1. National Institute of Health and Medical Research
7th Asia Pacific Biotech Congress July 13-15, 2015 Double Tree by Hilton, Beijing, China
« Combined Use of NIR Raman Spectroscopy and Baro-Bio Reactor to Correlate the Impact of Extreme Physico-Chemical Environments (D2O solvent, High Temperature and High Pressure) on the Viroid’s Structure and Function »
Gaston HUI BON HOA
National Institute of Health and Medical Research
(I.N.S.E.R.M., U779)

2. Plan of the Presentation
Introduction on plan-RNA Viroid, especially Avocado sunblotch viroid (ASBDd).
Bio-Technological Methods of Perturations:
NIR Raman Spectroscopy: Application to the Study of RNA’s Structures and Conformations.
Baro-Bio Reactor: Application to the Study of the ASBVd Viroid’s Selt-Cleavage Activity under High Pressure.
Conclusions.

3. Plant-RNA Viroids
Viroids are non encapsidated small non coding RNA plant pathogens .
They are able to infect dramatically important broad range of plants, including herbaceous and tree crops, by regulating the host gene expression.
Viroids are studied since the 1970s (Diener T.O., 1971). Unfortunatly, the ways by which viroids are able to induce diseases are actually unknown.
There is also a lack of understanding concerning the secondary and tertiary structures of theses pathogens, and how they are able to interact with host species and to use host machineries.
This situation prompts us to develop an adapted technology to elucidate the active structure of Viroid’s RNA and its interactions with small therapeutic agents and cell membranes.

4. Avocado Sunblotch Viroids (ASBVd)
The Avocado sunblotch viroids under studied are single-stranded, covalently closed circular RNA molecules with 246 and 401 n.t. chain length. They can adopt predicted branched secondary structure (Zuker, 1989), formed by hairpin:
The secondary structure is characterized by alternating double-stranded and single-stranded regions.
The conserved sequences in the hammerhead ribozyme are shown on green and blue.

5. Self-Cleavage Through Hammerhead Ribozyme Structure
The ASBVd with 2 polarities (+) and (-), belong to the Avsunviroidae species and are able to self-cleave through a hammerhead ribozyme structure and to replicate via a symmetric rolling circle pathway (Flores, R., et al., 2000; 2005).
Transesterification reaction:
5’,3’ diester
2’,3’ cyclic
phosphate
diester

6. NIR Raman Spectroscopy
Raman Spectroscopy has the great potentiel as a sensitive probe of molecular structure.
Vibrational spectra contain a great deal of information about the molecular dynamics of RNA.
It is well adapted to the study of viroids’s RNA, because the Raman signals are not contaminated by the capsid proteins.
One of the advantage of using NIR Raman Spectroscopy is because infra red excitation at 780 nm (a double photon excitation) avoids excitation of fluorescence emissions.
The goal in such investigations is to establish a reliable correlation between vibrational spectra and specific structural features of RNAs and their biologically important complexes.

7. Near Infra Red Laser Excitation for Raman Spectroscopy
N2 liquid
cooled
NIR CCD
Spectrograph
Acton SpectroPro 2500i
Argon-ion laser pump
Ti:Sapphire laser

8. Optical System for Raman Laser Excitation Vibrational Spectroscopy
Spectrograph
Backscattering objective

9. High Temperature Thermostated Cell for NIR Raman Spectroscopy
Quartz Raman sample cell.
Diam. 2X2X35 cm
H.T. thermostated bloc

10. Stainless-Steel High Pressure (400 MPa) Cell for Raman Spectroscopy
Pressure inlet fluid
thermostated fluid system (5-95°C)
4 Saphir Windows

11. 400 MPa High Pressure Cell for NIR Raman Spectroscopy
Stainless
steel
H.P. Cell
4 saphir windows

12. High Pressure Cell Connected to 400 MPa Generator
H.P. Cell
400 MPa manual generator
H.P. Valves
H. P. manometer
Reservoir
H.P. inlet fluid

