng of purified MS protein was coated onto a

373

NuncMaxiSorp™ 96 well-plate (Thermo Scientific)overnight at 4°C followed by blocking with 5%

374

Bovine Serum Albumin (BSA) at RT. The biotinylated aptamers pool of the archived population from

375

each round were heated at 92°C for 10 min, cooled on ice and brought to RT. One hundred ng of

376

biotinylated aptamer pool was then added to individual wells pre-coated with MS. Streptavidin-

377

Peroxidase Polymer Ultrasensitive (Sigma) was then added at1:2000 dilution followed by washing

378

with SB to remove unbound streptavidin-HRP conjugate and finally TMB (3, 3’, 5, 5’

379

Tetramethylbenzidine) (BD OptEIA™), was added. The reaction was stopped by 5% sulfuric acid

380

followed by absorbance measurement at 450 nm.

381

Screening of Aptamers via Aptamer Linked Immobilized Sorbent Assay (ALISA)

382

Briefly, 500 ng of MS was coated in 100 µL (0.1 mol/L) of sodium bicarbonate buffer (pH 9.6) on a

383

96 well-plate and incubated at 4°C overnight. The wells were blocked with 5% BSA solution for 2 hr

384

at RT. The rest of the ALISA procedure was followed as described previously.39

385

17

386

Circular dichroism (CD) spectra analysis

387

The CD spectra was obtained on Jasco J-815 Spectropolarimeter (Jasco Hachioji, Tokyo, Japan)

388

equipped with Peltier junction temperature controller with constant supply of nitrogen gas just to avoid

389

water condensation around the cuvette. The path length of the used cuvette was 0.2 cm, whereas,

390

spectral interval of each scan was set as 0.1 nm and the scanning rate was fixed at 20 nm/min. A blank

391

spectrum was recorded with SB only and subtracted from the CD spectrum of sample containing DNA

392

aptamer or DNA aptamer +MS. In each case, three scans were recorded in a range of 200-340 nm, and

393

the average of three scans was plotted to examine the target-dependent change in aptamer structure.

394 395

Surface plasmon resonance (SPR) analysis of aptamer interactions with MS.

396

The binding affinity of ssDNA aptamers to MS was investigated using surface plasmon resonance

397

(SPR) spectroscopy, where MS was immobilized on a CM5sensor chip (GE Healthcare). Briefly, the

398

carboxylic group on the sensor chip was activated with 1-ethyl-3-(3-dimethylaminopropyl)

399

carbodiimide (EDC) and N-hydroxysuccinimide (NHS) (BIAcore, Separations Scientific). Using

400

EDC-NHS chemistry, MS was coupled to the flow cell and the remaining carboxy site on the chip

401

surface was blocked using 1 M ethanolamine.41 MS specific ssDNA aptamer at different

402

concentrations (25, 50, 100, 200, 400 and 800 nM) was injected over the activated flow cells at a flow

403

rate of 10 µl/min for 5 min with a dissociation time of 5 min using Biacore 3000 instrument (GE

404

Healthcare). The blank surface of CM5sensor chip was used for background subtraction. The results

405

were obtained and analyzed using BiaEvaluation Software (Version 4.1.Biacore) to determine the

406

equilibrium dissociation constant Kd for each aptamer.

407 408 409 18

410

Aptamer-based inhibition of catalytic activity of MS

411

MS inhibition assay was performed at room temperature in a 96 well Corning® UV-Transparent

412

microplate (Corning, Inc. USA).The reaction mixture consisted of 20 mM Tricine buffer pH 7.5, 5

413

mM MgCl2, 0.8 mM EDTA, 2 mM glyoxylate, and 2 mM acetyl-CoA and 0.5 µg purified MS protein

414

was added and incubated at RT for 30 min. The reaction was stopped by the addition of 5,5'-dithiobis-

415

2-nitrobenzoic acid (DTNB) to a final concentration of 2 mM in Tris-HCl, pH-8.0. The amount of

416

CoA-SH released was measured by titrating the free thiol groups with DTNB and measuring the

417

change in absorption at 412 nm.10 Enzyme activity unit was represented as µmol/min. The screening

418

of ssDNA aptamers inhibiting MS enzyme activity was performed by using the same protocol

419

mentioned above with a slight modification. DNA aptamers (2 µM final concentration) and purified

420

MS protein (0.5 µg) was pre-incubated for 30 min in buffer containing 20 mM Tricine buffer (pH 7.5),

421

5 mM MgCl2 and 0.8 mM EDTA followed by the addition of 2 mM glyoxylate, and 2 mM acetyl-CoA.

