dnap protein review

8/2/2019 DNAP Protein Review

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WHY DNA BINDINGPROTEINS ?

Akhil Nair Megha Bhaskar Shinjini Chowdhary


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Main Paper:Dynamic proteinDNA recognition: beyond

what can be seenMonika Fuxreiter, Istvan Simon2and Sarah

Bondos

Motive : Accomplishes; mechanisms of DNA readout and protein DNA interactions and theories for

the same.

Other papers Discussed:1. SURVEY AND SUMMARY How do

site-specific DNA-binding proteins bind their

targets?Stephen E. Halford*and John F. Marko1

Motive: Addresses the thermodynamic and energy

aspects and existing models

** my inference,* cross reference, TF-Transcription

factor, DNAP DNA binding protein


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Classical logic : DNA protein interaction isstatic!

New hypothesis supported by proof but far fromcomplete theory:

Dynamic transient interactions betweenprotein and DNA.

Layout ofdiscussion:


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4/29/12*1 DNA READ OUT MECHANISMTHOUGHT TO BE

DIRECT: MEANS OF H BONDING INTERACTIONS

INDIRECT: ELECTROSTATIC INTERACTIONS WITH PHOSPHATES

Major factor affecting the reading:

*2 DNA shape dependent electrostatic potential

This about DNA what in protein effects or decide recognition of target sequence.

ID Regions: Intrinsically disordered regions :

Characteristic segment of protein which responds with conformational change in response toDNA contact.

Cross reference:*1 Luscombe, N.M. et al. (2001) Amino acid-base interactions: a threedimensional analysis of protein-DNAinteractions at an atomic level.Nucleic Acids Res. 29, 28602874

*2 Gromiha, M. et al. (2004) Intermolecular and intramolecular readout mechanisms in proteinDNArecognition. J. Mol. Biol. 337, 285294


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ID regions:*3Facilitate diffusion along DNAPlays role in transition from non specific to specific complex

Modulate selectivity

What are ID regions ?Basic principle:

*4 Change in folding pattern change in conformationalentropy!Improves mobility and binding affinity.

**DNAP:Two parts

DNA binding domainID regions

ID regions outside the binding contextcan modulate conformational ofresidues for DNA binding.

And how do they do that ?

Cross reference:*3 Laity, J.H. et al. (2000) DNA-induced alpha-helix capping in conserved linker sequences is a determinantof binding affinity in Cys(2)-His(2) zinc fingers. J. Mol. Biol. 295, 719727

*4 Spolar, R.S. and Record, M.T., Jr (1994) Coupling of local folding to site specific binding of proteins toDNA. Science 263, 777784


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*51. Influence thermodynamics of the binding process.2. Adjust the spacing between the protein and DNA.3. Adjust half- life of DNAP complex.

Example: DNA READ OUT MECHANISMS

Non specific to specific transition.**DNAP and P-P:Charged Id tails facilitate target search.**Concentration of protein and diffusion rateincreases substantially.Lock DNA binding motifs to target sequence by snaplock mechanism.*5

DYNAMIC MECHANISMS OF DNARECOGNITION

Modulation by four mechanisms:

1. Flexibility modulation2. Conformational modulation3. Competitive binding4. Tethering.Cross reference:

*5 Pontius, B.W. (1993) Close encounters: why unstructured, polymeric domains can increase rates ofspecific macromolecular association. Trends Biochem. Sci. 18, 181186


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Conformational modulation :

ID regions shift conformational equilibrium in favor of

binding.

