Evidência: Biociências, Saúde e Inovação - ISSN: 1519-5287 | eISSN 2236-6059 DOI: https://doi.org/10.18593/evid.34577
1
Saúde
Alessandra Nunes Duarte Miranda1, Mariane Izabella Abreu de Melo1, Camila Maria de Sousa Lacerda1, Antero Silva Ribeiro de Andrade1
1 Centro de Desenvolvimento da Tecnologia Nuclear (CDTN), Programa de Pós-Graduação em Ciência e Tecnologia das Radiações, Minerais e Materiais – Belo Horizonte, MG, Brazil.
Miranda, A. N. D. alessandranunes@hotmail.com https://orcid.org/0009-0005-8559-0293
Melo, M. I. A. de* abreu.mariane@gmail.com https://orcid.org/0000-0001-7150-9435
Lacerda, C. M. de S. cmslacerda@gmail.com https://orcid.org/0000-0003-4522-3737
Andrade, A. S. R. de antero@cdtn.br
https://orcid.org/ 0000-0003-0562-1757
* Corresponding author: Av. Pres. Antônio Carlos, 6627 – S/N – Pampulha, Belo Horizonte – MG, 31270-901.
Abstract: The clinically important viruses in the genus Flavivirus include Zika virus (ZIKV), dengue virus (DENV), and yellow fever virus (YFV). Accurately diagnosing flaviviral infection is challenging, especially in regions where multiple flaviviruses co-circulate. Flavivirus nonstructural protein 1 (NS1) is a glycoprotein of approximately 48 kDa whose presence in patient sera is an early marker of infection. The aim of this study was to select a capture aptamer for flavivirus NS1 proteins capable of binding with high affinity to the NS1 in all medically important flaviviruses, with the goal of applying it in aptamer-based diagnostic assays. Aptamer selection was performed using a variation of the SELEX technique, termed CESELEX, which uses capillary electrophoresis (CE) in the separation step. ssDNA sequences obtained after the third CESELEX cycle were sequenced. Quantitative real-time polymerase chain reaction (qPCR) was used to quantify DNA in affinity and specificity assays. An aptamer (Flav8) with high affinity (Kd = 3.58 nM ± 0.22) was obtained that could bind to the NS1 proteins of YFV, ZIKV, and DENV (serotypes 1, 2, 3, and 4) equally. The Flav8 aptamer, owing to its high affinity and ability to recognize the NS1 of all clinically important flaviviruses, is suitable as a capture aptamer for use in Enzyme-Linked Oligonucleotide Assay (ELONA) sandwiches or similar assays.
Recebido: 26/02/2024 | Aceito: 29/04/2024 | Publicado: 11/12/2025
Editor: Marcos Freitas Cordeiro
Evidência, 2025, v. 25, p. 1-10
https://periodicos.unoesc.edu.br/evidencia
CC BY-NC 4.0
Mosquito-transmitted viruses pose significant public health problems in several subtropical and tropical countries. Fifty-three virus species that are transmitted by mosquitoes belong to the genus Flavivirus. Among them, clinically important viruses in this genus include Zika virus (ZIKV), dengue virus (DENV), and yellow fever virus (Hitakarun et al., 2020).
In Brazil, the diagnosis of flaviviral infection is challenging because more than one flavivirus cocirculates and vaccination against YFV is extensive. During the initial stages and mild and medium phases of symptoms, DENV, ZIKV, and YFV infections exhibit very similar clinical presentations, including headaches, rash, fever, and fatigue, making it challenging to distinguish between them. Moreover, traditional serological diagnostic approaches also show relatively high antigenic cross-reactivity. Molecular techniques have been successfully applied for diagnosing flaviviral infections, offering advantages such as speed, sensitivity, and virus-specific identification. However, these techniques require professional expertise and expensive laboratory equipment and reagents, which are often not available in endemic regions (Moreli & Da Costa, 2013). Therefore, simpler, cheaper, and more accurate diagnostic methods are required.
Nucleic acid aptamers are single-stranded DNA or RNA oligonucleotides that bind target molecules with high affinity and specificity. Aptamers are selected using an in vitro selection process termed Systematic Evolution of Ligands by Exponential Enrichment (SELEX). SELEX is performed using selection cycles comprising target incubation with a random synthetic oligonucleotide library, partitioning, and amplification by polymerase chain reaction (PCR) (Tuerk & Gold, 1990). Usually, 6–15 rounds are reported to enable the selection of the best binders. Their low immunogenicity, batch-to-batch reproducibility, capacity for chemical modification, small size, and low production cost have enabled aptamers to replace antibodies in many applications. Consequently, aptamer-based approaches are being extensively investigated for diagnosing several viral diseases (Argondizzo et al., 2020; Chakraborty et al., 2022; Lou et al., 2022; Wandtke et al., 2022).
