Nucleotide Identification and Orientation Discrimination of DNA Homopolymers Immobilized in a Protein Nanopore

Abstract

Nanopores have been used as extremely sensitive resistive pulse sensors to detect analytes at the molecular level. There has been interest in using such a scheme to rapidly and inexpensively sequence single molecules of DNA. To establish reference current levels for adenine, cytosine, and thymine nucleotides, we measured the blockage currents following immobilization of single-stranded DNA polyadenine, polycytosine, and polythymine within a protein nanopore in chemical orientations in which either the 3' or the 5' end enters the pore. Immobilization resulted in low-noise measurements, yielding sharply defined current distributions for each base that enabled clear discrimination of the nucleotides in both orientations. In addition, we find that not only is the blockage current for each polyhomonucleotide orientation dependent, but also the changes in orientation affect the blockage currents for each base differently. This dependence can affect the ability to resolve polyadenine and polythymine; with the 5' end entering the pore, the separation between polyadenine and polythymine is double that observed for the 3' orientation. This suggests that, for better resolution, DNA should be threaded through the 5' end first in nanopore DNA sequencing experiments.

Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore

“…Recently, individual modified nucleotide bases have been observed ''on the fly'' (19), but these structures were very bulky. When DNA is immobilized within the ␣HL pore, by using a 5Ј-or 3Ј-terminal hairpin or biotin-streptavidin complex, better resolution of homopolymer sequences can be achieved because of the prolonged observation time (13,(28)(29)(30)(31) and in the present work we have extended the biotin-streptavidin approach.…”

“…In this state, the DNAs were captured and immobilized by ␣HL pores in an applied potential, but they were not translocated into the trans compartment (Fig. 1A) (30)(31)(32). The immobilized DNA molecules caused a sequence-dependent decrease in the current flow through the pore (Fig.…”

“…The value for 3Ј threading was similar. Interestingly, Purnell et al (31), using biotin-streptavidin immobilization, found that ⌬I RES depends on whether the 5Ј or 3Ј end of the DNA enters the pore first (5Ј entr y, ⌬I RES poly(dA)Ϫpoly(dC) ϭ ϩ1.2%; 3Ј entry, ⌬I RES poly(dA)Ϫpoly(dC) ϭ Ϫ2.9%). Our results (5Ј entry: ⌬I RES poly(dA)Ϫpoly(dC) ϭ ϩ0.6%) are in approximate agreement with the latter work.…”

Controlling polymer capture and translocation by electrostatic polymer-pore interactions

“…In the rest of the article, we will consider the case of a vanishing pressure gradient ∆P = 0 which yields a vanishing streaming velocity v str = 0 in Eq. (15). We also note that unless otherwise stated, all results will be obtained from the improved Donnan approach of Eqs.…”

Controlling molecular transport through nanopores

“…This allowed determination of the minimum voltage required to keep a DNA -avidin complex trapped in an a-haemolysin nanopore to be around 70 mV [33]. A very similar approach can be used to immobilize a DNA strand and thus identify which of the four bases of DNA is located in the constriction [34,35]. Another successful idea is the introduction of doublestranded DNA molecules into the pore, as indicated by the sketch in figure 1c, since the a-haemolysin nanopore only allows the passage of single-stranded DNA.…”