422

The enzyme activity in the presence of aptamer was determined as stated above.

423 424

Post-SELEX optimization of aptamer MS10

425

Post-SELEX truncation and mutation studies were performed with the best-performing aptamerMS10

426

to determine the minimal functional unit within it. For this, the MS10 sequence was analyzed using a

427

web-based tool QGRS mapper (http://bioinformatics.ramapo.edu/QGRS/index.php) and G4IPDB

428

(http://bsbe.iiti.ac.in/bsbe/ipdb/index.php) to determine the high propensity G4 forming region.42,43

429

Based on the output, truncated and mutant aptamers were designed and procured from IDT.

430

Determination of IC50 and inhibition constant (Ki) of aptamer candidates

431

The inhibitory activity of aptamers was assessed by determining IC50 values for aptamer-mediated

432

inhibition of enzymatic activity of MS. In this assay, 2-10 mM glyoxylate and 2 mM acetyl-CoA was 19

433

used in the reaction. Purified MS protein (0.5 µg) was incubated with aptamers for 30 min at room

434

temperature followed by the addition of glyoxylate and acetyl-CoA and measurement of enzyme

435

activity. Also, MS protein was incubated with substrate for 30 min at room temperature followed by

436

addition of aptamer and measurement of enzyme activity. The IC50 values for individual aptamer

437

inhibition were determined by fitting the data into a sigmoidal dose-response curve in GraphPad Prism

438

software (version 5.01). All the kinetic measurements were performed at room temperature. The mode

439

of enzyme inhibition was measured as a function of substrate concentration at a fixed concentration of

440

aptamer and obtained data were plotted using double reciprocal Lineweaver-Burk (LB) plot. Ki values

441

were determined from linear replots of Lineweaver-Burk slopes versus aptamer or mentioned inhibitor

442

concentrations. Experimental data were analyzed using GraphPad Prism (version 5.01).

443

444

Aptamer binding to Mtb cell surface

445

Log phase 107 CFU of Mtb H37Rv was treated with 10 mM vitamin C for 24 hr to overexpress the

446

glyoxylate shunt enzymes44,45. Cultures were washed with SB and incubated with 0.5 µM of 5’-FAM-

447

labeled aptamers (C2 and MS10-Trunc) for 1 hr at RT. After removal of unbound aptamers, bacilli

448

were washed, fixed in 2% paraformaldehyde at 4ºC for 4 hrs and acquired on BD LSRII

449

flowcytometer, to assess aptamer binding under FITC-A channel. Overlay plots were prepared and

450

data was analyzed using FlowJoTM 10.4.1 software.

451

Inhibition of Mtb invasion into cells

452

Mtb H37Rv was cultured in DTA medium (Dubos containing 0.5% BSA, 0.75% Dextrose and 0.085%

453

NaCl plus 0.1% Tween-80) with shaking at 220 rpm at 37°C till OD595 ~0.1-0.2 and treated with 10

454

mM vitamin C for 24 hr. The bacteria were washed with SB and then exposed to 8 µg aptamer (~2.2

455

µM) for 1hr in SB. Bacteria were washed to remove excess of aptamer and infected into 5 x 105 THP20

456

1 cells at a MOI 10:1 for 2 hr. The extracellular Mtb were removed by treatment with amikacin (200

457

µg/ml) for 2 hr following which, the Mtb infected THP-1 cells were lysed with 0.025% SDS. Bacterial

458

colony forming units (CFUs) were enumeratedby plating THP-1 lysates on Middlebrook 7H11 agar

459

supplemented with 1.5% glycerol (MB agar) and the counts were finalized at 5 weeks.

