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Single molecule electrical recording and fluorescence-force spectroscopy

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Many flavors of single molecule techniques

Force

Electrical

Fluorescence

In silico

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All-atom molecular dynamics (MD) simulations:

the computational microscope

Atoms move according to

classical mechanics (F=ma)

Interaction between atoms is

defined by molecular force field

Massive parallel computer

> 200,000 CPUs

Time scale: ~ 0.1-100 μs

Size scale: 10K - 100M atoms or (< 50 nm)3

Time resolution: 2 fs

Spatial resolution: 0.1 A

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Patch Clamp Technique

Single channel current

The Nobel Prize in Physiology or Medicine 1991�Erwin Neher, Bert Sakmann

for their discoveries concerning "the function of single ion channels in cells".

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MD simulations of α-hemolysin

Instantaneous current

Biophys. J. 88:3745 (2005)

slides from Alek Aksimentiev @UIUC

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Current-voltage curve of α-hemolysin

Instantaneous current

Ionic current at 120mV:

Experiment: 120 pA

Simulations: 130 pA

Biophys. J. 88:3745 (2005)

slides from Alek Aksimentiev @UIUC

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The ionic current blockade may reveal

the sequence of the confined nucleotides

Sequencing DNA by measuring ionic current

Nature Reviews Drug Discovery 1, 77-84 (January 2002)

This did not become a reality until ten years later. Why?

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Oxford Nanopore Technologies

MinION: ~800 parallel detection wells

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Read length: up to 100,000 nucleotides

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A pore protein with a narrow constriction

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DNA helicase

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Accuracy: 96%

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NANOPORE SEQUENCING

https://nanoporetech.com/applications/dna-nanopore-sequencing

  • Most other sequencing methods (Sanger, Illumina, PacBio etc) use sequencing by synthesis
  • Nanopore sequencing here uses sequencing by translocation

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Need for measuring multiple observables

  • Traditionally, measure one quantity from a single molecule or one number as a function of time
    • Ion channel: current vs time
    • Magnetic tweezers: extension vs time
    • FRET: distance vs time
  • But, biology is complex and measuring a single observable may not give us enough information

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Hybrid single-molecule techniques

Fluorescence

Force

Electrical

In silico

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Myosin

Power-stroke

Fluorescent

ATP

Time (s)

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optical tweezers

measure conformational change via fluorescence

as a function of applied force.

prior works: Yanagida, Kinosita, Block, Lang, Liphardt, …

single molecule FRET

Ha,…, Selvin, Weiss PNAS (1996)

Sungchul Hohng et al, science (2007)

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  • 3’ to 5’ DNA helicases
  • a poor unwinder

Rep/UvrD/PcrA helicases

-what is the functional form?

-closed vs open

-how is unwinding regulated?

closed

open

Comstock et al, nature methods (2011)

Comstock et al, science (2015)

FRET

velocity (bp/s)

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Single-Molecule Fluorescence

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Single-Molecule Fluorescence

Introduce kymograph in a separate slide

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C-Trap G2

Force-Fluorescence Correlative Measurements

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Laminar flow microfluidics

  • Flow in all the reagents and move between them
  • Construct the single-molecule assay directly in situ
  • Speed, ease and reproducibility

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CONFIDENTIAL

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CONFIDENTIAL

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kymograph