A Suggestion for Mutagenicity Assessment with Conformational RNA Structure. (ICAW2015, ベルギー) 伊純明寛

ISUMI [email protected]

International Comet Assay Workshop 2015

A Suggestion for Mutagenicity Assessment

with Conformational RNA Structure.

[1] Aptamer-based Mapping.- Monospecific Analysis.

[2] Riboswitch-based Mapping.- Bispecific Analysis.

ISUMI AKIHIRO

伊純明寛



Purposes.

Comet assay is a simple and strong method.

But it’s not suitable for the tissue-location specific assessmentsand also for the drug-interaction assessments.

Let’s try RNA aptamersand riboswitches !



What’s the Mutagenicity ?

Mutagens attack DNA sequencethat result in -

・ random base pair・ cross linkage・ internal cleavage etc…

H

HOH

H H

BaseCH2P O

O-

O-

O

Mutagen+

Pray, L. (2008) Discovery of DNA structure and function: Watson and Crick. Nature Education 1(1):100



Comet Assay -- A General Technique to Assess the Mutagenicity.

MARTINEZ, Carlos Augusto Real et al. The importance of oxygen free radicals in the etiopathogenesis of diversion colitis in rats.Acta Cir. Bras. [online]. 2010, vol.25

Comet Assay Criteriafrom Score 0 to 4 / cell

Score 0 – 400 / tissue

Not For the Detection of Drug-Specific Mutageneicity Localization.

Suggestion to use aptamersfor a visualization tool.

x 100 (cells) / tissue.



[1] Aptamer. What Are the Aptamers ?

PegaptanibThe Most Famous Aptamer as the First Anti-VEGF Therapy

for the Treatment of Exudative AMD

Edge, A. Safety. "The Role of PegaptanibIn the Treatment of Exudative AMD And Diabetic Retinopathy."

Ng, E. W., Shima, D. T., Calias, P., Cunningham, E. T., Guyer, D. R., & Adamis, A. P. (2006). Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease. Nature reviews drug discovery, 5(2), 123-132.

Excessive VEGF stimulation.

VEGF

The Artificial RNAsBinding the Specific Ligands.



[1] Aptamer. What Are the Aptamers ?

PegaptanibThe Most Famous Aptamer as the First Anti-VEGF Therapy

for the Treatment of Exudative AMD

Edge, A. Safety. "The Role of PegaptanibIn the Treatment of Exudative AMD And Diabetic Retinopathy."

Ng, E. W., Shima, D. T., Calias, P., Cunningham, E. T., Guyer, D. R., & Adamis, A. P. (2006). Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease. Nature reviews drug discovery, 5(2), 123-132.

Excessive VEGF stimulation.

VEGF



Why the Aptamers ? -- Aptamers than Antibodies.

The Aptamers Can Detect Haptens Easily.

It is difficult to make the anti-hapten antibodies due to the incomplete antigenicity.

The Aptamers Can Bind the Target Molecules With High Affinity.

Dissociation constant (Kd)

Aptamers - 10-9 M

Antibodies 10-9 - 10-6MThe more lower, the more better.

The instability rather contributesto the high affinity.

E S ES+ ⇄Ka

Kd



How to Make Aptamers ? -- SELEX method.

NNNNN

NNNNN

1．Making random RNA library.(10 ~ 25 N bases)

2. Removingnon-specific sequences.

NNNNN

3. Isolating specific sequencesbinding target-molecules labeled metal beads.

4. PCR

Probe 1 Probe 2

NNNNN

NNNNN

Repeat 10 – 15 times.

5. Sequencing 6. PEG and Fluo labeling. (See APPENDIX)



How to Use Aptamers ? -- (1) in vitro Normalization.

1. Making the aptamer standard curve against known conc. of mutagenswith an image analyzer.

012345

0μ

M

100μ

M

20

0μ

M

30

0μ

M

40

0μ

M

50

0μ

M

Flu

o.

2. Making the comet assay standard curvewith cultural cells.

012345

0μ

M

100μ

M

20

0μ

M

30

0μ

M

40

0μ

M

50

0μ

M

Sco

re

3. Normalizing the graph.012345

0 1 2 3 4 5

Sco

reFluo.



How to Use Aptamers ? -- (2) Pathological Application.

1. Medication.

2. Cryosection.

3. Applycation.

4. Detection and

Analysis. 012345

0 1 2 3 4 5

Sco

re

Fluo.

It’s simple as immunostaining or ISH.



What Can We Know ?

Location specific mutagenicity. Fluorescence

≒ Net Concentration.

Comet Assay Score.

Analysis relationship

between the Net Concentrations and the Mutagenicities.

It can be the indicator for the relationshipbetween the location specific accumulation and the mutagenicity.

0.70.70.7

0.7

0.8 0.70.70.7

0.6



[2] Riboswitch. What Are the Riboswitches ?

Aptamers can bind their targets.

Target.

Riboswitches can bind their secondary targetsonly when they bind their primary targets.

PrimaryTarget.

SecondaryTarget.

Screening tools for drug interactions.

Aptamer.

Riboswitch.



How to Make Riboswitches ? -- Additional Steps of Aptamers.

1．Making aptamers. 2. Removingnon-specific sequences.

3. Isolating specific sequencesbinding secondary target-molecules labeled metal beads.

4. PCR

Repeat 10 – 15 times.

5. Sequencing 6. PEG and Fluo labeling. (See APPENDIX)



How to Use Riboswitches ? -- (1) Preparing Patterns.

Primary Site. Secondary Site.

Patterns.



How to Use Riboswitches ? -- (2) Preparing Patterns.

1. Medication.

2. Cryosection.

3. Applycation.

4. Detection and

Analysis. 012345

0 1 2 3 4 5

Sco

re

Fluo.

It’s the same as the application of aptamers.



What Can We Know?

Suppressed by Enhanced by Activated by

0

1

2

3

4

5

0 1 2 3 4 5

Sco

re

Fluo.

Aptamer

0

1

2

3

4

5

0 1 2 3 4 5

Sco

re

Fluo.

Aptamer

0

1

2

3

4

5

0 1 2 3 4 5

Sco

re

Fluo.

Riboswitch

0

1

2

3

4

5

0 1 2 3 4 5

Riboswitch

Sco

re

Fluo.0

1

2

3

4

5

0 1 2 3 4 5

Riboswitch

Sco

re

Fluo.

It is suitable for screening the drug interactions.

. . .



Conclusion.

[2] Riboswitch-based Bispecific Mapping.

Fluorescence

≒ Net Concentration.

Mutageneicity Score.

0.70.70.7

0.7

0.8 0.70.70.7

0.6Relationship between the location specific mutagen accumulation and the mutagenicity.

[1] Aptamer-based Monospecific Mapping.

Screening the drug interactions.

Suppression. Enhancement. Co-factor.



Thanks.

Thank you so much !

Riboswitch Search.

If you are interested in these methods,please contact me. Or

ISUMI AKIHIRO

伊純明寛

Science

A Suggestion for Mutagenicity Assessment with Conformational RNA Structure. (ICAW2015, ベルギー) 伊純明寛