460 461

Diagnostic application of MS10-Trunc

462

The diagnostic utility of MS10-Trunc aptamer was evaluated through ALISA on 86 archived

463

cerebrospinal fluid samples.8,39 The samples belonged to pediatric TBM subjects (n=38) and disease

464

controls subjects (n=48). The ALISA procedure was same as described above except the CSF samples

465

were diluted 1:20 in coating buffer. Data was plotted after background subtraction [(O.D.450 of CSF

466

sample) - (O.D.450 of mean + 3SD of control wells)].

467

Structure determination by Nuclear Magnetic Resonance (NMR)

468

To validate the aptamer structure, 1H NMR spectra were acquired on AVANCE 500 MHz BioSpin

469

International AG, Switzerland equipped with a 5 mm broadband inverse probe. All the NMR spectra

470

were acquired using solvent containing H2O/D2O at 9:1 ratio and spectral data were processed,

471

integrated and analyzed on Topspin (1.3 version) software. NMR samples were referenced with 3 -

472

(Trimethylsilyl) propionic-2, 2, 3, 3-d4 acid sodium salt (TSP).

473

Molecular modeling of MS10-Trunc and docking on MS

474

To simulate the structure and binding of MS10-Trunc aptamer (5’-GGTGGTGGTGG-3’) to MS, a

475

model of aptamer structure was first generated and used as the input for molecular docking on MS

476

[Protein Data Bank (PDB) code:2GQ3]. A well-known intra-molecular G4 formed by the SV40 repeat

477

sequence (PDB code: 1EVO), was taken as the initial input for modeling the 3D structure of MS10-

478

Trunc aptamer and further needful mutations were performed on Discovery studio 3.5. Protein 1EVO 21

479

was chosen as initial input model due the presence of a similar kind of G4 topology and close

480

sequence homology between 1EVO and MS10-Trunc aptamer. The resulting structure was subjected

481

to energy minimization and optimized using various iterations of conjugate gradient and steepest

482

descent method. Electrostatics was set as ‘particle mesh Ewald’ and apply SHAKE constraints were

483

chosen for every minimization run. The molecular docking studies were carried out using Hex 8.0.0

484

tool in which G4 DNA was treated as a rigid body. Correlation type was set as “Shape+Electro” and

485

FFT mode was set as 3D for every docking iteration. Sampling methods were set as “Rang angles” and

486

docking output was ranked according to their free energy.

487 488

AUTHORS’ CONTRIBUTIONS

489

J.S.T., T.K.S., A.K., T.P.S designed and supervised the experiment. A.D., C.K., K.S., I.D., S.M., P.S.

490

performed experiments, J.S.T., T.K.S., A.K., A.D., C.K., K.S., I.D., S.M., P.S. collected and analyzed

491

the data. J.S.T., T.K.S., A.D., C.K., K.S., S.M., and A.K. wrote the manuscript. All authors reviewed

492

the manuscript.

493

Competing financial interests: The authors declare no competing financial interests.

494

Acknowledgments

495

This work was supported through National Biodesign Alliance project by Department of

496

Biotechnology, Govt. of India awarded to J.S.T. (BT/PR14638/MED/12/483/2010) and an Innovation

497

Award and THSTI core grant to T.K.S. by the Department of Biotechnology, Govt. of India. J.S.T. is

498

thankful to the Department of Science and Technology, Govt. of India for the J. C. Bose National

499

Fellowship during part of the study. T.K.S. is thankful to the Department of Biotechnology, Govt. of

500

India for the Innovative Young Biotechnologist Award (BT/010/IYBA/2016/10). A.D. acknowledges

501

the Indian Council of Medical Research, Govt. of India for a Senior Research Fellowship. K.S. is 22

502

thankful to Department of Science and Technology, Government of India (PDF/2016/000745) for

503

funding. The aptamer sequences reported in this study are proprietary reagents and are deposited in

504

the Indian Patent Office as part of Indian Patent application no. 201611021901. We thank Dr. Priya

505

Kalra for her help during FACS experiment. The admirable technical assistance of Sanjay Kumar and

506

Jitendra Singh is duly acknowledged. We sincerely thank Sarin Prakash and Sunil Kumar Gupta for

