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Characterization of DNA aptamers generated against the soft-shelled turtle iridovirus with antiviral effects.

Li P, Zhou L, Yu Y, Yang M, Ni S, Wei S, Qin Q - BMC Vet. Res. (2015)

Bottom Line: Soft-shelled turtle iridovirus (STIV) causes severe systemic disease in farmed soft-shelled turtles (Trionyx sinensis).Electrophoretic mobility shift assays and fluorescent localization showed that the selected aptamers had high binding affinity for STIV.Aptamer QA-36 had the highest calculated binding affinity (K d ) of 53.8 nM.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou, 510301, China.

ABSTRACT

Background: Soft-shelled turtle iridovirus (STIV) causes severe systemic disease in farmed soft-shelled turtles (Trionyx sinensis). More efficient methods of controlling and detecting STIV infections are urgently needed. 

Methods: In this study, we generated eight single-stranded DNA (ssDNA) aptamers against STIV using systematic evolution of ligands by exponential enrichment (SELEX).

Results: The aptamers formed representative stem-loop secondary structures. Electrophoretic mobility shift assays and fluorescent localization showed that the selected aptamers had high binding affinity for STIV. Aptamer QA-36 had the highest calculated binding affinity (K d ) of 53.8 nM. Flow cytometry and fluorescence microscopy of cell-aptamer interactions demonstrated that QA-12 was able to recognize both STIV-infected cells and tissues with a high level of specificity. Moreover, the selected aptamers inhibited STIV infection in vitro and in vivo, with aptamer QA-36 demonstrating the greatest protective effect against STIV and inhibiting STIV infection in a dose-dependent manner.

Discussion: We generated DNA aptamers that bound STIV with a high level of specificity, providing an alternative means for investigating STIV pathogenesis, drug development, and medical therapies for STIV infection.

Conclusions: These DNA aptamers may thus be suitable antiviral candidates for the control of STIV infections.

No MeSH data available.


Related in: MedlinePlus

SELEX isolated eight aptamers that specifically targeted STIV particles. a Binding to STIV was enhanced in the higher selected pools, as demonstrated by ELISA. b Cluster analysis of the selected aptamers. c Secondary structures of aptamers QA-36, QA-92, QA-9, and QA-12. The stabilities of aptamer secondary structures were calculated as the free energy (ΔG)
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Fig1: SELEX isolated eight aptamers that specifically targeted STIV particles. a Binding to STIV was enhanced in the higher selected pools, as demonstrated by ELISA. b Cluster analysis of the selected aptamers. c Secondary structures of aptamers QA-36, QA-92, QA-9, and QA-12. The stabilities of aptamer secondary structures were calculated as the free energy (ΔG)

Mentions: Specific enrichment of each selected pool was monitored by ELISA. ssDNA binding increased as the selection cycles progressed, confirming that the selected pools were specific to STIV. ssDNA binding to STIV peaked at the 8th pool (Figure 1a), and eight ssDNA aptamers were isolated from the 8th pool (Table 1) based on their highly-specific STIV binding. We did an alignment of the selected aptamers, the consensus regions within the individual aptamers were also shown in Table 1, all aptamers held the specific motifs of “-GG-------------------------G----------T-”. The QA-92 aptamer comprised 26 % of the aptamer pool, while the other aptamers were less prevalent in the pool. Cluster analysis showed that QA-92 was a single clade that did not cluster with the other seven aptamers (Fig. 1b). QA-92, QA-9, QA-12 and QA-36 comprised 88 % of the aptamer pool and were selected for further analysis.Fig. 1


Characterization of DNA aptamers generated against the soft-shelled turtle iridovirus with antiviral effects.

Li P, Zhou L, Yu Y, Yang M, Ni S, Wei S, Qin Q - BMC Vet. Res. (2015)

SELEX isolated eight aptamers that specifically targeted STIV particles. a Binding to STIV was enhanced in the higher selected pools, as demonstrated by ELISA. b Cluster analysis of the selected aptamers. c Secondary structures of aptamers QA-36, QA-92, QA-9, and QA-12. The stabilities of aptamer secondary structures were calculated as the free energy (ΔG)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4588899&req=5

Fig1: SELEX isolated eight aptamers that specifically targeted STIV particles. a Binding to STIV was enhanced in the higher selected pools, as demonstrated by ELISA. b Cluster analysis of the selected aptamers. c Secondary structures of aptamers QA-36, QA-92, QA-9, and QA-12. The stabilities of aptamer secondary structures were calculated as the free energy (ΔG)
Mentions: Specific enrichment of each selected pool was monitored by ELISA. ssDNA binding increased as the selection cycles progressed, confirming that the selected pools were specific to STIV. ssDNA binding to STIV peaked at the 8th pool (Figure 1a), and eight ssDNA aptamers were isolated from the 8th pool (Table 1) based on their highly-specific STIV binding. We did an alignment of the selected aptamers, the consensus regions within the individual aptamers were also shown in Table 1, all aptamers held the specific motifs of “-GG-------------------------G----------T-”. The QA-92 aptamer comprised 26 % of the aptamer pool, while the other aptamers were less prevalent in the pool. Cluster analysis showed that QA-92 was a single clade that did not cluster with the other seven aptamers (Fig. 1b). QA-92, QA-9, QA-12 and QA-36 comprised 88 % of the aptamer pool and were selected for further analysis.Fig. 1

Bottom Line: Soft-shelled turtle iridovirus (STIV) causes severe systemic disease in farmed soft-shelled turtles (Trionyx sinensis).Electrophoretic mobility shift assays and fluorescent localization showed that the selected aptamers had high binding affinity for STIV.Aptamer QA-36 had the highest calculated binding affinity (K d ) of 53.8 nM.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou, 510301, China.

ABSTRACT

Background: Soft-shelled turtle iridovirus (STIV) causes severe systemic disease in farmed soft-shelled turtles (Trionyx sinensis). More efficient methods of controlling and detecting STIV infections are urgently needed. 

Methods: In this study, we generated eight single-stranded DNA (ssDNA) aptamers against STIV using systematic evolution of ligands by exponential enrichment (SELEX).

Results: The aptamers formed representative stem-loop secondary structures. Electrophoretic mobility shift assays and fluorescent localization showed that the selected aptamers had high binding affinity for STIV. Aptamer QA-36 had the highest calculated binding affinity (K d ) of 53.8 nM. Flow cytometry and fluorescence microscopy of cell-aptamer interactions demonstrated that QA-12 was able to recognize both STIV-infected cells and tissues with a high level of specificity. Moreover, the selected aptamers inhibited STIV infection in vitro and in vivo, with aptamer QA-36 demonstrating the greatest protective effect against STIV and inhibiting STIV infection in a dose-dependent manner.

Discussion: We generated DNA aptamers that bound STIV with a high level of specificity, providing an alternative means for investigating STIV pathogenesis, drug development, and medical therapies for STIV infection.

Conclusions: These DNA aptamers may thus be suitable antiviral candidates for the control of STIV infections.

No MeSH data available.


Related in: MedlinePlus