13. Raman Spectra of free ASBVd(-) (blue) and (+) (red) Species
669 (G)
Sugar pucker
N-type
727 (A)
785 Pyrimidine
(C,U) breathing
813 uS (O-P-O)
Sugar
866
917
928
1100
u PO2-
1233 (U/C)
1250
1460
d-CH2 deform.
1485
stretching
A-G ring
(A,G) H-bonding
C=O

14. Phospodiester conformations
Wavenumber (cm-1)
In ( cm-1) region:
- 2 nucleotide heterocyclic rings:
- 1) (G) breathing vibration line at (669 cm-1) which appears
in the N-type sugar pucker conformation.
- 2) (A) strong purine breathing line at (727 cm-1)
Raman Markers for Nucleotide heterocycle rings and
Phospodiester conformations
- Existence of 2 strong and sharp characteristic lines at:
785 cm-1: ring breathing of pyrimidine (C,U) bases
813 cm-1: symmetric stretching vibration of phosphodiester
linkage = us (C-O-P-O-C) : A-type viroide RNA.
in B-DNA: a weak broad line with shoulder at : us = 835cm-1.
. Doublet is sensitive to backbone’s bond angle deformation.
. Degree of double helical content can be defined as:
r2 = I813/I1100.
. Nucleic Acid’s conformations can be defined as: rconf. = I785/I813

15. Raman Markers for Sugar and Phosphodioxy group
Wavenumber (cm-1)
Raman Intensity (a.u.)
Raman Markers for Sugar and Phosphodioxy group
B) In ( cm-1) region:
3 line at cm-1 (sugar vibrational lines) are also
sensitive to backbone geometry and secondary structure.
C) In ( cm-1) region:
- A strong line at 1100 cm-1 assignable to the symmetric
stretching vibration of the phosphodioxy group: uS (PO2-).
- Sensitive to changes in the electrostatic environment,
but insensitive to the bases composition of RNA.
. It serves as a useful internal marker.
. Normalization on this intensity line, allows comparison
between:
- Raman spectra of various nucleic acids.
- Strength of several base-specific vibrational modes
in the same RNA structure.

16. Raman Markers for Base-Base Stacking and Sugar Puckering
D) In ( cm-1) region:
- Base-stacking modes have several lines around cm-1.
a) 1300 cm-1 and 1378 cm-1 are composite vibrations of purine
(A) and (G) ring systems.
b) 1338 cm-1 arises from imidazole ring vibration alone of the
pyrimidines.
Normalized intensities of these lines was use to evaluate the
degree of stacking of A/G bases:
rstack = (I I1378) / I1338 .
- Strong line at 1250 cm-1 participates to the backbone geometry
and rotation of bases with respect to the sugar:
- N-Type: C-3’ atom of the sugar in «endo» position
base in «anti» rotation geometry: ASBVd(-)
- Strong line at 1267 cm-1 characterizes the
- S-Type: C-2’ atom of the sugar in «endo» position base in «anti» rotation geometry: Not ASBVd(-)
A/G Stacking
1300
1338
1378
Wavenumber (cm-1)
Raman Intensity (a.u.)

17. Raman Markers for Base-Pairing and H-Bonding
Wavenumber (cm-1)
Raman Intensity (A.u.)
1574
1686
Raman Markers for Base-Pairing and H-Bonding
E) In cm-1) region:
. Two strong Raman lines appear at 1485 cm-1 and
1574 cm-1
which result from (A-G) stretching vibrations (I = 0.9),
and the (A,G) H-bonding (0.7).
. The H-bonding in the carbonyl C=O stretching modes of
pyrimidines manifests a broad band centered
at 1686 cm-1, (I = 0.5).
. The intensity and position of this composite band is
sensitive to its coupling with the N-H deformation mode
of the bases, to thermal denaturationn and D2O
perturbation.
. The typical line at 1460 cm-1 is attributed to methylene
twisting ut (d-CH2)