507

their active involvement in data management and sample collection. BEI Resources, NIAID, NIH,

508

USA is acknowledged as below for providing various proteins that were utilized in this study. “The

509

following reagents were obtained through BEI Resources, NIAID, NIH: Plasmid pMRLB.8

510

Containing Gene Rv1837c (Protein GlcB), from Mycobacterium tuberculosis, NR-13281, Plasmid

511

pMRLB.38 Containing Gene Rv3803c (Protein Mpt51) from Mycobacterium tuberculosis, NR-

512

13291, Plasmid pMRLB.12 Containing Gene Rv1980c (Protein mpt64) from Mycobacterium

513

tuberculosis, NR-13273, Plasmid pMRLB.46 Containing Gene Rv3874 (Protein Cfp10)

514

from Mycobacterium tuberculosis, NR-13297 and Ag85 Complex, Purified Native Protein

515

from Mycobacterium tuberculosis, Strain H37Rv, NR-14855, HspX, Recombinant Protein Reference

516

Standard, NR-49428, GroES (Gene Rv3418c), Purified Native Protein from Mycobacterium

517

tuberculosis, Strain H37Rv, NR-14861, Mycobacterium tuberculosis, Strain H37Rv, Purified

518

Lipoarabinomannan (LAM), ESAT-6, Recombinant Protein Reference Standard, NR-49424 and

519

Mycobacterium tuberculosis, Strain H37Rv, Culture Filtrate Proteins, NR-14825."

520

ADDITIONAL INFORMATION

521

Supplementary Information.

522 523

23

524

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Platella, C., Riccardi, C., Montesarchio, D., Roviello, G. N. & Musumeci, D. G-quadruplex-based aptamers against protein targets in therapy and diagnostics. Biochimica et biophysica acta. General subjects 1861, 1429-1447, doi:10.1016/j.bbagen.2016.11.027 (2017). Sharma, T. K., Bruno, J. G. & Dhiman, A. ABCs of DNA aptamer and related assay development. Biotechnology advances 35, 275-301, doi:10.1016/j.biotechadv.2017.01.003 (2017). Kypr, J., Kejnovska, I., Renciuk, D. & Vorlickova, M. Circular dichroism and conformational polymorphism of DNA. Nucleic acids research 37, 1713-1725, doi:10.1093/nar/gkp026 (2009). Choi, E. W., Nayak, L. V. & Bates, P. J. Cancer-selective antiproliferative activity is a general property of some G-rich oligodeoxynucleotides. Nucleic acids research 38, 1623-1635, doi:10.1093/nar/gkp1088 (2010). Tucker, W. O., Shum, K. T. & Tanner, J. A. G-quadruplex DNA aptamers and their ligands: structure, function and application. Curr Pharm Des 18, 2014-2026 (2012). He, X., Guo, L., He, J., Xu, H. & Xie, J. Stepping Library-Based Post-SELEX Strategy Approaching to the Minimized Aptamer in SPR. Analytical chemistry 89, 6559-6566, doi:10.1021/acs.analchem.7b00700 (2017). Tawani, A., Amanullah, A., Mishra, A. & Kumar, A. Evidences for Piperine inhibiting cancer by targeting human G-quadruplex DNA