Dynamics of Trapped Oligonucleotides

Transcription

1 Dynamics of Trapped Oligonucleotides Joel H. Parks The Rowland Institute at Harvard Cambridge, MA

2 Support Allison S. Danell Joseph T. Khoury Amit Meller Jim Foley Jean-Marc Fournier Wolfgang Wenzel Funding Rowland Institute

3 Outline Fluorescence of Gas Phase Biopolymers FRET fluorescence and mass spectrometry measurements of trapped oligonucleotides DNA duplex dissociation: intermediate states Sequential Charge Loss in Oligonucleotides Charge-state dependence Sequence dependence Conclusions and future Work

4 Fluorescence Analysis of Trapped Biopolymers Use fluorescence measurements to monitor conformational changes Fluorescence Resonance Energy Transfer Correlate changes in fluorescence intensity with changes in average conformation

5 Electrospray Ionization Ion Trap Mass Spectrometer

6 Illumination and Detection Optics

7 Ion Cloud Fluorescence Overlap Volume δv hν hν V cloud

8 FRET Pair BODIPY TMR BODIPY TR

9 BODIPY Solvent Dependence abs em abs em

10 BODIPY Temperature Dependence abs em abs em

11 Fluorescence 0.9 µj ~ 350 ions in δvhν

12 Fluorescence vs. N ions (BODIPY-TMR-5'-AATTAATCCGGCCG-3') 6-

13 Dissociation of DNA Duplexes Mass Spectrometry Thermal Dissociation Rates Watson-Crick Pairing Schnier, P. D., Klassen, J. S., Strittmatter, E. F., Williams, E. R. J. Am. Chem. Soc. 1998, 120, Collision-induced Dissociation Rates multistep process Gabelica, V., De Pauw, E. J. Am. Soc. Mass Spectrom. 2002, 13,

14 Intermediate States: Fraying/Bubble Dynamics Modify single strands with fluorophores Heat trapped duplexes to induce dissociation Monitor changes in fluorescence Fluorescence Mass Spectrometry

15 Dissociation Intermediate States

16 Effect of <R> on Donor Fluorescence Donor Fluorescence Mass Spectrum Intensity of ss

17 Nanospray Mass Spectrum BODIPY-TR-3'-TTAATTAGGCCGGC-5' BODIPY-TMR-5'-AATTAATCCGGCCG-3 60/20/20 Acetonitrile/water/isopropanol (ss) 3- /(ds) 6- Solution Absorbance 2.5 Intensity (ss) 6- /(ds) 12- (ss) 5- /(ds) 10- (ss) 4- /(ds) 8- (ds) 7- (ds) m/z

18 Isolated 14-mer Duplex Fluorescence (ds) 7- T=117 C 60 s ~ 400 ions in δv hν

19 Threshold Fluorescence

20 Intermediate State Model: Data Comparison

21 Nanospray Mass Spectrum BODIPY-TR-5'-AAAAAAAGGCCGGC-3' BODIPY-TMR-3'-TTTTTTTCCGGCCG-5 70/20/10 Methanol/Water/Trifluoroethanol

22 Duplex Fluorescence Fluorescence Intensity (per ion) µj s T ( C)

23 min. T 160 C q z (ds 7- ) 0.27 ds 7- Isolation MS/MS MS/MS Acc-a 7 Acc-w 7 BODIPY-TR-5'-AAAAAAAGGCCGGC-3' 0.2 Acc-w 7 2- / Don-a7 2- Acc-a 7 2- Don-w 7 Don-a 7 BODIPY-TMR-3'-TTTTTTTCCGGCCG Acc-w Na Acc-w 7 1-Don-a 7 1- Dissociation conformation MS/MS ds A D Acc-a Na + K Isolation MS/MS A MS/MS FRET 0.1 Don-w D

24 Observation of Charge Loss (ds 14 ) n- (ds 14 ) n- (ds 14 ) (n-1)- m/z - No indication of heavy ion fragmentation - No pressure dependence - Constant total ion number (MS, fluorescence)

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26 (T 7 ) n- Temperature Dependence

27 Multi-Charged Anions: Repulsive Coulomb Barriers Stable Anions Metastable Anions (PtCl 4 ) 2- [CuPc(SO 3 ) 4 ] 4- Energy b n Repulsive Coulomb Barrier AB (n-1)- +e - Energy b n e n Detachment Energy AB n- R-e - AB n- R-e - -e n k Detachment = F(b n, e n ) AB (n-1)- +e - Lai-Sheng Wang and Xue-Bin Wang, J.Phys.Chem. A104, 1978 (2000)

28 3-2-

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30 MD Simulation: C K, 2 ns

31 Temperature Dependence ln (k 32 ) A 7 ACACACA C 7 T T (ºC)

32 Charge Loss Model sequence of repulsive coulomb barriers

33 Research Plans FRET Fluorescence Measurements DNA duplex intermediate states DNA hairpin closure rates Alpha helix stability Trp-cage unfolding Oligonucleotide Charge Loss Mechanism Sequence sensitivity

34 Summary/Conclusions Fluorescence measurements of trapped biopolymers provide sufficient sensitivity for dynamics studies FRET fluorescence correlated with mass spectrometry shows promise as a probe of the intermediate states of DNA duplexes Sequential charge loss in oligonucleotides exhibits a strong dependence on sequence base stacking