Generation and application of DNA aptamers against HspX for accurate diagnosis of tuberculous meningitis

Dhiman, Abhijeet; Haldar, Sagarika; Mishra, Subodh Kumar; Sharma, Neera; Bansal, Anjali; Ahmad, Yusra; Kumar, Amit; Sharma, Tarun; Tyagi, Jaya Sivaswami

doi:10.1016/j.tube.2018.07.004

Cited by 36 publications

(43 citation statements)

References 38 publications

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“…A synthetic 80-mer single-stranded (ss) random DNA library (RDL) containing a random region of 44 nt flanked by fixed primer binding sequences of 18-mer each that was used in our previous study 40 was employed to screen MS-specific aptamers. The fixed primer binding sequences allow DNA amplification by PCR using DNA Random Forward primer (DRF) (forward 5′-GTC TTG ACT AGT TAC GCC-3′) and DNA Random Reverse primer (DRR) (reverse 5′-GAG GCG CCA ACT GAA TGA-3′) primers.…”

Section: Methodsmentioning

confidence: 99%

“…PCR cycle numbers were optimized in order to avoid formation of any undesired high-molecular-weight amplicon. Subsequently, ssDNA was prepared using alkaline treatment as described previously 40 , 41 and used in the next round of SELEX. The rigorousness of selection was intensified in every successive round of SELEX by increasing the strength of Tween 20 during washing and by decreasing the interaction time with the target during selection.…”

Section: Methodsmentioning

confidence: 99%

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

Dhiman

Kumar

Mishra

et al. 2019

Molecular Therapy - Nucleic Acids

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The successful management of tuberculosis (TB) requires efficient diagnosis and treatment. Further, the increasing prevalence of drug-resistant TB highlights the urgent need to develop novel inhibitors against both drug-susceptible and drug-resistant forms of disease. Malate synthase (MS), an enzyme of the glyoxylate pathway, plays a vital role in mycobacterial persistence, and therefore it is considered as an attractive target for novel anti-TB drug development. Recent studies have also ascribed an adhesin function to MS and established it as a potent diagnostic biomarker. In this study, a panel of Mycobacterium tuberculosis (Mtb) MS-specific single-stranded DNA aptamers was identified by Systematic Evolution of Ligands by EXponential enrichment (SELEX). The best-performing G-quadruplex-forming 44-mer aptamer, MS10, was optimized post-SELEX to generate an 11-mer aptamer, MS10-Trunc. This aptamer was characterized by various biochemical, biophysical, and in silico techniques. Its theranostic activity toward Mtb was established using enzyme inhibition, host cell binding, and invasion assays. MS10-Trunc aptamer exhibited high affinity for MS (equilibrium dissociation constant [KD] ∼19 pM) and displayed robust inhibition of MS enzyme activity with IC50 of 251.1 nM and inhibitor constant (Ki) of 230 nM. This aptamer blocked mycobacterial entry into host cells by binding to surface-associated MS. In addition, we have also demonstrated its application in the detection of tuberculous meningitis (TBM) in patients with sensitivity and specificity each of >97%.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Dhiman

Kumar

Mishra

et al. 2019

Molecular Therapy - Nucleic Acids

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“…Regarding the HspX protein, its increased representation in the 3955 strain compared to RUS_B0, which went down in the strain 2093, was observed. Previously, it was proposed that HspX can be used as a marker of the disease, including its latent form [21,22]. A reduced representation of this protein in the resistant strain casts doubts on its usefulness.…”

Section: Analysis Of Drug Resistance Genes On Multi-omics Levelmentioning

confidence: 99%

Metabolic Changes of Mycobacterium tuberculosis during the Anti-Tuberculosis Therapy

et al. 2020

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Tuberculosis, caused by Mycobacterium tuberculosis complex bacteria, remains one of the most pressing health problems. Despite the general trend towards reduction of the disease incidence rate, the situation remains extremely tense due to the distribution of the resistant forms. Most often, these strains emerge through the intra-host microevolution of the pathogen during treatment failure. In the present study, the focus was on three serial clinical isolates of Mycobacterium tuberculosis Beijing B0/W148 cluster from one patient with pulmonary tuberculosis, to evaluate their changes in metabolism during anti-tuberculosis therapy. Using whole genome sequencing (WGS), 9 polymorphisms were determined, which occurred in a stepwise or transient manner during treatment and were linked to the resistance (GyrA D94A; inhA t-8a) or virulence. The effect of the inhA t-8a mutation was confirmed on both proteomic and transcriptomic levels. Additionally, the amount of RpsL protein, which is a target of anti-tuberculosis drugs, was reduced. At the systemic level, profound changes in metabolism, linked to the evolution of the pathogen in the host and the effects of therapy, were documented. An overabundance of the FAS-II system proteins (HtdX, HtdY) and expression changes in the virulence factors have been observed at the RNA and protein levels.

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“…3 Both intraspinal tuberculosis and metastatic cancer may be rare, but they are difficult to differentiate because of their similar presentations and the high negative incidence of microbiological test for tuberculosis in cerebrospinal fluid (CSF) and their prognoses are completely different. 1,4 Most patients with intraspinal tuberculosis can achieve remarkable recovery after early diagnosis and treatment, whereas intraspinal metastatic cancers have a poor treatment response and poor prognosis. [5][6][7] To the best of our knowledge, although the optimal treatment of both diseases remains controversial, early accurate diagnosis can be helpful to improve neurological deficits and quality of life.…”

Section: Introductionmentioning

confidence: 99%

<p>Differentiation of Intraspinal Tuberculosis and Metastatic Cancer Using Magnetic Resonance Imaging</p>

Song

et al. 2020

IDR

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This study aimed to explore the differences in the magnetic resonance imaging (MRI) findings between intraspinal tuberculosis and metastatic cancer, which may aid in making the correct diagnosis. Patients and Methods: The clinical features and MRI findings of 15 patients with intraspinal tuberculosis and 11 patients with intraspinal metastatic cancers were retrospectively analyzed. Results: The mean ages of the patients with intraspinal tuberculosis and metastatic cancer were 26.3 (15-42) and 52.1 (38-67) years, respectively. All intraspinal tuberculosis cases were secondary to primary extraspinal tuberculosis, including tuberculous meningitis (11/15), as well as pulmonary (9/15), vertebral (5/15), urinary tract (1/15), abdominal (1/15), cervical lymph node (1/15), and multisystem tuberculosis (9/15). The intraspinal metastases originated from the breast (5/11), lung (3/11), kidney (1/11), ovarian (1/11), and nasopharyngeal cancers (1/11). Both intraspinal tuberculosis and metastatic cancers presented with multiple intra-and extramedullary lesions throughout all regional segments of the spinal canal, accompanied by irregularly thickened meninges. Intraspinal tuberculous lesions had indistinct edges that integrated with each other, most of them exhibiting obvious enhancement on MRI. Conversely, intraspinal metastatic lesions were distinctly separated with clear edges and exhibited lesser enhanced MRI than intraspinal tuberculosis. Conclusion: A combined analysis of clinical features and MRI findings may be helpful in differentiating intraspinal tuberculosis from metastatic cancer.

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Generation and application of DNA aptamers against HspX for accurate diagnosis of tuberculous meningitis

Cited by 36 publications

References 38 publications

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

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

Metabolic Changes of Mycobacterium tuberculosis during the Anti-Tuberculosis Therapy

<p>Differentiation of Intraspinal Tuberculosis and Metastatic Cancer Using Magnetic Resonance Imaging</p>

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