sequences. Scientific reports 6, 39239, doi:10.1038/srep39239 (2016). Kondrashov, F. A., Koonin, E. V., Morgunov, I. G., Finogenova, T. V. & Kondrashova, M. N. Evolution of glyoxylate cycle enzymes in Metazoa: evidence of multiple horizontal transfer events and pseudogene formation. Biol Direct 1, 31, doi:10.1186/1745-6150-1-31 (2006). Keefe, A. D., Pai, S. & Ellington, A. Aptamers as therapeutics. Nature reviews. Drug discovery 9, 537550, doi:10.1038/nrd3141 (2010). Nimjee, S. M., White, R. R., Becker, R. C. & Sullenger, B. A. Aptamers as Therapeutics. Annual review of pharmacology and toxicology 57, 61-79, doi:10.1146/annurev-pharmtox-010716-104558 (2017). Zhou, J. & Rossi, J. Aptamers as targeted therapeutics: current potential and challenges. Nature reviews. Drug discovery 16, 440, doi:10.1038/nrd.2017.86 (2017). Macdonald, J., Houghton, P., Xiang, D., Duan, W. & Shigdar, S. Truncation and Mutation of a Transferrin Receptor Aptamer Enhances Binding Affinity. Nucleic Acid Ther 26, 348-354, doi:10.1089/nat.2015.0585 (2016). Bates, P. J., Laber, D. A., Miller, D. M., Thomas, S. D. & Trent, J. O. Discovery and development of the G-rich oligonucleotide AS1411 as a novel treatment for cancer. Experimental and molecular pathology 86, 151-164, doi:10.1016/j.yexmp.2009.01.004 (2009). Nadal, P., Svobodova, M., Mairal, T. & O'Sullivan, C. K. Probing high-affinity 11-mer DNA aptamer against Lup an 1 (beta-conglutin). Analytical and bioanalytical chemistry 405, 9343-9349, doi:10.1007/s00216-013-7385-0 (2013). Hotoda, H. et al. Biologically active oligodeoxyribonucleotides. 5. 5'-End-substituted d(TGGGAG) possesses anti-human immunodeficiency virus type 1 activity by forming a G-quadruplex structure. J Med Chem 41, 3655-3663, doi:10.1021/jm970658w (1998). Squeglia, F., Ruggiero, A., De Simone, A. & Berisio, R. A structural overview of mycobacterial adhesins: Key biomarkers for diagnostics and therapeutics. Protein science : a publication of the Protein Society 27, 369-380, doi:10.1002/pro.3346 (2018). Chen, F., Zhang, X., Zhou, J., Liu, S. & Liu, J. Aptamer inhibits Mycobacterium tuberculosis (H37Rv) invasion of macrophage. Molecular biology reports 39, 2157-2162, doi:10.1007/s11033-011-0963-3 (2012). Liao, J. et al. Cell-specific aptamers and their conjugation with nanomaterials for targeted drug delivery. Expert opinion on drug delivery 12, 493-506, doi:10.1517/17425247.2015.966681 (2015). AlShamaileh, H. & Veedu, R. N. Next-Generation Nucleic Acid Aptamers with Two-Base-Modified Nucleotides Have Improved Binding Affinity and Potency. Chembiochem : a European journal of chemical biology 18, 1565-1567, doi:10.1002/cbic.201700276 (2017). 25