18. 20 mM Mg2+ binding perturbation:
- No effect on Raman Phosphodioxy line 1100 cm--1,
- Purine (A) at 727 cm-1 increased by +7%
- Small effect on N-type sugar pucker at 669 cm-1 (+4%)
- Doublet at 785 and 813 cm-1 increased by 8%:
rconf = 1.14 and rstack = 1.4
- Changes in nucleotide geometry and ribose conformation were observed.
Deuteration effects on mobile H-bonding:
- Huge changes in 727 cm-1 and 669 cm-1 purine (A) and (G)
stretching, with frequency downshift (Du = -11 cm-1):
- It is a onsequense of the removal of coupling between
ribose and purine bases upon deuteration.
- Frequency downshift in 785 cm-1 line indicates that a new
A-type phosphodiester structure is appearing.
rconf = 0.9 in D2O (decreases by 25 %)
r increases from 1.28 to 1.40.
- Interesting, Phosophidioxy 1100 cm-1 line is not perturbed.
Wavenumber (cm-1)
Results: Mg2+ Binding, D2O Perturbations on Phosphodiester backbone’s frequency region

19. Results: Temperature Perturbations on Phosphodiester Backbone Frequency’s Region
Temperature Perturbation at 65°C:
- The phosphodiester mode at 813 cm-1 decreases in
intensity and is transformed into a broad shoulder at
779 cm-1
- Frequency downshift (Du = -15 cm-1) was observed.
- Pyrimidine stretching mode at 785 cm-1 downshifts
by Du = - 5 cm-1.
- r2 decreases from 1.2 to 1.79 indicating a
decrease of 27% ordered double helical content.
- rconf increases from 1.2 to 1.85
- There is a loss of the A-type structure for ASBVd(-) and
a loss of nucleotide conformation (671 cm-1 line decreases).
Wavenumber (cm-1)

20. Results: Mg2+ Binding, D2O Perturbations on
Base stacking and H-Bonding Frequency
Region
20 mM Mg2+ binding perturbation:
- No big perturbation of the Raman spectrum, only
small increase in intensity of several lines.
- no changes in the base stacking ratio, H-bonding
and C=O double bonds.
(B) Deuteration effects on mobile H-bonding:
- Raman marker in 1233 cm-1 (U/C stretching vibrations)
desapears upon deuteration, while an intense line at
1302 cm-1 of purine (A) appears, indicating that an
external «in plane» C-N stretching vibrations of purine
are sensitive to D2O perturbation.
- The stacking parameter increases from 1.32 in H2O till
1.66 in D2O (34%);
- The line at 1485 cm-1 assignable to (A/G) purine ring
stretching is also very sensitive to D2O perturbation.
It is suggested that D2O perturbs some internal loops,
base-base interactions and incresases double helical
rearrangement to a new conformation and rigidity.
Wavenumber (cm-1)
Raman Intensity (a.u.) A B C

21. Results: Temperature Perturbations on
Base Stacking and H-Bonding Frequency
Region
Wavenumbers (cm-1)
Raman intensity (a.u.) R
An hyperchromism of the 1228 cm-1 line is observed,
associated with an increase of the rstack parameter from
1.36 to 1.45, c
- Both results are indicative of a loss of base-stacking and
the destabilization of the double-helical structure.
- A moderate intensity increase in the carbonyl region
around 1690 cm-1 reflects the rupture of hydrogen
bonds between bases at 65°C.
- Note that the effects of temperature are quite different
from that of solvent deuteration. A B C

22. Resutlts: Effect of RNA’s Self-Cleavage Activity on Raman Structural Markers
1) Experimental conditions:
. All Raman spectra were recorded at 20°C,
. Sample was incubated at 45°C, in the presence of 20 mM
Mg2+ during 4 hours and then bring back to 20°C.
2) In the phosphodiester frequency region:
. There were weak Raman intensity increases.
. Interestingly, several Raman band downshifts were
observed upon Mg2+-induced self-cleavage:
D u= - 10cm-1 (pyrimidine at 784 cm-1 (- 6%)).
. The intensity of the symmetric phosphodiester Raman line
increased by 4% and its frequency downshifted by –10 cm-1.
. The phosphodioxy PO2- line also downshifted by – 9 cm-1.
. All these frequency downshifts are indicative of vibrational
energy decrease of the Raman Markers. It is a consequence
of the self-cleavage sites leading to change from:
5’,3’ phosphate diester to 2’,3’ cyclic phosphate diester.
. Stretching mode of (A) at 727 cm-1 were also perturbed.
3) In the stacking and H-bonding frequency region:
rstack was not changed
Wavenumber (cm-1)
Raman Intensity (a.u.)
Wavenumber (cm-1)