Flaviviruses are enveloped viruses with a positive-sense RNA genome that encodes seven nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5) and three structural proteins (C, M, and E). Flavivirus nonstructural protein 1 (NS1) is a glycoprotein (with a molecular weight of approximately 48 kDa) found in the inner and outer membranes of infected cells and is secreted into the extracellular space. The presence of NS1 in serum is an early marker of infection in patients with DENV, ZIKV, and YFV (Lee & Zeng, 2017; Mok et al., 2023; Thevendran et al., 2023).
The objective of this study was to select a capture aptamer for flavivirus NS1 proteins capable of binding with high affinity to the NS1 protein of all medically important flaviviruses, with the goal of applying it in aptamer-based diagnostic assays. The capture aptamer functions by attaching the NS1 protein present in a biological sample to a substrate. Following this, another aptamer or antibody capable of distinguishing between the NS1 of different flaviviruses (DENV, ZIKV, and YFV) can be added for differential diagnosis.
Aptamer selection was performed using CESELEX, a variation of the SELEX technique that utilizes capillary electrophoresis (CE) to separate oligonucleotides that bind to the target during the separation step (Mendonsa & Bowser, 2004).
NS1 Protein
Recombinant yellow fever virus NS1 protein expressed in mammalian HEK293 cells (Native Antigen Company, UK) was used.
Oligonucleotide Library Design
A random oligonucleotide library was purchased from IDT DNA Technologies (USA). The 80-mer library sequence (Berezovski et al., 2006) is as follows: 5´-CTTCTGCCCGCCTCCTTCC-(39N)-
GGAGACGAGATAGGCGGACACT-3´, where the central 39N represents the random part of the sequence.
CE-SELEX
Three CE-SELEX selection cycles were performed. Initially, 800 µM of ssDNA was heated at 95 ºC for 5 min, snap-cooled on ice for 15 min, and left at room temperature for 10 min. Subsequently, it was incubated with 80 pmol of YFV NS1 protein in 60 mM of borate buffer (Sigma-Aldrich, USA) for 30 min at 25 ºC under shaking. A sample of the incubation solution was subjected to CE separation using a neutral capillary in a P/ACE MDQ capillary electrophoresis system (Beckman Coulter, Inc., USA). The mixture was separated under reverse polarity (26 kV) in borate buffer at 25 ºC and monitored at 214 nm.
The NS1-aptamer complexes were eluted at different positions in relation to the oligonucleotide library and collected in a vial containing 8 µL of separation buffer. After collecting material from five runs, the bound sequences were amplified using PCR. The PCR amplification was performed using 0.2 µM forward primer (5´-CTTCTGCCCGCCTCCTTCC-3´),
0.2 µM reverse primer (5´-AGTGTCCGCCTATCTCGTCTCC-3´), 1 U Taq DNA polymerase (Invitrogen, USA), 50 mM MgSO4 in 1× PCR buffer (Invitrogen), and a reaction master mix containing 200 µM dNTPs. The PCR thermocycling parameters were as follows: 94 ºC for 2 min for denaturation, followed by 17 cycles of denaturation at 94 ºC for 15 s, annealing at 72 ºC for 30 s (-1 ºC per cycle), and an extension at 68 ºC for 30 s. Subsequently,
cycles were completed, involving denaturation at 94 ºC for
s, annealing at 55 ºC for 30 s, and an extension at 68 ºC for 30 s. Following the last cycle, a final extension step was performed at 68 ºC for 2 min.
The single-stranded DNA of interest was obtained from the conventional PCR products using asymmetric PCR. This was achieved using a Master Mix reaction kit containing 0.2 µM forward primer, 0.02 µM reverse primer, 200 µM dNTPs, 1 U Taq DNA polymerase (Invitrogen), and 50 mM MgSO4 in 1× High Fidelity PCR buffer (Invitrogen). The PCR thermocycling conditions are described above. Conventional and asymmetric PCR were performed using a Veriti Thermal Cycler (Applied Biosystems, USA). PCR amplification was verified by electrophoresis on 2% agarose gels (in Tris-borate-EDTA buffer) stained with ethidium bromide and imaged using a UV transilluminator BioDoc-It® Imaging Systems (Analytik Jena US LLC,USA).