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46 47 48 49

50 51

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Dhiman, A. et al. Generation and application of DNA aptamers against HspX for accurate diagnosis of tuberculous meningitis. Tuberculosis 112, 27-36, doi:10.1016/j.tube.2018.07.004 (2018). Kalra, P. et al. G-Quadruplex-Forming DNA Aptamers Inhibit the DNA-Binding Function of HupB and Mycobacterium tuberculosis Entry into Host Cells. Molecular therapy. Nucleic acids 13, 99-109, doi:10.1016/j.omtn.2018.08.011 (2018). Rotherham, L. S., Maserumule, C., Dheda, K., Theron, J. & Khati, M. Selection and application of ssDNA aptamers to detect active TB from sputum samples. PloS one 7, e46862, doi:10.1371/journal.pone.0046862 (2012). Kikin, O., D'Antonio, L. & Bagga, P. S. QGRS Mapper: a web-based server for predicting G-quadruplexes in nucleotide sequences. Nucleic acids research 34, W676-682, doi:10.1093/nar/gkl253 (2006). Mishra, S. K., Tawani, A., Mishra, A. & Kumar, A. G4IPDB: A database for G-quadruplex structure forming nucleic acid interacting proteins. Scientific reports 6, 38144, doi:10.1038/srep38144 (2016). Sikri. K et al. Multifaceted remodeling by vitamin C boosts sensitivity of Mycobacterium tuberculosis subpopulations to combination treatment by anti-tubercular drugs. Redox Biology (2018). Taneja, N. K., Dhingra, S., Mittal, A., Naresh, M. & Tyagi, J. S. Mycobacterium tuberculosis transcriptional adaptation, growth arrest and dormancy phenotype development is triggered by vitamin C. PloS one 5, e10860, doi:10.1371/journal.pone.0010860 (2010). Thakur, R., Goyal, R. & Sarma, S. Laboratory diagnosis of tuberculous meningitis - is there a scope for further improvement? Journal of laboratory physicians 2, 21-24, doi:10.4103/0974-2727.66705 (2010). Thwaites, G. et al. Tuberculous meningitis. Journal of neurology, neurosurgery, and psychiatry 68, 289299, doi:10.1136/jnnp.68.3.289 (2000). Rufai, S. B. et al. Diagnostic usefulness of Xpert MTB/RIF assay for detection of tuberculous meningitis using cerebrospinal fluid. The Journal of infection 75, 125-131, doi:10.1016/j.jinf.2017.04.010 (2017). Solomons, R. S. et al. Commercial nucleic acid amplification tests in tuberculous meningitis--a metaanalysis. Diagnostic microbiology and infectious disease 78, 398-403, doi:10.1016/j.diagmicrobio.2014.01.002 (2014). Pai, M. et al. Diagnostic accuracy of nucleic acid amplification tests for tuberculous meningitis: a systematic review and meta-analysis. The Lancet. Infectious diseases 3, 633-643 (2003). Haldar, S., Sharma, N., Gupta, V. K. & Tyagi, J. S. Efficient diagnosis of tuberculous meningitis by detection of Mycobacterium tuberculosis DNA in cerebrospinal fluid filtrates using PCR. J Med Microbiol 58, 616-624, doi:10.1099/jmm.0.006015-0 (2009). Flores, L. L. et al. Systematic review and meta-analysis of antigen detection tests for the diagnosis of tuberculosis. Clinical and vaccine immunology : CVI 18, 1616-1627, doi:10.1128/CVI.05205-11 (2011).

653 654

26

655

FIGURE LEGENDS

656

Figure 1. Systematic enrichment of aptamers against MS. Negative selection was performed by

657

incubating the random ssDNA library (RDL) with nitrocellulose membrane (NCM) (step

658

a);partitioning of bound and unbound sequences (step b); the unbound DNAs were collected and then

659

incubated with immobilized MS for positive selection (step c); after washing, the bound DNAs were

660

eluted and amplified by PCR for the next round of SELEX (step d); after enrichment, the DNA

661

population was cloned and sequenced to identify the sequences of individual monoclonal aptamers

662

(step e).

663

Figure 2. (A) Relative binding of various aptamer pools (Round 2, 4, 6, 7, 8, 9, 10 and RDL) to target

664

MS as determined by Aptamer Linked Immobilized Sorbent Assay (ALISA). (B) Relative binding of

665

various monoclonal aptamers with MS. The aptamers highlighted in blue color were selected for

666

further characterization based on an arbitrary cut-off O.D.450nm ≥0.6. (C) CD spectra of MS aptamer

667

candidates.

668

Figure 3. (A) Effect of aptamers on the catalytic activity of Mtb MS at a fixed aptamer concentration

669

(2 µM). (B) Binding ability of truncated and different mutant versions of MS10 aptamer to target MS.

670

The binding of individual mutant aptamers to MS was compared with MS10-Trunc binding to MS

671

using ALISA. Individual base deletions are represented as cyan color bars in the graph. (C) Effect of

672

MS10 and MS10-Trunc aptamers on catalytic activity of Mtb-MS. C1 is the control aptamer (sequence

673

given in Table S2, used in panels A and C).

674

Figure 4. (A) Determination of IC50of MS10and MS10-Truncaptamers. The relative activity of the

675

Mtb-MS as a function of the various concentrations of inhibitory aptamers MS10 and its truncated

676

form MS10-Trunc. Ki value and inhibition mechanism of aptamer MS-10 and its derivative MS10-

677

Trunc was determined using double-reciprocal Line weaver Burk (LB) plot (B and C).

