Mass spectrometry Laboratory Liege University · 2 Workshop on electronic recognition of DNA molecules Liège, September 1-3 ToolsTools • Sensors, bioassays, rush methods for screening

11

Molecular recognition based on nucleic acids structures: from gas

phase to solution

Molecular recognition based on nucleic acids structures: from gaMolecular recognition based on nucleic acids structures: from gas s

phase to solutionphase to solution

V.V.GablelicaGablelica, F. , F. RosuRosu, E. De Pauw, E. De Pauw

Mass spectrometry LaboratoryMass spectrometry Laboratory

Liege UniversityLiege University

Workshop on Workshop on electronic electronic recognition of DNA recognition of DNA molecules molecules Liège, Liège, September September 11--33

ContextContextContext

•• Development Development of of multilevel analytical strategiesmultilevel analytical strategies

– Screening

– Semi-quantitation

– Confirmation

•• RequirementsRequirements

– Identification

– Quantification

– ISO 17025 compatible in terms of false positive,

negative samples

22


ToolsToolsTools

•• SensorsSensors, , bioassaysbioassays, rush , rush methods methods for for screeningscreening

•• Confirmation Confirmation often requires often requires mass mass spectrometryspectrometry, ,

which was problematic which was problematic for non volatile, polar for non volatile, polar

compoundscompounds


Electrospray ESI (nanoESI)Electrospray Electrospray ESI (ESI (nanoESInanoESI))

Cone de Taylor

•• Concentrations: 10Concentrations: 10--6 6 MM

Solvent: HSolvent: H22O O

(+ MeOH, CH(+ MeOH, CH33CN)CN)

•• Multiply charged ionsMultiply charged ions

•• Quasi no fragmentsQuasi no fragments

intact non covalent assemblies

33


MALDIMALDIMALDI

From Leonid V. From Leonid V. Zhigilei Zhigilei web siteweb site

Direct identification of solid phase compoundsDirect identification of solid phase compounds


QuadrupolesQuadrupolesQuadrupoles

•• Filters: trajectory stabilityFilters: trajectory stability MS

CC

A/m

B/m

44


Quadrupolar ion trapsQuadrupolar Quadrupolar ion trapsion traps

•• Selective instabilitySelective instability


FTMSFTMSFTMS

55


Time of flights (TOF)Time of flights (TOF)Time of flights (TOF)

QuickTime™ et un

décompresseur codec YUV420

sont requis pour visionner cette image.

The Mass Spectrometry ToolboxThe Mass Spectrometry ToolboxThe Mass Spectrometry Toolbox

•• M/Z determination at the nucleonic levelM/Z determination at the nucleonic level

•• Direct sampling of the solution (ESIDirect sampling of the solution (ESI--MS)MS)

•• Kinetics of fragmentation (breakdown curves MSKinetics of fragmentation (breakdown curves MS--MS)MS)

•• Ion trappingIon trapping

•• Modes of energy transfer: CID, SID, ECD, IRMPDModes of energy transfer: CID, SID, ECD, IRMPD

•• Structural information: full sequencing facility, fragmentation Structural information: full sequencing facility, fragmentation

mechanisms, H/D exchangemechanisms, H/D exchange

•• Database searching algorithmsDatabase searching algorithms

66


Measuring the affinity of a chemical for the molecular targetMeasuring the affinity of a chemical for the molecular targetMeasuring the affinity of a chemical for the molecular target

+ +A ABB

A AB B

Electrospray

BABFull scan MS

m/z

The basic hypothesisThe basic hypothesis

DNA as an exampleDNA as an exampleDNA as an example

1.1. Potentially important target for the design of new Potentially important target for the design of new

drugs, fast screening tests requireddrugs, fast screening tests required

2.2. Oligomers Oligomers are (seems to be) good models for larger are (seems to be) good models for larger

structuresstructures

3.3. Reference methods for structures well established Reference methods for structures well established

in solution and solid phasein solution and solid phase

4.4. Increasing importance of DNA in sensors as Increasing importance of DNA in sensors as

capture elementscapture elements

77

1:1or

2:1

Minor groove binding

Major groove binding

Stacking

« Historical » DNA duplexes (Watson-Crick)«« HistoricalHistorical » DNA duplexes (» DNA duplexes (WatsonWatson--CrickCrick))

Stoechiometry

Caution: non Caution: non specific specific complexescomplexes


IntercalationIntercalationIntercalation

Random stacking between the base pairspreferential GC stacking

intercalation of planar compound

88

G-QuadruplexesGG--QuadruplexesQuadruplexes

Q1: (TG4T)4 Q2: (G4T4G4)2

G

G

G

G


Sampling solution to the gas phaseSampling Sampling solution to solution to the gas the gas phasephase