Eg:*6 Max TF, interacts with E-Box (CACGTG), bHLH, LZ

Max free dorm Dimer in which LZ domain unstable : folding unfolding transitions ;Nterminal mask destabilizing electrostatic potential at LZ.followed by recognition loops decreases Kd by

100 fold. effects Myc gene expressionFlexibility modulationRemote regions interact with dynamism maintaining flexibility at DNAP interface:

Eg :

*7Ets 1 TF, regulated by auto-inhibitory region. Requires H1 helix to unfold Serinerich region regulates that. Phosphrylation in Srr reduces binding affinity. Removal of Srrregion mobility increases due to H1 unfolding.**Id region removal of SRR from that

specific region

Cross reference:*6 Naud, J.F. et al. (2005) Structural and thermodynamical characterization of the complete p21 gene product

of Max.Biochemistry 44, 1274612758*7 Pufall, M.A. et al. (2005)Variable control of Ets-1 DNA binding by multiple phosphates in an unstructured


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3. Competitive binding :

Rapidly fluctuating ID chain can screen electrostatic

attraction in DNAP. Or repulsion resulting inunavailability of binding region.

Eg:*7 Human positive factor PC4, recruits TF and stimulates RNAP II activity

regulated by Lys rich region NTD alone lacks affinity to ss or ds DNA .ID region interaction based onunwinding the fuzzy complex with DNA and C terminal competes with binding motifthereby **increasing selectivity of binding.

4. Tethering:Multidomain motif DNAP sets of linker ID region present in between those act to preserve the

conformational stability of all domains.Eg:

*8Human replication protein(RPA),contains weak and high affinity DNA binding domainsDBD, **Initial contact by High affinity followed by contraction of the linkers thereby increasing the local

concentration of DBD.

Cross reference:*7Jonker, H.R. et al. (2006) The intrinsically unstructured domain of PC4 modulates the activity of thestructured core through inter- and

intramolecular interactions. Biochemistry 45, 50675081*8NMR chemical shift and relaxation measurements provide evidence for the coupled folding and binding ofthe p53 transactivation domain. Nucleic Acids Res. 33, 20612077


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REGULATION OF DNA BINDINGVIA ID REGIONS:

By means of:

1.Post translational modifications2.Protein-Protein Interaction3.Alternative Splicing

1.Post translational modifications:

Phosphorylation perturbs the DNA binding

interface:

Eg: *9 FACT, displaces nucleosomes and facilitates RNAPII uses HMG domainflanked by and acidic and basic ID regions.Acidic region: maintains intermolecular attraction between with HMG domainsBasic Region: competes for DNA contact

Strengthened directly by phosphorylation due to inc in electron cloud acidic region

Cross reference:*9Tsunaka, Y. et al. (2009) Phosphorylated intrinsically disorder edregion of FACT masks its nucleosomal DNA

binding elements. J.Biol. Chem. 284, 2461024621.


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2.Protein-Protein Interaction :

ID regions competing with Binding motifs forDNA :

Eg:*8 p53 tumor suppressor activation depends on interaction with hRPA70.Affinity of

hRPA70 is higher compared to p53. The interaction controlled by linker ID.ID increases the localconcentrations of DBD on signal from hRPA70 to increase affinity of p53.

3.Alternative Splicing :

Modulate the DNA expression by altering the IDresidue composition:

Eg:**Change in sequence of residues would result in

change in interaction with the DNA sequence thetarget sequence of interaction and the pronouncedeffect of the same. Alternate translation site inMeCP2 increases the length and flexibility of


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How do site-specific DNA-bindingproteins find theirtargets?


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Facilitated Diffusion

Some proteins locate their target sites veryrapidly, much more rapidly than can seeminglybe accounted for by diffusional collisionsbetween the protein and the DNA molecule.Hence, these proteins must find their targets by`facilitated diffusion.

Proposals:

v One-dimensional diffusion

v Hopping through 3-D space by dissociatingfrom one site re-associating elsewhere in the

same chain

v Intersegmental transfer

Relevant only to proteins with two DNAbinding surfaces.

e.g. Lac repressor or Sfil

endonuclease

Figure 1. Courtesy: Nucleic Acids Research, 2004,Vol. 32, No. 10

Cross referencesBerg,O.G. and Blomberg,C. (1976) Association kinetics with coupled diffusional ows. Special application to

the lac repressoroperatorsystem. Biophys. Chem., 4, 367381


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q Berg and Blomberg - theoretical analysis of how 1-D diffusion along a DNAcontour can facilitate specific site targeting

Protein will alternatively undergo 1-D sliding and 3-D diffusive hops

Characteristic sliding length covered by 1-D sliding

Sliding length determined by the lifetime of a non-specific DNA - proteininteraction and by the effective 1-D diffusion constant

q Berg et al. experiments on Lac repressor protein

Time required for the repressor to find its target in a large DNA can bechanged

q Sliding length was originally estimated to be 100 bp under physiologicalconditions.