678

27

679

Figure 5. Inhibition of Mtb infection with MS10-Trunc in THP-1 cell infection model. (A) Binding of

680

MS aptamer to Mtb H37Rv determined by flow cytometry using 5’-FAM labeled aptamers. C2 was

681

used as control aptamer (sequence given in Table S2, used in panels A). (B) Aptamer treated Mtb was

682

infected in THP-1 monocytes and intracellular bacteria plated on MB agar post-infection for CFU

683

determination. The graph shows the percentage infectivity calculated with respect to the untreated

684

(UT, no aptamer treatment) control. ***P <0.0001 (calculated using one way ANOVA). C1 and C2

685

were used as control aptamer (sequence given in Table S2, used in panel B). The difference in

686

infectivity between UT, C1 and C2 was found to be non-significant.

687

Figure 6. MS10-Trunc aptamer-based detection of MS antigen by ALISA in archived CSF samples

688

categorized as TBM patients (n=38) and Non-TBM patients (n=48).

689

Figure 7. (A) Circular Dichroism titration spectrum for MS10-Truncaptamer (Red) and aptamer in

690

the presence of increasing concentration of MS upto Protein (P):Aptamer(A) ratio = 2.0. (B)

691

Temperature dependent 1H NMR spectra for MS10-Trunc showing imino and base region.

692

Figure 8. (A) A model of G4 structure formed by MS10-Trunc aptamer and (B) Hydrogen Bonds in

693

each G and T quartet of G4structure.

694

Figure 9. The interaction of PEP (red) and MS10-Trunc (multicolor) with MS. The interactions

695

between residues in MS protein and bases in aptamer are shown in cartoon representation and the

696

regions in the box are shown enlarged in ball and stick model. The interactions are shown as blue

697

dashes.

698 699 700 701 28

702 703

Table 1: Surface Plasmon Response (SPR) binding kinetics data of aptamers.

704

Aptamer

Kon(1/Ms)

Koff(1/s)

KA (1/M)

KD (M)

MS4

2.4 x 105

2.4 x 10-3

1 x 108

9.9 x 10-9

MS5

3.7

1 x 10-5

3.7 x 105

2.7 x 10-6

MS6

3.6 x 104

1 x 10-5

3.6 x 109

2.8 x 10-10

MS10

2.8 x 105

3.2 x 10-3

8.7 x 107

1.2 x 10-8

MS20

11

1 x 10-5

1.1 x 106

9.1 x 10-7

MS10-Trunc

5.2 x 105

1.6 x 10-5

5.2 x 1010

1.9 x 10-11

R0 (Naïve library)

35

1 x 10-5

3.4 x 106

2.9 x 10-7

705

29

706 707

Table 2: Performance of various tests in diagnosis of TBM Diagnostic Assays for TBM Diagnosis

Comments

References

Sensitivity (%)

Specificity (%)

TB culture

27

100

Poor sensitivity, long turnaround time.

Smear microscopy

10-20

100

Poor sensitivity.

47

Moderate sensitivity, but an excellent rule-in test.

48

It requires DNA isolation and amplification steps, timeconsuming with Suboptimal sensitivity.

49

It requires DNA isolation and amplification steps and timeconsuming.

8

46

DNA-based Tests Xpert MTB/RIF assay

55.1

94.8

Commercial NAATs metaanalysis

64

98

Nucleic acid commercial amplification tests meta-analysis

56

98

100 (definite TBM) 98 (probable and possible TBM) 87.6 (filtrate) 53.1(sediment)

96-97 (definite TBM) 98 (probable and possible TBM)

MS (current study)

97.44

97.96

HspX (Aptamer-based ALISA)

100

91

100 (definite TBM)

96-97 (definite TBM)

92-95 (probable and possible TBM)

93-96 (probable and possible TBM)

In-house qPCR

In-house qPCR

50

51

92

Antigen Detection Tests

GlcB, HspX& PstS1 (Antibody-based ELISA)

GlcB, HspX& MPT51 (Antibody-based ELISA) LAM, Ag85 complex, 65 kDa& ESAT-6 (Antibody-based ELISA)

87

Excellent diagnostic tool to detect TBM with high accuracy. Excellent diagnostic accuracy, laboratory-based format.

-

Excellent diagnostic accuracy, laboratory-based format, timeconsuming, uses antibody which might have batch to batch variation.

8

Laboratory-based format, timeconsuming, uses antibody which might have batch to batch variation.

84

708

30

39

8

52