Plots of the relative intensities of the different speciesPlots of the relative intensities of the different species(duplex, (duplex, 1:1 complex1:1 complex, , 2:1 complex2:1 complex)versus the drug molar fraction)versus the drug molar fraction

Direct Direct titrationtitration

99


Manipulating the ions in gas phase: MS/MSManipulating the Manipulating the ions in ions in gas gas phase: MS/MSphase: MS/MS

Gas Gas phase phase denaturation curve denaturation curve

for for the the duplex GCduplex GC55--, ,

GCGC55-- + + HoechstHoechst, GC, GC55-- ++Netropsin Netropsin

compared compared to solution thermal to solution thermal

denaturation monitored denaturation monitored by UVby UV

V. V. GabelicaGabelica, E. De Pauw, , E. De Pauw, Rapid Comm Rapid Comm 14, 464, 200014, 464, 2000

TTeff eff ??

Easy differentiation Easy differentiation of of binding binding sitessites


Molecular recognition by MS-MSMolecular Molecular recognition by MSrecognition by MS--MSMS

Intercalators: even with« random » intercalation, specificity Is conserved in the gas phase

NH

N

CH3+

NH

NCH3

+

Cryptolepine

Neocryptolepine

ds+C5-

ds+NeoC5-

1010


Structure is largely maintainedStructure Structure is largely maintainedis largely maintained

5’-AAATCGCGGCGCTAAA-3’3’-TTTAGCGCCGCGATTT-5’

∆∆Hn-n = -136.8 kcal mol-1

5’-GGGCTATAATATCGGG-3’3’-CCCGATATTATAGCCC-5’

∆∆Hn-n = -125.3 kcal mol-1

5’-AGACTGTGAGTCAGTG-3’3’-TCTGACACTCAGTCAC-5’

∆∆Hn-n = -122.6 kcal mol-1

5’-GGGCTTTTAAAACGGG-3’3’-CCCGAAAATTTTGCCC-5’

∆∆Hn-n = -135.9 kcal mol-1

! not ! not expressed expressed in in the the CMCM

VoltageVoltage

Not a proof, Not a proof, just just a a reasonable guess reasonable guess !!


ApplicationsApplicationsApplications

Part I:Part I: Screening DrugScreening Drug--DNA InteractionsDNA Interactions

Effect of the DrugEffect of the Drug Telomestatin Telomestatin onon Telomeric Telomeric

Quadruplex Quadruplex UnfoldingUnfolding

Part II:Part II: H/D exchange on H/D exchange on QuadruplexesQuadruplexes

Part III:Part III: Modified Modified DNA: DNA: Molecular nanoprobesMolecular nanoprobes

Part IV :Part IV : hyphenationhyphenation

1111

Part I:

Screening Drug-DNA Interactions with sequence specificity

Part I:Part I:

Screening DrugScreening Drug--DNA Interactions with sequence specificityDNA Interactions with sequence specificity

Human Telomeres: (TTAGGG)nHuman Telomeres: (TTAGGG)Human Telomeres: (TTAGGG)nn

143D: NMRStructure (1993) 1: 263

1KF1: X-rayNature (2002) 417: 876

Q3: GGGTTAGGGTTAGGGTTAGGG

1212

=> Search for drugs that stabilize the G-quadruplex conformation

Quadruplexes in Telomerase InhibitionQuadruplexes Quadruplexes in Telomerase Inhibitionin Telomerase Inhibition

ApoptosisApoptosis

ImmortalityImmortality

Targeting G-Quadruplexes with LigandsTargeting GTargeting G--Quadruplexes with LigandsQuadruplexes with Ligands

Groove bindingIntercalation

External Stacking

Loop Recognition

1313

Some drug candidatesSome drug candidatesSome drug candidates

QuickTime™ et undécompresseur TIFF (LZW)


Screening Example 1Screening Example 1Screening Example 1

1350 1400 1450 1500 1550 1600 1650 1700 1750 1800m/z

1499.7

1613.3

1550 1600 1650 1700 1750 1800 1850 1900 1950 2000m/z

1804.3

1661.9

1946.5

1350 1400 1450 1500 1550 1600 1650 1700m/z

1457.4

1684.9d(CGTA3T3ACG)2

d(CGCG3C3GCG)2

(TG4T)4

(G3T2A)3GGG

(G4T4G4)2

d(CGCGA2T2CGCG)2

Amount bound (µM)

Only (2:1)5-

complex!