Aptamer Sequencing
The ssDNA sequences obtained after the third cycle of the CE-SELEX procedure were sequenced on an Illumina MiSeq platform (Illumina Inc., USA). Sequencing and bioinformatic analyses were performed using GenXPro GmbH (Germany). Post-sequencing analyses were performed using the Galaxy server available at https://usegalaxy.org/ (Thiel, 2016).
Aptamer similarity analysis and secondary structure prediction
The phylogenetic tree of the selected aptamers was constructed using the Maximum Likelihood method and the Tamura-Nei model (Tamura & Nei, 1993). The initial tree(s) for the heuristic search were obtained automatically by applying the neighbor-joining and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura-Nei model and then selecting the topology with the highest log likelihood value. All analyses were performed using MEGA 11 software under default parameters (Tamura et al., 2021), available at https://www.megasoftware.net/. The predicted secondary structure and Gibbs free energy of the selected aptamers were determined using the Mfold web server, available at http://unafold.rna.albany.edu (Zuker, 2003); under room temperature (25 °C) and 60 mM Na+ conditions.
Equilibrium Dissociation Constant (Kd) Determination
The YFV NS1 protein (40 pmol) was incubated with different aptamer concentrations in 60 mM borate buffer at 25 ºC for 30 min under shaking. The sequences bound to the target were separated from the unbound sequences through filtration using Amicon 0.5 mL Filters for DNA Purification and Concentration – 50K (Merck, Germany), according to the manufacturer’s instructions (Ramos et al., 2007).
The amount of bound sequences in the samples was quantified by qPCR using Go-Taq® qPCR Master Mix (Promega, USA), according to the manufacturer’s instructions. The amplification parameters were as follows: 95 ºC for 2 min for
denaturation, followed by 40 cycles of denaturation at 94 ºC for 15 s and annealing at 60 ºC for 1 min, and a final step at 95 °C for 15 s, followed by the melting stage. The reactions were performed using a qTOWER3 Real-time Thermal Cycler (Analytik Jena, Germany). A standard curve was constructed for each aptamer ranging from 0.05 ng to 0.00005 ng, with a dilution factor of 1:50. The samples were processed in triplicates.
The aptamer mass (ng) in each elution sample was calculated. The results were used to plot saturation curves. Data were analyzed by nonlinear regression analysis (one-site-specific binding with Hill slope) using GraphPad Prism 5 software (GraphPad Software, USA).
Specificity Assay
50 nM of the aptamer in the selection buffer was incubated with 30 pmol of each NS1 protein (DENV serotypes 1, 2, 3, 4, YFV, or ZIKV) in 60 mM borate buffer for 30 min, under shaking, at 25 ºC. Bound and unbound sequences were separated as previously described by filtration using
Figure 1
Illustrative electropherogram of CE-SELEX separation
Amicon 0.5 mL filters. The number of bound sequences in the samples was quantified using qPCR, as previously described.
CE-SELEX was used for aptamer selection in the present study. An illustrative CE electropherogram is shown in Fig. 1. The elution conditions used allowed the separation of peaks corresponding to oligonucleotides that did not bind to the target (as indicated by peak B) and NS1-aptamer complexes (as indicated by peak A), allowing them to be collected individually. Four runs were performed to accumulate oligonucleotides for the next PCR amplification step, and these runs were overlaid and presented in Figure 1. After the third selection cycle, the obtained ssDNA pools were sequenced.
Figure 2 shows a phylogenetic tree of the 27 most prevalent sequences obtained during the sequencing step. Seven sequences were chosen for further characterization based on the number of reads obtained in the sequencing, the lowest ΔG value at 25 °C, and their distance in the phylogenetic tree.
Note: Peak A corresponds to the NS1-aptamer complexes, while peak B represents oligonucleotides that did not bind to the target. Four runs, displayed using distinctly colored lines, were performed to accumulate sufficient DNA for PCR amplification. The runs are overlaid on the graph. The x axis on the graph represents minutes, while the y axis represents absorbance units (A.U.)
Figure 2
Phylogenetic tree of selected aptamers
Note: The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 27 nucleotide sequences. A total of 81 positions were used in the final dataset. The chosen sequences are highlighted in boxes. Evolutionary analyses were conducted using MEGA 11 software. (*Number of reads, **Gibbs free energy of the secondary structure).
