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High-affinity five/six-letter DNA aptamers with superior specificity enabling the detection of dengue NS1 protein variants beyond the serotype identification.

ABSTRACT: Genetic alphabet expansion of DNA by introducing unnatural bases (UBs), as a fifth letter, dramatically augments the affinities of DNA aptamers that bind to target proteins. To determine whether UB-containing DNA (UB-DNA) aptamers obtained by affinity selection could spontaneously achieve high specificity, we have generated a series of UB-DNA aptamers (KD: 27-182 pM) targeting each of four dengue non-structural protein 1 (DEN-NS1) serotypes. The specificity of each aptamer is remarkably high, and the aptamers can recognize the subtle variants of DEN-NS1 with at least 96.9% amino acid sequence identity, beyond the capability of serotype identification (69-80% sequence identities). Our UB-DNA aptamers specifically identified two major variants of dengue serotype 1 with 10-amino acid differences in the DEN-NS1 protein (352 aa) in Singaporeans' clinical samples. These results suggest that the high-affinity UB-DNA aptamers generated by affinity selection also acquire high target specificity. Intriguingly, one of the aptamers contained two different UBs as fifth and sixth letters, which are essential for the tight binding to the target. These two types of unnatural bases with distinct physicochemical properties profoundly expand the potential of DNA aptamers. Detection methods incorporating the UB-DNA aptamers will facilitate precise diagnoses of viral infections and other diseases.

SUBMITTER: Matsunaga KI

PROVIDER: S-EPMC8599795 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: Early diagnosis of dengue infection is crucial for better management of the disease. Diagnostic tests based on the detection of dengue virus (DENV) Non Structural Protein 1 (NS1) antigen are commercially available with different sensitivities and specificities observed in various settings. Dengue is endemic in Indonesia and clinicians are increasingly using the NS1 detection for dengue confirmation. This study described the performance of Panbio Dengue Early NS1 and IgM Capture ELISA assays for dengue detection during our surveillance in eight cities in Indonesia as well as the genetic diversity of DENV NS1 genes and its relationship with the NS1 detection. METHODS: The NS1 and IgM/IgG ELISA assays were used for screening and confirmation of dengue infection during surveillance in 2010-2012. Collected serum samples (n?=?440) were subjected to RT-PCR and virus isolation, in which 188 samples were confirmed for dengue infection. The positivity of the ELISA assays were correlated with the RT-PCR results to obtain the sensitivity of the assays. The NS1 genes of 48 Indonesian virus isolates were sequenced and their genetic characteristics were studied. RESULTS: Using molecular data as gold standard, the sensitivity of NS1 ELISA assay for samples from Indonesia was 56.4% while IgM ELISA was 73.7%. When both NS1 and IgM results were combined, the sensitivity increased to 89.4%. The NS1 sensitivity varied when correlated with city/geographical origins and DENV serotype, in which the lowest sensitivity was observed for DENV-4 (19.0%). NS1 sensitivity was higher in primary (67.6%) compared to secondary infection (48.2%). The specificity of NS1 assay for non-dengue samples were 100%. The NS1 gene sequence analysis of 48 isolates revealed the presence of polymorphisms of the NS1 genes which apparently did not influence the NS1 sensitivity. CONCLUSIONS: We observed a relatively low sensitivity of NS1 ELISA for dengue detection on RT-PCR-positive dengue samples. The detection rate increased significantly when NS1 data was combined with IgM. In our study, the low sensitivity of NS1 antigen detection did not relate to NS1 genetic diversity. Rather, the performance of the NS1 antigen test was affected by the infection status of patients and geographical origin of samples.

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High-affinity five/six-letter DNA aptamers with superior specificity enabling the detection of dengue NS1 protein variants beyond the serotype identification.

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