Kinetics of T3-DNA Ligase-Catalyzed Phosphodiester Bond Formation Measured Using the α-Hemolysin Nanopore

Tan, Cherie S.; Riedl, Jan; Fleming, Aaron M.; Burrows, Cynthia J.; White, Henry S.

doi:10.1021/acsnano.6b05995

Cited by 20 publications

(20 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Solid-state nanopores were used to fingerprint nucleic acid nanoparticles 24 , and for protein detection and quantitation 25 . α-HL was successfully used for polymer sizing 26 , for metallic nanoparticle characterization 27 , and to measure the kinetics of enzymatic phosphodiester bond formation 1,28 . A modified MspA was shown to measure the movement of enzymes along DNA in real time 29 , wt aerolysin to resolve a series of deoxyriboadenylates 30 , and an engineered Fragaceatoxin C to discriminate among peptides 31 .…”

Section: Introductionmentioning

confidence: 99%

Nanopore device-based fingerprinting of RNA oligos and microRNAs enhanced with an Osmium tag

Sultan¹,

Kanavarioti²

2019

Sci Rep

View full text Add to dashboard Cite

Protein and solid-state nanopores are used for DNA/RNA sequencing as well as for single molecule analysis. We proposed that selective labeling/tagging may improve base-to-base resolution of nucleic acids via nanopores. We have explored one specific tag, the Osmium tetroxide 2,2′-bipyridine (OsBp), which conjugates to pyrimidines and leaves purines intact. Earlier reports using OsBp-tagged oligodeoxyribonucleotides demonstrated proof-of-principle during unassisted voltage-driven translocation via either alpha-Hemolysin or a solid-state nanopore. Here we extend this work to RNA oligos and a third nanopore by employing the MinION, a commercially available device from Oxford Nanopore Technologies (ONT). Conductance measurements demonstrate that the MinION visibly discriminates oligoriboadenylates with sequence A15PyA15, where Py is an OsBp-tagged pyrimidine. Such resolution rivals traditional chromatography, suggesting that nanopore devices could be exploited for the characterization of RNA oligos and microRNAs enhanced by selective labeling. The data also reveal marked discrimination between a single pyrimidine and two consecutive pyrimidines in OsBp-tagged AnPyAn and AnPyPyAn. This observation leads to the conjecture that the MinION/OsBp platform senses a 2-nucleotide sequence, in contrast to the reported 5-nucleotide sequence with native nucleic acids. Such improvement in sensing, enabled by the presence of OsBp, may enhance base-calling accuracy in enzyme-assisted DNA/RNA sequencing.

show abstract

Section: Introductionmentioning

confidence: 99%

Nanopore device-based fingerprinting of RNA oligos and microRNAs enhanced with an Osmium tag

Sultan¹,

Kanavarioti²

2019

Sci Rep

View full text Add to dashboard Cite

show abstract

“…15–18 The α-HL protein nanopore has been harnessed as a nanoreactor to follow the reaction evolution for the click reaction, 19 1,4-Michael additions, 20 and an enzyme-catalyzed reaction on DNA. 15,21 The sensitivity of the α-HL to monitor molecular structure allowed determination of epimers of carbohydrates, 22 amino acid enantiomers, 23 isomers of heteropolytungstates, 24 and diastereomers in duplex DNA. 25 Additionally, the latch zone of the α-HL protein was identified by the Burrows/White laboratories to possess high sensitivity for monitoring base pairs in duplex DNA.…”

Section: Resultsmentioning

confidence: 99%

Nanopore Analysis of the 5-Guanidinohydantoin to Iminoallantoin Isomerization in Duplex DNA

et al. 2018

Self Cite

View full text Add to dashboard Cite

In DNA, guanine oxidation yields diastereomers of 5-guanidinohydantoin (Gh) as one of the major products. In nucleosides and single-stranded DNA, Gh is in a pH-dependent equilibrium with its constitutional isomer iminoallantoin (Ia). Herein, the isomerization reaction between Gh and Ia was monitored in duplex DNA using a protein nanopore by measuring the ionic current when duplex DNA interacts with the pore under an electrophoretic force. Monitoring current levels in this single-molecule method proved to be superior for analysis of population distributions in an equilibrating mixture of four isomers in duplex DNA as a function of pH. The results identified Gh as a major isomer observed when base paired with A, C, or G at pH 6.4-8.4, and Ia was a minor isomer of the reaction mixture that was only observed when the pH was >7.4 in the duplex DNA context. The present results suggest that Gh will be the dominant isomer in duplex DNA under physiological conditions regardless of the base-pairing partner in the duplex.

show abstract

“…2,3,7 Monitoring the current flow through a nanopore under an applied transmembrane potential (voltage) enables the real-time, in situ characterization of the interaction between the pore and other molecular entities: any changes in the recorded current can provide information about the molecular structure of an analyte, [8][9][10][11] the kinetics and dynamics of non-covalent binding, 8,[12][13][14][15] or chemical reactivity. [16][17][18][19][20][21][22][23] Biological nanopores have even served as the scaffold for constructing molecular machines. [24][25][26] Compared to solid-state nanopores, 6,[27][28][29][30] the possibilities for tuning the properties of biological protein pores via chemical modification are more limited.…”

Section: Take Down Policymentioning

confidence: 99%

Synthetically Diversified Protein Nanopores: Resolving Click Reaction Mechanisms

et al. 2019

View full text Add to dashboard Cite

Nanopores are emerging as a powerful tool for the investigation of nanoscale processes at the singlemolecule level. Here, we demonstrate the methionineselective synthetic diversification of α-hemolysin (α-HL) protein nanopores and their exploitation as a platform for investigating reaction mechanisms. A wide range of functionalities, including azides, alkynes, nucleotides, and single-stranded DNA, were incorporated into individual pores in a divergent fashion. The ion currents flowing through the modified pores were used to observe the trajectory of a range of azide−alkyne click reactions and revealed several short-lived intermediates in Cu(I)-catalyzed azide−alkyne [3 + 2] cycloadditions (CuAAC) at the singlemolecule level. Analysis of ion-current fluctuations enabled the populations of species involved in rapidly exchanging equilibria to be determined, facilitating the resolution of several transient intermediates in the CuAAC reaction mechanism. The versatile pore-modification chemistry offers a useful approach for enabling future physical organic investigations of reaction mechanisms at the single-molecule level.

show abstract

Kinetics of T3-DNA Ligase-Catalyzed Phosphodiester Bond Formation Measured Using the α-Hemolysin Nanopore

Cited by 20 publications

References 53 publications

Nanopore device-based fingerprinting of RNA oligos and microRNAs enhanced with an Osmium tag

Nanopore device-based fingerprinting of RNA oligos and microRNAs enhanced with an Osmium tag

Nanopore Analysis of the 5-Guanidinohydantoin to Iminoallantoin Isomerization in Duplex DNA

Synthetically Diversified Protein Nanopores: Resolving Click Reaction Mechanisms

Contact Info

Product

Resources

About