Development of a DNA aptamer for screening neisseria meningitidis serogroup B by cell SELEX

Background: Artificial oligonucleotides like DNA or RNA aptamers can be used as biodiagnostic alternatives for antibodies to detect pathogens. Comparing to antibodies, artificial oligonucleotides are produced easily at lower costs and are more stable. Neisseria meningitidis, the causative agent of meningitis, is responsible for about 1% of infections in an epidemic period. Specific DNA aptamers that bind to N. meningitidis serogroup B were identified by whole-cell Systemic Evolution of Ligands by EXponential Enrichment [SELEX]

Methods: The SELEX begins with a library of labeled ssDNA molecules. After six rounds of selection and two rounds of counter-selection, 60 clones were obtained, of which the binding efficiency of 21 aptamers to the aforementioned bacterium was tested by flow cytometry

Results: The aptamers K3 and K4 showed the highest affinity to N. meningitidis serogroup B and no affinity to N. meningitidis serogroups Y, A, and C, or to other meningitis causing bacteria. The dissociation constant [Kd value] for K3 and K4 were calculated as 28.3 +/- 8.9 pM and 39.1 +/- 8.6 pM, respectively. K3 aptamer with the lowest Kd was chosen as the main aptamer. K3 could detect N. meningitidis in patients' cerebrospinal fluid [CSF] samples and in CSF from healthy volunteers inoculated with N. meningitidis serogroup B [ATCC 13090] at 200 and 100 CFU ml[-1], respectively

Conclusion: The findings suggest the application of the developed aptamer in specific detection of N. meningitidis serogroup B amongst a group of meningitis causing bacteria

Development of a single stranded DNA aptamer as a molecular probe for LNCap cells using Cell-SELEX

Background: Nowadays, highly specific aptamers generated by cell SELEX technology[systematic evolution of ligands by exponential enrichment] are being applied forearly detection of cancer cells. Prostate Specific Membrane Antigen [PSMA], over expressedin prostate cancer, is a highly specific marker and therefore can be used fordiagnosis of the prostate cancer cells. The aim of the present study was to select singlestrandedDNA aptamers against LNCap cells highly expressing PSMA, using cell-SELEX method which can be used as a diagnostic tool for the detection of prostatecancer cells

Methods: After 10 rounds of cell-SELEX, DNA aptamers were isolated against PSMAusing LNCaP cells as a target and PC-3 cell lines for counter SELEX. Five DNA aptamerswith more than 70% affinity were selected up on flow cytometry analysis of positiveclones

Results: Dissociation constants of two selected sequences [A12-B1] were estimated inthe range of 33.78-3.77 and 57.49-2.214 pmol, respectively. Conserved secondary structuresof A12 and B1 sequences suggest the necessity of these structures for binding withhigh affinity to native PSMA. Comparison of the secondary structures of our isolatedaptamers and aptamer A10 obtained by protein SELEX showed similar stem-loopstructures which could be responsible for the recognition of PSMA on LNCap cell surface

Conclusion: Our results indicated that selected aptamers may turn out to be idealcandidates for the development of a detection tool and also can be used in targeteddrug delivery for future smart drugs