Direct In Vitro Selection of Hemin-Binding DNA Aptamer with Peroxidase Activity

Abstract

A novel hemin-binding DNA was synthesized using in vitro selection (SELEX) method. The selected DNA not only bound to the hemin, but also had peroxidase activity when complexed with hemin. A pool of 104-nt single-stranded DNA (ssDNA) molecules containing a randomized sequence of 60 nt was synthesized. The DNA pool having random sequences was incubated with a hemin-immobilized affinity column. Bound DNAs were eluted with hemin solution and amplified by PCR with biotin-labeled primers. ssDNAs were isolated from the biotin-labeled double-stranded DNA (dsDNA) molecules after each selection process. After four rounds of selection process, the selected DNAs were cloned and sequenced. Four DNA aptamers were chemically synthesized from the sequenced clones. Two aptamers exhibited binding affinity to hemin and peroxidase activity. A 21-nt oligonucleotide was designed from the aptamer sequence that formed a complex with hemin and exhibited high peroxidase activity.

Prediction of aptamer-protein interacting pairs using an ensemble classifier in combination with various protein sequence attributes

“…Figure 1 depicts the structures of two aptamers binding to specific targets. With a deeper understanding of aptamers in terms of their conformational and protein-binding properties, aptamer-protein interacting pairs have a potential to perform a variety of functions [ 8 , 9 ]. Aptamers exhibit significant advantages over antibodies in flexibility of selection, chemical stability [ 4 ], and post-modifications [ 10 ].…”

Recent advances in electrochemical biosensing schemes using graphene and graphene-based nanocomposites

“…For the former, they consist of short strands of oligonucleotides known as DNA or RNA with robust binding affinities for different target targets [168]. In some cases, DNA and RNA aptamers have been selected for the same target, e.g., human immunodeficiency virus trans-acting responsive element [169], dopamine [170], lysozyme [171], thrombin [172], hemin [173], interferon c [174], vascular endothelial growth factor [175], prostate specific antigen [176], etc.…”

Expansion of the aptamer library from a “natural soup” to an “unnatural soup”

“…An aptamer-based biosensor typically comprises two basic elements: a biological recognition element (an aptamer) and a transducer element. In recent years, several promising transducers have been reported including fluorescence detection, [61][62][63][64] chemiluminescence detection, [65][66][67] colorimetric detection, [68][69][70] and electrochemical detection. [71][72][73] There are several outstanding reviews of this application.…”