(Re)discovering missing physics in Rosetta, through RNA Rhiju Das Baker lab.

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(Re)discovering missing physics in Rosetta, through RNA Rhiju Das Baker lab

Understanding structure Watson & Crick (1953)

Beyond a single structure? Work with Pehr Harbury and Rebecca Fenn (Stanford) “Remeasuring the Double Helix”

The basic idea Thomas Young (1773-1829)

 = /d d The basic idea Thomas Young (1773-1829)

A new x-ray ruler Sugar coated gold clusters 86 Å = 68 Å + 2 x 9 Å

The double helix as a length standard 15bp 10bp 25bp 20bp 35bp 30bp 15bp 10bp 25bp 20bp 35bp 30bp

The double helix as a length standard Rise per base pair: 3.29±0.07 Å Crystal avg: 3.32±0.19 Å

Wait a minute… 15bp 10bp 25bp 20bp 35bp 30bp Variance in length of 10 bp duplex: 1.4 Å 2 (predicted) 8.5 Å 2 (measured) Variance in length of 35 bp duplex: 4.8 Å 2 (predicted) 51.0 Å 2 (measured)

Wait a minute… 15bp 10bp 25bp 20bp 35bp 30bp Conventional model [elastic rod, S=1000 pN] Best fit elastic rod model Quadratic fit

What does it mean? Uncorrelated (drunken walk) variance  length Correlated (marching in lockstep) variance  length 2

Are you sure? (How a rotation project got stretched 7 years) 1.Statistical errors 2.Sample impurities 3.Multiple DNA’s per gold particle 4.Truncation products from oligo synthesis 5.Perturbations of DNA by gold probes 6.Poorer signal-to-noise in longer constructs 7.End-effects 8.Long-range electrostatics between gold probes

Are you sure? (How a rotation project got stretched 7 years) 1.Statistical errors 2.Sample impurities 3.Multiple DNA’s per gold particle 4.Truncation products from oligo synthesis 5.Perturbations of DNA by gold probes 6.Poorer signal-to-noise in longer constructs 7.End-effects 8.Long-range electrostatics between gold probes

Are you sure? (How a rotation project got stretched 7 years) 1.Statistical errors 2.Sample impurities 3.Multiple DNA’s per gold particle 4.Truncation products from oligo synthesis 5.Perturbations of DNA by gold probes 6.Poorer signal-to-noise in longer constructs 7.End-effects 8.Long-range electrostatics between gold probes

Are you sure? (How a rotation project got stretched 7 years) 1.Statistical errors 2.Sample impurities 3.Multiple DNA’s per gold particle 4.Truncation products from oligo synthesis 5.Perturbations of DNA by gold probes 6.Poorer signal-to-noise in longer constructs 7.End-effects 8.Long-range electrostatics between gold probes

Are you sure? (How a rotation project got stretched 7 years) 1.Statistical errors 2.Sample impurities 3.Multiple DNA’s per gold particle 4.Truncation products from oligo synthesis 5.Perturbations of DNA by gold probes 6.Poorer signal-to-noise in longer constructs 7.End-effects 8.Long-range electrostatics between gold probes

Are you sure? (How a rotation project got stretched 7 years) 1.Statistical errors 2.Sample impurities 3.Multiple DNA’s per gold particle 4.Truncation products from oligo synthesis 5.Perturbations of DNA by gold probes 6.Poorer signal-to-noise in longer constructs 7.End-effects 8.Long-range electrostatics between gold probes

Are you sure? (How a rotation project got stretched 7 years) 1.Statistical errors 2.Sample impurities 3.Multiple DNA’s per gold particle 4.Truncation products from oligo synthesis 5.Perturbations of DNA by gold probes 6.Poorer signal-to-noise in longer constructs 7.End-effects 8.Long-range electrostatics between gold probes 10 mM NaCl 100 mM NaCl 1 M NaCl -- indistinguishable variances

Why hasn’t this been detected before? As for crystallography, single molecule FRET, electron paramagnetic resonance: The “stiffness” of DNA was previously measured by single molecule measurements under high tension.

Wanted: a mechanism for correlations

Remeasuring the double helix A new x-ray ruler for measuring distance distributions [see also work of Alivisatos+colleagues] Unexpected and unexplained Soft, correlated stretching fluctuations in DNA. [see also: unusually high “kink” rate (Widom), twist/stretch coupling (Bustamante), more]

Acknowledgments Pehr Harbury Becky Fenn Josh Silverman Peter Walker Tim Fenn Gold advice Chris Ackerson DNA advice Buzz Baldwin Ed Boas Zev Bryant Stirling Churchman Alex Dunn Dave Halpin Jim Havranek Dan Herschlag Lance Martin Bruce Robinson Mickey Schurr Alyssa Smith Rebecca Weisinger Jarret Wrenn SAXS advice Yu Bai Seb Doniach Kazuki Ito Jan Lipfert Ian Millett Soenke Seifert P. Thiyagarajan Hiro Tsuruta Rosetta advice Phil Bradley David Baker Data collected at: SSRL APS

Where do the X-rays come from? s = 

SAXS data looks like this. Fourier transform

SAXS data looks like this. Fourier transform

Since the biomolecule scatters, how to extract probe-probe interference? ––+ Need to subtract out: Biomolecule scattering Biomolecule-probe interference (Probe scattering)

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