The affinity of the aptamer for the target is represented by the equilibrium dissociation constant (Kd). The dissociation constants of the seven selected aptamers were calculated using saturation binding assays. Specific binding curves representing the quantity (ng) of aptamers bound to the NS1 protein relative to the corresponding aptamer concentration are shown in Figure 3. For Flav3 (C), it was not possible to
Figure 3
Dissociation constant (Kd) determination of NS1 aptamers
obtain a saturation curve, indicating low or no affinity for the target. Flav8 (D) and Flav10 (E) aptamers exhibited the best affinity, with Kd values of 3.58 ± 0.22 nM and 4.42 ± 0.19 nM, respectively. The remaining Kd values in ascending order were: 11.01 ± 0.69 nM (Flav2, B), 22.42 ± 2.78 nM (Flav25, G),
76.99 ± 31.1 nM (Flav1, A), and 176.3 ± 25.9 nM (Flav22).
Note: The saturation curve was obtained by plotting the aptamer concentration (nM) versus the total ssDNA (ng) bound to NS1 protein. Protein-bound aptamer was quantified using a quantitative real-time polymerase chain reaction (qPCR). Non-linear regression analysis (one-site-specific binding with hill slope (GraphPad Prism 5) was used to determine the Kd. A) Flav1 (76.99 ± 31.1 nM); B) Flav2 (11.01 ± 0.69 nM); C) Flav3 (undetermined); D) Flav8 (3.58 ± 0.22 nM); E) Flav10 (4.42 ± 0.19 nM); F) Flav22 (176.3 ± 25.9 nM); and G) Flav25 (22.42 ± 2.78 nM).
A specificity assay (Fig. 4) was performed for the aptamer with the highest affinity (Flav8), which revealed that Flav8 could bind equally to the NS1 proteins of DENV (serotypes 1, 2, 3, and 4), ZIKV, and YFV.
Figure 4
Specificity assay
Note: The binding of the Flav8 aptamer to flavivirus NS1 proteins was assessed using qPCR. The graph shows the amount of ssDNA (ng) recovered after incubating 50 nM of Flav8 aptamer with 30 pmol of each protein. Statistical analyses were performed using GraphPad Prism 5 software (**p < 0.01). The control represents the Flav8 aptamer without NS1 protein incubation.
These results demonstrate that Flav8 is suitable as a capture aptamer for use in enzyme-linked oligonucleotide assay (ELONA) sandwiches or similar assays, owing to its high affinity and ability to recognize the NS1 protein of all clinically important flaviviruses. The Flav8 sequence and its predicted secondary structure are shown in Figure 5.
Figure 5
The Flav8 sequence and its predicted secondary structure
Note: (A) The Flav8 aptamer sequence with the conserved regions shown in bold. (B) The secondary structure of the Flav8 aptamer predicted using the Mfold server (ΔG = -10.06 kcal/mol).
Nucleic acid aptamers are usually developed in vitro using a molecular evolution process termed SELEX, which is based on iterative selection-amplification steps (Tuerk & Gold, 1990). This methodology involves exposing a chemically synthesized library of random sequences (1014–1015 sequences) to the target molecule, enabling the interaction of all target-binding oligonucleotides. The next step involves separating sequences that bind to the target from the unbound sequences for subsequent PCR amplification. Following this, the PCR products are exposed to the target again. The selection, separation, and amplification steps comprise one SELEX round. After the last round, the PCR products are cloned and sequenced to identify the binding sequences (Wang et al., 2019).
CE-SELEX is a powerful SELEX variant in which capillary electrophoresis (CE) is used to separate the oligonucleotides that bind to a target from free oligonucleotides (Mendonsa & Bowser, 2004) based on the difference in mobility between these molecules under specific conditions. CE separations
are based on the presence of an electrically induced flow, called electroosmotic flow (EOF). The capillary is filled with a buffer solution and its ends are immersed in the same buffer. An electric field is applied at the ends, generating current inside the capillary with consequent movement of the analytes towards the electrodes. It is essential to standardize CE conditions so that free and bound oligonucleotides elute in separate positions in the electropherogram, allowing their collection for subsequent PCR amplification. To this end, variables such as type of capillary, buffer, pH and polarity (standard or reverse) can be explored. In the present study, the best results were obtained using a neutral capillary associated with reverse polarity in borate buffer.
