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RNA structure probing

M. R. Brøgger-JensenM. LückmannV. Valintėlis

2012

Outline

•RNA structure determination

•Base-selective probing

•OH· probing

•Investigating RNA flexibility

•Space-neighbours interaction

•Future outlook

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RNA structure determination

Physical methods

•X-ray crystallography

•NMR spectroscopy

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Biochemical methods

• Enzymes

• Chemicals

Probe has to be specific to RNA structure !

Base-selective probingRNA modification sites

4Weeks, K.M. (2010). Advances in RNA structure analysis by chemical probing. Current opinion in structural biology 20, 295-304.

Base-selective probingRNA secondary structure prediction

5http://smg.nichd.nih.gov/put_structure.jpg

put RNA

OH· probing

Determines nucleotide accessibility

Random cleavage

Folding analysis

Quantitative information

6Edited from: Tullius, T.D., and Greenbaum, J.A. (2005). Mapping nucleic acid structure by hydroxyl radical cleavage. Current opinion in chemical biology 9, 127-134

RNA nucleotide dynamicsIn-Line probing

Investigating local nucleotide flexibilitywithout the use of chemicals

7Direct structure analysis of modified RNA by fluorescent in-line probing; Benjamin Strauss, Alexander Nierth, Marco Singer and Andres Jäschke, Nucleic Acids Research, 2012, Vol 40, No 2, 861-870

RNA nucleotide dynamicsSHAPE

Shape reactivity – a measure of local nucleotide flexibility

Different cDNA‘s are created by acylating the RNA backbone and performing primer extension

SHAPE reactivities are obtained by quantifying the cDNA‘s and normalizing the results

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Top figure: Probing Retroviral and Retrotransposon Genome Structures: The "SHAPE" of things to come; Joanna Sztuba-Solinska and Stuart F.J. Le Grice; Molecular Biology International, Vol 2012Bottom figure: Accurate SHAPE-directed RNA structure determination; Katherine E. Deigan, Tian W. Li, David H. Mathews and Kevin M. Weeks; PNAS, 2009, Vol 106, No 1, 97-102

RNA nucleotide dynamicsMutate-and-map

Mutating nucleotides and mapping the fragments in a 2D plot provides information RNA structure.

9A two-dimensional map-and-mutate stradegy for non-coding RNA struckture; Wipapat Kladwang, Christopher C. Vanlang, Pablo Cordero and Rhiju Das; Nature Chemistry, 2011, Vol 3, 954-962

Through-space connectivity information facilitates conformational space-search

10Judit E. Šponer, Kamila Réblová, Ali Mokdad, Vladimír Sychrovský, Jerzy Leszczynski and Jiří Šponer, J. Phys. Chem. B, 2007, 111 (30), pp 9153–9164Eizadora T. Yu, Arie Hawkins, Julian Eaton, and Daniele Fabris, PNAS, 2008, vol. 105, no. 34

•Radical cleavage analysis

•Site-directed cleavage

•Bifunctional crosslinking

Three major approaches

RNA through-space neighboursMultiplexed Hydroxyl Radical Cleavage Analysis (MOHCA)

1.Random incorporation of cleavage agents

2.Fragments analysis in 2D gel

11Rhiju Das, Madhuri Kudaravalli, Magdalena Jonikas, Alain Laederach, Robert Fong, Jason P. Schwans*, David Baker, Joseph A. Piccirilli, Russ B. Altman, and Daniel Herschlag, Structural inference of native and partially folded RNA by high-throughput contact mapping, PNAS, 2008, vol. 105, no. 11

RNA through-space neighboursSite-directed cleavage

12Costin M. Gherghe, Christopher W. Leonard, Feng Ding, Nikolay V. Dokholyan, and Kevin M. Weeks, Native-like RNA Tertiary Structures Using a Sequence-Encoded Cleavage Agent and Refinement by Discrete Molecular Dynamics, J. AM. CHEM. SOC. 2009, 131, 2541–2546

1.Systematic incorporation of cleavage agent (mutagenesis)

2.Fragment subjection to primer extension

3.cDNA separation by PAGE

4.Cleavage intensity quantification using phosphorimaging

RNA through-space neighboursBifunctional crosslinking

13A.E. Hawkins and D. Fabris, RNA Structure Determination by Structural Probing and Mass Spectrometry: MS3D, Nucleic Acids and Molecular Biology 27, Springer-Verlag Berlin Heidelberg 2012

1.Bridging of proximal bases using bifunctional reagents

2.Nuclease digestion

3.Fragments analysis in MS3D

Future outlook

•Probing techniques especially usefull to study larger, more dynamic RNAs

•Experiments become easier to apply, more high-throughput approaches

•De novo structure prediction lacks accuracy when no experimental data is incorporated

•Challenge: develop „soft“ techniques to act under native-like conditions

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Questions

• How can MOCHA influence the structure of RNA?• DMS should be  dimethylsulfate, not …sulfide, or?• Why are the more flexible (unstructured) regions cleaved in an

alkaline environment, but less flexible (structured) regions are not? (Utilized in in-line probing)

• In page 6, the part for “Site-directed cleavage”, could you please explain how cleavage agent is inserted systematically?

• In in-line probing are all RNA nucleotides radioactive labelled? If not, how can they visualize all the cleavage fragments?

• Explain mutate-and-map figure in more details.• Elucidate on MOHCA, how does a randomly inserted cleavage

agent and a 2-D gel give info on through space neighbours? And what does the size of the reagent matter?

• What is 'soft' techniques and why are they a challenge to use?

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