Targeting Yellow-Fever Virus: Development of a specific aptamer to NS1 protein

Yellow Fever Virus (YFV), a mosquito-borne flavivirus, remains a significant public health concern despite the availability of effective vaccines. Accurate differential diagnosis continues to be challenging due to the high antigenic similarity among flaviviruses such as dengue and Zika, which compromises the specificity of current serological assays. Aptamers have emerged as promising alternatives to antibodies for diagnostic applications because of their high specificity, thermal stability, and ease of synthesis. In this study, we developed a DNA aptamer (Flav5) targeting the nonstructural protein 1 (NS1) of YFV using capillary electrophoresis-based SELEX (CE-SELEX). After three selection rounds, aptamer pools were sequenced and analyzed. Specificity assays demonstrated minimal cross-reactivity of Flav5 with NS1 proteins from dengue virus (serotypes 1-4) and Zika virus. The aptamer exhibited a high binding affinity for YFV-NS1, with a dissociation constant (Kd) of 34.5 ± 8.2 nM, as determined by quantitative PCR. The three-dimensional structure of Flav5 was modeled and docked to the tetrameric YFV-NS1 using the HDOCK server, revealing a stable binding interface within the inter-subunit cavity of NS1, supported by high confidence scores (>0.95). The Flav5 aptamer demonstrates strong potential for incorporation into YFV-specific diagnostic platforms, particularly in regions where multiple clinically relevant flaviviruses co-circulate. Its combination of high affinity, specificity, and favorable molecular docking characteristics, position it as a promising candidate for point-of-care diagnostic tools.