The CE technique has advantages such as speed, versatility, low cost per analysis, high separation power and minimum consumption of samples and solvents. However, the technique is not suitable for use with non-polar and volatile compounds. During the CE process, there is no steric impairment for the aptamers to bind to the target because the molecules remain in solution throughout the separation (Gao et al., 2020). Moreover, the protein targets retain their native conformations. The main advantage of CE separation is that high-affinity aptamers can be obtained in only three to five selection rounds (Mendonsa & Bowser, 2004). In this study, high-affinity aptamers were obtained using only three CE-SELEX rounds, thus demonstrating their efficiency.
The diagnosis of flaviviral diseases can be difficult due to several factors: presence of clinically asymptomatic or oligosymptomatic disease; clinical similarity with other diseases; phylogenetic cross-reactions that can occur between serological tests (especially in endemic regions) and difficulty in accessing reference laboratories that can perform differential diagnosis. Specificity is one of the main problems related to the diagnosis of flavivirus infection.
In the clinical setting, early diagnosis of flavivirus infection would be more effective for controlling the epidemic and for timely treatment. Reverse transcription PCR (RT-PCR) has become the gold standard for molecular detection of viruses due to its high selectivity and relatively high sensitivity. However, elaborate laboratories and highly qualified specialists are required, available only in large medical centers and central laboratory facilities. RT-PCR has
also some inherent disadvantages, including false-negative results from new strains or false-positive results due to sample contamination. Finally, molecular diagnosis is only possible in the acute phase of the disease (Moreli & Da Costa, 2013). The development of portable reverse transcription polymerase chain reaction (RT-PCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) devices are promising efforts for molecular diagnostics in the field, but applications of these assays remain limited. For these reasons, serological assays continue to be important alternatives for detection, particularly when focusing on field-ready assays (Varghese et al., 2023). However, serological tests are often unable to specifically identify viruses of the same genus, particularly in the case of immunoglobulin G (IgG), not allowing a specific diagnosis.
Antibodies are regularly used in current diagnostic strategies for NS1 detection (Hermann et al., 2014). Compared to antibodies, aptamers have lower production costs, reversible denaturing properties, and are stable at room temperature for long-term storage. In addition, aptamers typically do not exhibit batch-to-batch variation. The drawbacks associated with antibodies have motivated efforts to shift towards NS1 protein-aptamer-based diagnostics. The advantages of aptamers over antibodies allow them to serve as molecular recognition elements in various diagnostic platforms (Argondizzo et al., 2020; Chakraborty et al., 2022; Lou et al., 2022; Wandtke et al., 2022).
ELONA is a bioassay that employs oligonucleotides, such as aptamers, instead of antibodies to quantify a target using conventional plate readers (Drolet et al., 1996). The ELONA sandwich is a variation of this technique that uses a pair of aptamers, one for capture and the other for detection. Essentially, the capture aptamer retains and concentrates the protein target on the plate, and the detection aptamer detects and identifies the immobilized protein (Lee & Zeng, 2017; Mok et al., 2023). Although a similar diagnosis can be performed using the sandwich ELISA (Enzyme-Linked Immunosorbent Assay), once specific antibodies have been obtained, the use of aptamers is more promising for the reasons discussed above.
In a previous study using CE-SELEX, our research group selected the ZIKV60 detection aptamer (Almeida et al.,
2022). ZIKV60 exhibited high binding affinity to the ZIKV NS1 protein (Kd = 2.28 ± 0.28 nM) and could distinguish between ZIKV NS1 and NS1 from DENV (serotypes 1, 2, 3, and 4) and YFV. To achieve this specificity, counter-selection steps (negative selection) were included for NS1 proteins from other flaviviruses. The aim of the present study was to obtain a capture aptamer for use in combination with ZIKV60 in an ELONA sandwich assay for ZIKV diagnosis. The Flav8 aptamer that was successfully obtained met the affinity and specificity requirements for this application. Currently, we are in the process of selecting detection aptamers for YFV NS1 and DENV NS1 (serotypes 1, 2, 3, and 4) for use with Flav8 and ZIKV60 in a single diagnostic test kit capable of distinguishing between all the aforementioned diseases.
CONCLUSION
The present study presents an important step towards the development of a practical diagnostic test for the differential diagnosis of clinically relevant flaviviruses, which is vital for the adequate management of these infectious diseases.
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