Cross References:Berg,O.G., Winter,R.B. and von Hippel,P.H. (1981) Diffusion-driven mechanisms of protein translocation onnucleic acids. 1. Models and

theory. Biochemistry, 20, 6929-6948.


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Three Dimensional Diffusion-Limited Targeting

Diffusion of a protein

A single protein moves through buffer byBrownian motion (diffusion) trajectory is arandom walk.

Major feature Average of the distance squaredcovered by a diffusing particle grows linearlywith time.

where D is the diffusion constant

Einsteinss formula diffusion of a protein



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Probability of finding a nearby target bydiffusion

The diffusing protein, at an initial distance rfrom the target, will either collide with thetarget or diffuse off into bulk solution, neverfinding this target.

During its random walk within a distance r ofthe target, the protein visits a fraction a/r ofthe `voxels' of size a.

The probability of encountering the target is

thus a/r. This is the exact result for diffusionto capture.

The diffusion-limited reaction rate

The above result is used to compute the association rate for binding.



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Simple-minded View Of FacilitatedDiffusion

Main ideas

Non-specific association of proteins with DNA can reduce the timerequired for them to find their target sequences, essentially byrestricting their motion to along the DNA contour.

The association rate is increased by the non-specific DNA flankingthe target, which serves to greatly increase the effective target size.

Facilitated diffusion provides accelerated targeting essentially byincreasing the target size, without decreasing the diffusion constantof the protein that is doing the search.

Cross references:Shimamoto,N. (1999) One-dimensional diffusion of proteins along DNA. J. Biol. Chem., 274, 1529315296.


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The Sliding Length

A protein binds non-specificallyto the DNA double helix (left)and then undergoes slidingsteps randomly to the left andto the right, exploring the DNAcontour through 1-D diffusion.

Eventually, a dissociation eventoccurs; the characteristicdistance explored between

association and dissociationevents is the sliding length.

Due to the random nature of 1-D diffusion, the same DNA sites

will be sampled repeatedly.

The sliding length can be controlledby adjustment of the non-specificbinding affinity.



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Processivity As A Probe For FacilitatedDiffusion

Processivity in this context is defined as the number of reactions inwhich the enzyme acts at both sites during one DNA-binding eventrelative to the total number of reactions by the enzyme. It is a unit-less parameter, a ratio of rates.

In a processive reaction on a DNA with two sites separated by aknown distance, the distance travelled by a protein from an initialnon-specific site to its final specific site is indeterminate.

The relationships between processivity and the length of DNAbetween the sites are much simpler.

In a processivity experiment, one considers a protein that has justacted at one site (here acting means binding, catalysis and

dissociation) and then measures the probability of it acting again ata second site in cis. The targeting distance concept permits a simple


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Conclusions and Outlookv Fundamental Problem: How DNA-binding proteins find

specific sites amongst huge amounts of non-specific

(chromosomal) DNA

v Association rate measurements and facilitated diffusion

Association rate measurements on Lac repressor protein remain to this

time one of the main supports for the facilitated diffusion theory. Information about a spatial reaction pathway can only be extracted from

total reaction rate measurements by the use of a model.

v Processivity experiments

Direct information about the spatial pathway that is followed by a DNA-binding protein to its target site can be obtained.

Processivity experiments on catenated DNA molecules haveprovided further evidence for transfer of proteins through spacefrom one DNA segment to a nearby one, presumably via diffusionin three dimensions.

Cross references:Terry,B.J., Jack,W.E. and Modrich,P. (1985) Facilitated diffusion during catalysis by EcoRI endonuclease.

Nonspecic interactions in EcoRI catalysis. J. Biol. Chem., 260, 1313013137.