Duplex5-

Telomeric quadruplex4-(1:1)4-

(2:1)4-

(1:1)5-

Quadruplex5-



1414

Discovery of new Specific 2:1 ComplexDiscovery of new Specific 2:1 ComplexDiscovery of new Specific 2:1 Complex

12274

Semi-empirical model for dimer

Manual docking and molecular dynamics

Screening Example 2Screening Example 2Screening Example 2

11190: weaker binding, but very specific for telomeric quadruplexes

d(CGTA3T3ACG)2

d(CGCG3C3GCG)2

(TG4T)4

(G3T2A)3GGG

(G4T4G4)2

d(CGCGA2T2CGCG)2

Amount bound (µM)

1600 1650 1700 1750 1800 1850 1900 1950 2000m/z

1661.9

1801.3

1450 1500 1550 1600 1650 1700 1750m/z

1499.7

1420 1460 1500 1540 1580 1620 1660 1700m/z

1457.3

1:1

1:1

(1:1)4-

Duplex5-

Quadruplex5-

Telomeric quadruplex4-

1515

Part II:

Mechanisms of action: Effect of the Drug Telomestatin on

Telomeric Quadruplex Unfolding

Part II:Part II:

Mechanisms of action: Effect of the Drug Telomestatin onMechanisms of action: Effect of the Drug Telomestatin on

Telomeric Telomeric Quadruplex UnfoldingQuadruplex Unfolding

1:1

telomestatin

slowDuplex

(GGGTTA)3GGGG-quadruplex

(CCCAAT)3CCC

fast

2:1

telomestatin

slow

F. Rosu et al., Chem. Commun. (2003) 2702.

Objective: To mimick drug-induced telomerase inhibition

Quenching the hybridization kineticsQuenching the hybridization kineticsQuenching the hybridization kinetics

TelomeraseTelomerase

Complementary strandComplementary strand

1616

No drug With telomestatin

t = 200 s

t = 2000 s

t = 200 s

t = 2000 s

Effect of telomestatin on the hybridization kineticsEffect ofEffect of telomestatin telomestatin on the hybridization kineticson the hybridization kinetics

ôdupl = 532 ± 11 s

τ G4 532 ± 11 s

Relative intensity

Intensity of the duplex

Intensity of the quadruplex

ττ hybridize = 532 ± 11 shybridize = 532 ± 11 s

Hybridization kinetics: without drugHybridization kinetics: without drugHybridization kinetics: without drug

1717

ôdupl = 1010 ± 90 s

τG4 = 540 ± 30 s

1:1 cplx

2:1 cplx

τhybridize = 1010 ± 90 s

Rate-limiting step = decomplexation

Hybridization kinetics: with drugHybridization kineticsHybridization kinetics: : with drugwith drug

Part III:

Structure (?): H/D exchange on Quadruplexes

Part III:Part III:

Structure (?): H/D exchange onStructure (?): H/D exchange on QuadruplexesQuadruplexes

1818

Conformation of Quadruplexes in the Gas PhaseConformation ofConformation of Quadruplexes Quadruplexes in the Gas Phasein the Gas Phase

Preliminary experiments on H/D exchangePreliminary experiments on H/D exchangePrevious studies on DNA non-covalent assemblies:

general idea =

more compact structures have less H/D exchange sites

CD3OD (deuterated methanol) in the FTICR cell (p = 8 X10-9 T)Bruker Apex-Q (Bremen, Germany)

The Kinetic of deuterium incorporation was monitored (0-20 min)

-S.A. Hofstadler and R.H. Griffey, Chem. Rev. (2001), 101, 377-390. (duplex)

-M. Vairamani and M.L. Gross, JACS (2003), 125, 42-43. (quadruplex)

0 sec

2 sec

4 sec

50 sec

300 sec

20 H/DTypical: large spreadTypical: large spreadDifferent protons have Different protons have different exchange ratesdifferent exchange rates

Duplex (CGCGAATTCGCG)25-Duplex (CGCGAATTCGCG)Duplex (CGCGAATTCGCG)2255--

1919

0 sec

2 sec

4 sec

50 sec

300 sec

28 H/D

Quadruplex [(TGGGGT)4°3NH4+-8H+]5-Quadruplex Quadruplex [(TGGGGT)[(TGGGGT)4°4°3NH3NH44++--8H8H++]]55--

UnexpectedUnexpected: : FastFast, Quasi no, Quasi no spreadspread

Single strand2-

[TGGGGT-2H+]2-

Quadruplex5-

[(TGGGGT)4°3NH4+-8H+]5-

Quadruplex4-

[(TGGGGT)4°3NH4+-7H+]4-

2 componentsFast: 25.3 ± 0.5 H/D

(time constant: 3.01 ± 0.14 s)Slow: 6.2 ± 1.2 H/D

(time constant: 450 ± 250 s)

