Electrochemical Sensing for HIV Integrase Activity Detection Employing G4/Hemin Dnazyme
Authors: Inaba Shintaro , Le Thy , Ikebukuro Kazunori , Benhabbor Rahima , Sode Koji 
Published: December 2024

Introduction; HIV (human immunodeficiency virus) remains a major global public health issue, having claimed 40.4 million [32.9–51.3 million] lives so far with ongoing transmission in all countries globally. The World Health Organization (WHO) recommends a combination prevention approach [1], which uses a mix of biomedical, behavioral, and structural interventions, to have the greatest sustained impact on reducing new infections. Among the various strategies includes pre-exposure prophylaxis (PrEP), where people at risk of contracting HIV take antiretroviral medication in order to reduce the risk of HIV acquisition. To be effective, PrEP requires maintaining high adherence to antiretroviral medications, represented by HIV integrase inhibitors (INSTIs) [2]. Therefore, knowing an individual’s blood INSTI concentration is important to improve their adherence and to adjust individual dosage/administration methods accordingly for effective treatment. Currently, the HIV-1 Integrase assay kit is available, which is based on the monitoring of HIV-integrase DNA strand elongation activity. Using this assay, blood concentration of HIV-integrase inhibitors can be substantially monitored. However, the current procedures are complicated and require specific instruments, which make it difficult to apply for point-of-care testing and personal use. Accordingly, there is a growing interest in exploring alternative analytical methods to detect HIV-integrase activity that offer advantages such as reduced sample volume requirements, simplified procedures, and portability. In this presentation, we report the development of an electrochemical sensor for HIV integrase activity detection employing G4/hemin DNAzyme, focusing on the unique enzymatic reaction of HIV integrase, which will be utilized for the detection of inhibitors as well as the screening of novel inhibitors.

Methods; The sensing principle of this sensor is based on the unique properties of HIV integrase, which recognizes the terminal regions of HIV genomic DNA and can integrase a dsDNA derived from HIV genomic DNA (Donor DNA) with any dsDNA (target DNA) [3]. To detect this activity, we designed target DNA sequence harboring DNAzyme sequence in the 5’ end, which shows peroxidase activity [4]. Accordingly, the amount of target DNA integrated into donor DNA can be monitored by peroxidase activity derived from DNAzyme, which is the subject of the electrochemical monitoring.

Results and Discussion; Firstly, we evaluated the function of hemin aptamer-fused target DNA as a substrate of HIV integrase using fluorescence dye labeled hemin aptamer-fused target DNA. As a result, the fluorescence intensity increased depending on HIV integrase, so hemin aptamer-fused target DNA works as a substrate of HIV integrase. Secondly, we evaluated the peroxidase activity of the complex of hemin aptamer-fused target DNA. As a result, the complex retained peroxidase activity, indicating that the hemin aptamer-fused target DNA/hemin works as DNAzyme. The electrochemical detection of HIV integrase activity based on hemin aptamer-fused target DNA/hemin by chronoamperometry will be presented.

References

[1] World Health Organization, Global HIV Programme, (2024, April 18)

[2] Baeten,J.M., Donnell,D., Ndase,P., Mugo,N.R., Campbell,J.D., Wangisi,J., Tappero,J.W., Bukusi,E.A., Cohen,C.R., Katabira,E., et al. (2012) Antiretroviral prophylaxis for HIV prevention in heterosexual men and women. N. Engl. J. Med., 367, 399–410.

[3] Hazuda,D.J., Hastings,J.C., Wolfe,A.L. and Emini,E.A. (1994) A novel assay for the DNA strand-transfer reaction of HIV-1 integrase. Nucleic Acids Res., 22, 1121–1122.

[4] Travascio,P., Li,Y. and Sen,D. (1998) DNA-enhanced peroxidase activity of a DNA-aptamer-hemin complex. Chem. Biol., 5, 505–517.