Restriction Enzyme Analysis of Double-Stranded DNA on Pristine Single-Walled Carbon Nanotubes

Abstract

Nanoprobes such as single-walled carbon nanotubes (SWCNTs) are capable of label-free detection that benefits from intrinsic and photostable near-infrared fluorescence. Despite the growing number of SWCNT-based applications, uncertainty surrounding the nature of doublestranded DNA (dsDNA) immobilization on pristine SWCNTs has limited their use as optical sensors for probing DNA−protein interactions. To address this limitation, we study enzyme activity on unmodified dsDNA strands immobilized on pristine SWCNTs. Restriction enzyme activity on various dsDNA sequences was used to verify the retention of the dsDNA's native conformation on the nanotube surface and to quantitatively compare the degree of dsDNA accessibility. We report a 2.8-fold enhancement in initial enzyme activity in the presence of surfactants. Forster resonance electron transfer (FRET) analysis attributes this enhancement to increased dsDNA displacement from the SWCNT surface. Furthermore, the accessibility of native dsDNA was found to vary with DNA configuration and the spacing between the restriction site and the nanotube surface, with a minimum spacing of four base pairs (bp) from the anchoring site needed to preserve enzyme activity. Molecular dynamics (MD) simulations verify that the anchored dsDNA remains within the vicinity of the SWCNT, revealing an unprecedented bimodal displacement of the bp nearest to SWCNT surface. Together, these findings illustrate the successful immobilization of native dsDNA on pristine SWCNTs, offering a new near-infrared platform for exploring vital DNA processes.

Fluorescent Single-Walled Carbon Nanotubes for Protein Detection

“…Single-walled carbon nanotubes functionalized with nucleic acids or peptides form stable complexes, even in complex biological environments [99][100][101][102][103][104][105], with increased thermal stability up to 200 • C [105]. Moreover, SWCNTs functionalized with DNA sequences containing an endonuclease recognition site have been successfully used to study restriction enzyme activity by monitoring their fluorescent emissions [106]. The DNA-SWCNTs have shown increased fluorescence intensity in response to neurotransmitters and have successfully detected dopamine efflux in neuroprogenitor cell cultures [107][108][109][110][111] and in acute brain slices [112,113].…”

Non-covalent Methods of Engineering Optical Sensors Based on Single-Walled Carbon Nanotubes

“…DNA is one of the most extensively studied wrappings for optical sensing applications based on Raman, fluorescence, and absorption spectroscopies (Zheng et al, 2003a; Heller et al, 2006; Enyashin et al, 2007; Zhang et al, 2011; Bansal et al, 2013; Kupis-Rozmysłowicz et al, 2016; Wu et al, 2018). The non-covalent functionalization of ssDNA is based on π-stacking of the aromatic nucleotide bases with the sp 2 -hybridized side-wall of carbon nanotubes (Zheng et al, 2003a).…”

“…In addition to enabling solubilization and fluorescence, surface functionalization can also modify the nanotube surface to promote selective interactions with particular analytes of interest (Figure 1). The underlying mechanism for this selectivity depends on the wrapping and remains an ongoing area of research for many wrappings (Jeng et al, 2006; Hertel et al, 2010; Bisker et al, 2016; Polo and Kruss, 2016; Antonucci et al, 2017; Kruss et al, 2017; Mann et al, 2017; Zubkovs et al, 2017; Gillen et al, 2018; Wu et al, 2018; Lambert et al, 2019).…”

A simple micropreparative gel electrophoresis technique for purification of proteins, nucleic acids, and bioconjugates

“…The pore size in polyacrylamide gel (PAG) can be precisely controlled to separate biomolecules of a particular size with no need to use specialized equipment or columns for different size ranges, as performed in chromatography systems. In fact, PAGE serves as the gold standard for separating DNA with a single base pair resolution [12,13]. Due to its high resolution, low cost, and facile and rapid separation, PAGE is considered a standard and widespread analytical separation technique that can be found in most biological laboratories.…”