Electrochemical Quantitation of a Repeat Expansion Sequence (GGGGCC/CCCCGG) in Length-Mismatched Duplexes
Authors: Taki Motahareh , Gagnon Keith , Shamsi Mohtashim 
Published: February 2020

Repeat expansion disorders are a growing class of neurological diseases that are currently incurable and estimated to be 1 in 2,000 individuals worldwide. c9FTD/ALS is one of such classes, caused by a GGGGCC/CCCCGG hexanucleotide repeat expansion in the first intron of a gene called C9ORF72.
Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are thought to be members of c9FTD/ALS [1]. Laboratory testing may take several days for diagnosis, therefore a cost-effective and rapid detection method is urgently needed at early signs of neurological decline. Electrochemical methods have been extensively employed as rapid and sensitive tool to detect DNA hybridization for matched and mismatched sequences. Surface density of DNA on electrode surface plays a critical role in a sensor’s sensitivity, which can be affected by length mismatch between a target and its probe sequence. This situation may occur while detecting repeat expansions in a target RNA/DNA sequence. To best of our knowledge, the sensitivity of solution-based versus surface-bound DNA hybridization between a short target and a longer probe sequences has not been investigated by electrochemical methods before. Here, we report chronocoulometric study to compare solution versus surface hybridization sensitivity on gold electrode surfaces. Specifically, for solution hybridization method, an 18-mer ss-DNA (5′-HS-C6H12 -ATT AGA CCC CGG AGA TTA-3′) was hybridized with a hexanucleotide GC-rich complementary sequence (5′-CCG GGG-3′) in buffer solution followed by surface immobilization of the ds-DNA helix on gold macroelectrodes. For surface hybridization method, gold macroelectrodes were modified with single-stranded probe DNA (ss- DNA) followed by hybridization on surface by incubating the modified electrodes with the complementary sequence. Then, ds-DNA surface densities were measured using chronocoulometry in absence and presence of redox probe, [Ru(NH3)6]3+. Based on previous report [2], the number of phosphate residues and therefore the surface density of the oligonucleotide strands can be determined by electrostatic binding of the redox probe to the anionic DNA phosphate backbone. The DNA surface density was determined to be (3.84 ± 1.1)×1012 molecules/cm2 for surface hybridized ds-DNA versus (2.15 ± 1.1)×1012 molecules/cm2 for solution hybridized ds-DNA. The higher surface density by surface-bound hybridization was also confirmed by cyclic voltammetry that show lower current response for surface-bound hybridization compared to solution-hybridized immobilization. In conclusion, even though solution hybridization has been reported efficient compared to surface-bound hybridization, however, surface-bound hybridization favors higher ds-DNA surface density that may lead to higher sensitivity at very low sample concentrations.

1. Bible, E., Neurodegenerative disease: Researchers identify the protein in c9FTD/ALS inclusions. Nat Rev Neurol, 2013. 9(4): p. 183-183.

2. Steel, A.B., Herne, T.M., and Tarlov, M.J., Electrochemical Quantitation of DNA Immobilized on Gold. Analytical Chemistry, 1998. 70(22): p. 4670-4677.