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Engineering of repressor

protein into site-specific cutters

-an application of DNA-binding proteins


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Converting the protein into site-specific scission reagent

Generating TrpR mutants using PCR-based sitedirected mutagenesis.

Fig 1 and 2 :courtesy Branden, C. and Tooze, J. (1991) Introduction to Protein Structure, Garland

Publishing, New York Chapter 7


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Iodoacetamide chemical modification of cysteineresidue of mutant protein.

alkylation with 5-iodoacetamido-1,10-

phenanthroline.(IAAOP)

Fig 3 :courtesy Branden, C. and Tooze, J. (1991) Introduction to Protein Structure, Garland

Publishing, New York Chapter 7

The amino acid residue X must be in close proximity


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The amino acid residue X must be in close proximitywith the minor groove of DNA.

Binding of repressor to DNA takes place through helix-turn-helix motif .

Fig 3: courtesy Branden, C. and Tooze, J. (1991) Introduction to Protein Structure, GarlandPublishing, New York Chapter 7


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trpR E49C-OP chimeras scission ascission reagent- promotes positioningof trpR on trpEDCBA.

Factors which affect their nucleaseactivity :

amino acid position

the affinity of binding pH of the cleavage reaction

Cross reference:Lavoie.T.A. and CaryJ. (1994) In Bokstein.F. and Lilley.D.M.J. (eds). Nucleic Acids and MolecularBiology. Vol. 8. Springer-Verlag, Berlin, Heidelberg,184-188


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Optimizing the orientation of reactioncenter

Positioning of OP-Cu on the dyad axis

TrpRE49C-OP, places two OP-Cu residues rightat the centre of the dyad axis

constrained within the dyad axis in the minorgroove



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Fig 4. A Strathclyde minor groove binder in the double helix of DNA showing the antiparallel 2:1 bindingmode and the potential for hydrophobic interactions (grey patches).Courtesy: http://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_binders
http://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_binders


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Gel-retardation assay was used to studyDNA-protein interactions.

DNAse footprinting- protein binding site onDNA.

Fig 5:Tryptophan-dependent gel shift of a trpEDCBA containing end-labeled fragment by TrpR E49C-OP.Lanes I and 2, no protein (duplicated for scission purposes); lane 3, retardation by TrpR, gel-shift conditionsas described Courtesy:Protein Engineering vol.9 no.7 pp.603-610. 1996
http://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_binders


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Optimization of scission yield

Two methods have been determined:

carrying out the reaction between pH 7.2 and7.5 rather than pH 8.0.

using high-affinity mutants such as A77S.

http://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_binders


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CLEAVAGE OF LINEARISEDPLASMID DNA

Promotes specific background scission.

increases the affinity of the repressor for thetrp operator.

destabilizes the binding of the Trp repressorfor 'nonspecificprotein-DNA complexes'
http://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_binders


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PROPOSED APPLICATIONS OF CHIMERAS

treatment of disease

identifying a specific binding site in the DNA

chromosome mapping

Cloning

probes of macromolecular structure

http://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_binders


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CONCLUSIONS
http://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_binders


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MAIN REFERENCES:

1.Monika Fuxreiter Istvan Simon and Sarah Bondos 2011; Dynamic proteinDNArecognition: beyond what can be seen;Trends in Biochemical Sciences, August 2011,

Vol. 36, No. 8 Cell press

2. Stephen E. Halford* and John F. Marko1 SURVEY AND SUMMARY How do site-specificDNA-binding proteins and their targets?Nucleic Acids Research, 2004, Vol. 32, No. 10

3.Ralf Landgraf, Clark Pan, Christopher Sutton, Lori Pearson and David S.Sigman 1996;Engineering of DNA binding proteins into site-specific cutters:reactivity of Trprepressor-l,10-phenanthroline chimeras Protein Engineering vol.9 no.7 pp.603-610.
http://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_bindershttp://www.chem.strath.ac.uk/people/academic/colin_j_suckling/research/minor_groove_binders

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