23. Results: Effects of D2O solvent on the self-cleavage activity of ASBVd(-) and (+) strands in the presence of 20 mM Mg2+, (gel electrophoresis analysis at 45°C).
Solvent deuteration perturbation is a useful tool to study the accessibity
of mobile hydrogen in the viroids cleavage’s site:
a) The (-) strand is 3.5 time more active in H2O than in D2O solvents.
b) The ASBVd(-) is about 3.5 times more active than ASBVd(+) in H2O,
even in D2O solvent.
The results show that H-bonding in ASBVd(-) is more mobile and accessible
and its structure more flexible than ASBVd(+).
- A mechanism of acid/base catalysis is suggested, involving proton transfer.

24. Principe of Direct Sampling-In and -Out Bio-Reactor at Constant Pressures (400 MPa) and Controlled Temperatures
constant pressure’s sas
(v=35 ml)
(or N2 gas)
Products’s
collection
Sas lock 1
Reactor lock
Sas lock 2

25. High Pressure Bio-Reactor allowing the Sampling-in of Substrates and Sampling-out of Products
Electric high pressure generator
Driving piston system
N2 gas and valve
for purching the sas
HP transducer
Seringe and valve for
filling the reactor
Remoted Controlled
Driver for the generator
Termostated
400 MPa
bio-reactor
Thermostated fluid
-in and -out
Sas and valves for sampling_out products

26. Remoted controlled driver for the 400 MPa generator
Pushing piston
Electric driven H.P. generator
H.P. bio reactor
H.P. transducer

27. Results: Temperature-profile of ASBVd(-) Self-Cleavage activity at difference pressure
Temperature (°C)
V(i) (% cleaved species)
DV≠ = -+17 ml mol-1
t = 35°C
Ea ≈ 10 kcal.K-1.mol-1
. Experimantal conditions: Kinetics of the viroid cleavage was
follwed at 65°C in cacodylate buffer, at several pressure.
35 ml of the incubated solution was sampling out every 5 mn
till 45mn, then at 60, 90 and 120 mn.
- They were then quenched in a volume of stop solution (7 M
urea,50 mM EDTA, pH 7.5 and 0.01% xylene Cyanol) at 25°C.
- Each aliquot was then loaded onto a denaturing gel (6% SDS-
PAGE) for the determination of the fraction of the cleaved
products.
. Results show a bell-shape temperature dependence of the
cleavage activity (Max at 55°C) .
Increasing pressure inibited the activity without any changes of
the bell-shape.
. A plot of the rates: ln V(i) vs P(bar) shows a complexe pressure-
dependence reaction behavior. There are composite slopes.
. Howerver extraction of the initial slope permits to deduce the
activation volume of the reaction: DV≠ = + 17 ml.mol-1
. It is interpreted as the implication of one molecule of water (one
H+) in the mechanism of the transesterification reaction in the
cleavage site.

28. Conclusions
Our results emphasize the power of the combined use of Raman structural markers and physico-chemical perturbations methods to analyze in details the Raman spectra of ASBVd viroids.
The specific Raman markers provide quantitative informations revealing some dynamic aspects of the viroid’s structure.
The next steps are to study the interactions of such viroids with small therapeutic agents and cell membranes in order to elucidate better the mechanism action of such viroids to infect plants and to find efficients inhibitors.
High pressure will be another interesting perturbation tool to unfold the structure and to shift all chemical as well as biochemical equilibriums and kinetics. The determination of the volumes(DV°) of reaction and activation volumes (DV≠) of reaction, allows us to interpret the results in term of structural dynamics, the compressibility of the biomolecules and the implication of non traditional role of water molecules in all these reactions.

29. Acknowledgements
Marie-Christine Maurel UMR 7205, Sorbonne, Universités,
Jacques Vergne UPMC, Univ_Paris 6, France
Hussein Kaddour
Sergei G. Kruglik Laboratoire Jean-Perrin, UPMC
Pierre et Marie Curie, Paris 6
Gaston HUI BON HOA INSERM, U779, CHU Kremlin Bicêtre, France
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