Detection of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Using Aptamers as Probes in Affinity Capillary Electrophoresis

Abstract

An affinity capillary electrophoresis/laser-induced fluorescence (CE/LIF) assay was developed for direct and specific detection of reverse transcriptase (RI) of the type 1 human immunodeficiency virus (HIV-1) using fluorescently labeled single-stranded DNA aptamers as probes. The aptamer used (RT 26) is specific for HIV-1 RT, and it exhibited no cross-reactivity with RTs of the enhanced avian myeloblastosis virus (AMV), the Moloney murine leukemia virus (MMLV), or denatured HIV-1 RT. An affinity complex of RT 26-HIV-1 RT was readily formed, and calibration curves were linear up to 50 nM (6 microg/mL) HIV-1 RT concentration, with both the free probe and complex peak usable for analytical quantitation. Cell culture media (RPMI with 10% fetal bovine serum) interfered with the assay and aptamer-HIV-1 RT binding. Nonspecific binding was observed in low or undiluted culture, necessitating at least 100-fold dilution for analysis of raw culture samples.

Affinity capillary electrophoresis with laser induced fluorescence detection for thrombin analysis using nuclease-resistant RNA aptamers

“…When HEPES (20 mM, pH 7.4) solution was used as sample buffer in CE analysis of thrombin with Toggle-25-TMR, poor CE separation was observed [7,15]. We chose Tris-HCl as sample buffer in our experiments as previous work showed this condition allowed for good separation of aptamer and aptamerprotein complex [8,16,18,19,24]. We then investigated the effects of metal ions in sample buffer (10 mM Tris-HCl, pH 7.5) on the performance of separation of complex of thrombin and Toggle-25-TMR.…”

“…Aptamer affinity capillary electrophoresis applies aptamers as affinity ligands in CE separation [7,8]. The aptamers are single stranded nucleic acid selected from random library, which can bind with the targets with high binding affinity and selectivity [9,10].…”

“…These properties make aptamer favorable for affinity CE analysis as affinity probe. In aptamer affinity CE-LIF assay for proteins [7,8], the aptamer is usually labeled with a fluorescent dye. As the aptamers usually have many negative charges and smaller molecular weight than proteins, the binding of aptamer on proteins with large size and less charged can significantly change the ratio of charge to mass of the labeled aptamer.…”

“…Therefore, the aptamerprotein complex and the free aptamer has great differences in electrophoretic mobility, enabling the easy separation of aptamerprotein complex from the unbound aptamers in CE [13]. By using aptamer affinity CE-LIF, a few proteins and peptides have been successfully detected [7,8,[14][15][16][17][18][19][20][21][22][23][24], including thrombin [7,[14][15][16][17][18]24], immunoglobin E (IgE) [7,15,16], platelet derived growth factor BB (PDGF-BB) and its isomers [16,19], human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) [8,16,20], ricin [21], human erythropoietin-␣ [22], glucagon [23], amylin [23], and etc. Detection of multiple proteins has been achieved with aptamers in one CE separation [16].…”

Aptamer-based therapeutics and their potential in radiopharmaceutical design

“…Aptamers have been selected for various purposes, making use of their high specificity, versatility and affinity in target recognition. Examples include aptamers that recognize proteins, peptides, dyes, amino acids, nucleotides, and drugs among others, to act as biosensors (Potyrailo et al, 1998), probes (Pavski and Le, 2001) and anti-clotting agents (Bock et al, 1992), whilst the first aptamer has made it to the market gaining FDA approval.…”