Total: 31.5 ± 1.7 H/D

2 componentsFast: 16.8 ± 0.8 H/D

(time constant: 2.9 ± 0.5 s)medium: 23.2 ± 0.8 H/D


Total: 40 ± 1.6 H/D

1 componentSlow: 7.7 ± 1.6 H/D


Other duplexes and quadruplexes: typical time constant = 25-50 s

Effect of the charge and aggregation stateEffect of the charge and aggregation stateEffect of the charge and aggregation state

2020

G

G

G

G

Per backbone (4) : max 7 H

(5 phosphate + 2 sugar) 28

Per thymine (8) : max 1 H 8

Per guanine (4*3) : max 3H 36

Per NH4+ (3) : max 4 H 12

Per strand: max 18

Per G4: 84

First analysis of the numbersFirst analysis First analysis of of the numbersthe numbers

23.2+/23.2+/--0.80.8

00

00

mediummedium

16.8+/16.8+/--0.80.8G4 4G4 4--

25.3+/25.3+/--0.50.56.2+/6.2+/--1.21.2G4 5G4 5--

007.7+/7.7+/--1.61.6SS 2SS 2--

FastFastSlowSlow

Charge fluctuation ?Charge fluctuation ?

0 sec

2 sec

4 sec

8 sec

50 sec

The species still exchange to the same extent => NH4+ not exchanged

[(TGGGGT)4 °(3-n)NH4+ ° nK+-8H+]5-

1 23

Effect of Cation replacementEffect ofEffect of Cation Cation replacementreplacement

2121

0 sec

0.4 sec

1 sec

2 sec

4 sec

[(TGGGGT)4+3NH4++2H+]5+ : fast[(TGGGGT)[(TGGGGT)44+3NH+3NH44+++2H+2H++]]5+ 5+ : fast: fast

[(TGGGGT)4+5H+]5+ :slow[(TGGGGT)[(TGGGGT)44+5H+5H++]]5+ 5+ :slow:slow⇒The inner cations are essential

for fast H/D exchange

Role of the cationRole Role of of the the cationcation


Structural informationStructural informationStructural information

Inner cations are essential to provoke fast exchange (both positive and negative ion mode!). But the inner NH4

+

hydrogens are not essential for fast exchange.

Hypothesis: the cations included confer a rigid conformation to the quadruplex, which is highly favorable for

H/D exchange.

Next stepNext step: influence of : influence of the drugsthe drugs

2222


Part III:Part III:

nanoprobesnanoprobes: Fullerene functionalized DNA: Fullerene functionalized DNA


Fullerene modified DNAFullerene modified Fullerene modified DNADNA

+

2323


Part IV: building Part IV: building the sensorsthe sensors

The The bridge bridge between solidbetween solid, , liquid and liquid and gaz phasegaz phase


Affinity capture, SPR and MS Affinity Affinity capture, SPR capture, SPR and and MS MS

Surface Surface Plasmon resonancePlasmon resonancebased detection based detection coupled with coupled with MS identificationMS identification

Challenge: Challenge: development development of capture of capture

elementselements

2424


Biacore-MS couplingBiacoreBiacore--MS MS couplingcoupling



From: R. From: R. KarlssonKarlsson, J. Mol. , J. Mol. RecogniRecogni. 2004, 17, 151. 2004, 17, 151--161161


New ligands: aptamers (aptare=to fit)New ligands: New ligands: aptamers aptamers ((aptare=to aptare=to fit)fit)

Molecular Molecular «« wrappingwrapping »»

Molecular imprinting Molecular imprinting in solutionin solution

2525


Aptamer capture and MALDI MSAptamer Aptamer capture and MALDI MScapture and MALDI MS






Direct MALDI fingerprinting of bacteria Direct MALDI Direct MALDI fingerprinting fingerprinting of of bacteria bacteria

2626

MASS SPECTROMETRY LABORATORYDr. V. GabelicaF. RosuJF Greisch (fullerenes)

Funding: FNRS, European Union, Walloon Region, University Concerted research actions

COLLABORATIONSScreening of DNA-drug interactions:Dr. P. Mailliet (Aventis Pharma, Vitry sur Seine, France)Dr. J.L. Mergny, L. Guittat (Laboratoire de Biophysique, INSERM UMR 8646,

Muséum National d’Histoire Naturelle, Paris, France)Telomestatin:Dr. K. Shin-ya (Institute of Molecular and Cellular Biosciences,

The University of Tokyo, Japan)H/D exchange in the gas phase:Drs. M. Witt and G. Baykut (Bruker Daltonik, Bremen, Germany)

AknowledgmentsAknowledgmentsAknowledgments

Documents

Mass spectrometry Laboratory Liege University · 2 Workshop on electronic recognition of DNA molecules Liège, September 1-3 ToolsTools • Sensors, bioassays, rush methods for screening