Solid-State Nanopore Analysis of Diverse DNA Base Modifications Using a Modular Enzymatic Labeling Process

Abstract

Many regulated epigenetic elements and base lesions found in genomic DNA can both directly impact gene expression and play a role in disease processes. However, due to their non-canonical nature, they are challenging to assess with conventional technologies. Here, we present a new approach for the targeted detection of diverse modified bases in DNA. We first use enzymatic components of the DNA base excision repair pathway to install an individual affinity label at each location of a selected modified base with high yield. We then probe the resulting material with a solid-state nanopore assay capable of discriminating labeled DNA from unlabeled. The technique features exceptional modularity via selection of targeting enzymes, which we establish through the detection of four DNA base elements: uracil, 8-oxoguanine, T:G mismatch, and the methyladenine analog 1,N6-ethenoadenine. Our results demonstrate the potential for quantitative nanopore assessment of a broad range of base modifications.

Modular affinity-labeling of the cytosine demethylation base elements in DNA

“…non-displacing) polymerase and a biotinylated dNTP are used to replace the excised base, yielding an affinity tag located at the precise location of the target base modification. In previous work by us 23 and others 24 , efficacy was demonstrated for a variety of bases that included uracil, 8-oxoguanine, and the methyladenine analog 1,N 6 -ethenoadenine. Here, we demonstrate a series of adaptations to enable recognition of all four elements of the cytosine demethylation pathway as well, including 5mC, 5hmC, 5fC, and 5caC.…”

“…We recently 23 reported on a general methodology for labeling single base modifications in DNA using elements of the BER pathway ( Fig. 2a).…”

“…Figure 2b-e shows the results of the sequential process for both of the modifications, as demonstrated by a denaturing gel that follows the single DNA strand featuring the 5′ fluorescent label. The initial 34 nt construct (lane 1) is first exposed to WT TDG glycosylase, along with AP endonuclease 1 (APE1 mutant D308A 25 with reduced exonuclease activity) to displace the glycosylase 23 , which is known to bind tightly to the DNA substrate 26 . After a subsequent treatment with EndoIV to nick the DNA 5′ to the remnant AP site, we observe a shorter 21 nt product (lane 2), consistent with the position of the modification at base 22.…”

Mapping shifts in nanopore signal to changes in protein and protein-DNA conformation

“…Hydroxymethylcytosine was thus detected in fragmented mouse genomic DNA following addition of a biotin linker to 5-methylcytosine sites and subsequent incubation with MSA. (13) A biotinylated DNA-MSA approach was also used to identify base modifications (14) and to quantify nanomolar levels of MSA bound to 7.2 kb dsDNA. (15) More recently, MSA acted as a critical component in a scheme to detect single nucleotide polymorphism, (16) and to map the positions of short sequences in longer dsDNA.…”

“…(19) Nanopore sensing of DNA-streptavidin complexes, however, is commonly performed over a range of pore sizes (7 to 9 nm) and under higher applied voltage (e.g., 600 mV). (11,14,15) Here, we report the behavior of this versatile protein and its DNA complex under varying experimental conditions, including salt concentration (0.5 to 4 M), salt type (KCl, LiCl, NaCl) and voltage (200-600 mV). We develop a simple geometric model that is broadly applicable and discuss the proposed physical origins of various translocation signals.…”