Human Cyclophilin B Nuclease Activity Revealed via Nucleic Acid‐Based Electrochemical Sensors

Abstract

Human cyclophilin B (CypB) is oversecreted by pancreatic cancer cells, making it a potential biomarker for early‐stage disease diagnosis. Our group is motivated to develop aptamer‐based assays to measure CypB levels in biofluids. However, human cyclophilins have been postulated to have collateral nuclease activity, which could impede the use of aptamers for CypB detection. To establish if CypB can hydrolyze electrode‐bound nucleic acids, we used ultrasensitive electrochemical sensors to measure CypB's hydrolytic activity. Our sensors use ssDNA and dsDNA in the biologically predominant d‐DNA form, and in the nuclease resistant l‐DNA form. Challenging such sensors with CypB and control proteins, we unequivocally demonstrate that CypB can cleave nucleic acids. To our knowledge, this is the first study to use electrochemical biosensors to reveal the hydrolytic activity of a protein that is not known to be a nuclease. Future development of CypB bioassays will require the use of nuclease‐resistant aptamer sequences.

Explaining the Decay of Nucleic Acid-Based Sensors under Continuous Voltammetric Interrogation

“…14 As model systems, this work uses NBEs functionalized with ssDNA oligos; however, the decay mechanisms revealed here should also affect, to varying extents, NBEs based on duplex DNA 6 or other oligo types. 13,23 Alkylthiol-Modified DNA Monolayers Are Stable for Weeks if Left Undisturbed. We first investigated the stability of freshly fabricated NBEs when stored in phosphate-buffered saline under minimal voltage perturbations for 2 weeks.…”

“…As a complementary technique, we use square wave voltammetry to monitor with high sensitivity changes in electron transfer originating from the loss of reporter-modified oligos from the electrode surface . As model systems, this work uses NBEs functionalized with ssDNA oligos; however, the decay mechanisms revealed here should also affect, to varying extents, NBEs based on duplex DNA or other oligo types. , …”

“…To discard the possibility that NBE signal decay is due to the loss of methylene blue reporters and not to the loss of nucleic acids, we employed the method developed by Steel, Herne, and Tarlov to quantitatively measure surface DNA concentrations. , This method is based on exposing gold surfaces functionalized with nucleic acids to a solution of ruthenium hexamine at low ionic strength. The ruthenium complex is positively charged (3+) and strongly binds to the negatively charged backbone of the nucleic acids (not in the case of PNA).…”

Os(ii/iii) complex supports pH-insensitive electrochemical DNA-based sensing with superior operational stability than the benchmark methylene blue reporter

“…Seventeen years after the first report on this technology, 3 most published E-AB sensors still rely on the use of a single redox reporter which undergoes pH-dependent electron transfer: methylene blue (MB). 14,15…”

“…Seventeen years after the first report on this technology, 3 most published E-AB sensors still rely on the use of a single redox reporter which undergoes pH-dependent electron transfer: methylene blue (MB). 14,15 Alternative redox reporters such as anthraquinone or ferrocene have been used for the fabrication of E-AB sensors; however, these reporters are not suitable for continuous monitoring applications. On the one hand, anthraquinone presents a formal redox potential of E °′ AQ $ À0:50 V vs. Ag/AgCl, 16,17 which overlaps with the reduction of molecular oxygen, making the electrochemical response of this reporter oxygen concentration-dependent.…”