James Stanek, OMS II Jocelyn Powell, OMS II

James Stanek, OMS II

Jocelyn Powell, OMS II

Learning Objectives:

1) What are the current limitations to viral detection and why do we need to develop new methods?

2) What are morpholino probes and why are they beneficial over DNA probes?

3) How can the morpholino technique be applied to improve viral detection methods?

What are the current techniques for detecting viruses?

Polymerase Chain Reaction(PCR)

Cell Culture

Antibody/Antigen Testing

Limitations:

• Time

• Equipment

• Laboratory

• Training

Example: Past Ebola Outbreaks

Better method of viral detection would include:

• The ability to test outside of a laboratory and in the field

• Low cost for use in rural and underserved areas

• Detection even with very small amounts of DNA

• Ex. Saliva, low viral count, incubation period

• Limited training needed

• Fast

• Without need for transport of samples

Morpholino Anatomy

Dye Sequence

complementary

to target DNA

A B

Molecular structure of phosphorodiamidate morpholino oligomer (PMO) probes.

Target DNA made based on

conserved sequence of Ebola

genome.

Two morpholinos differ in the following ways:

1) Attached dye(donor, acceptor)

2) Pairing Sequence (Region A vs B)

3) Orientation of binding (upstream vs downstream)

Excitation: 470 nm

Emission for Alexa488(donor): 520 nm

Emission for TMR(acceptor): 575 nm

Donor Morpholino(Alexa 488)

Excitation @

470 nmEmission @

520 nm

Excitation @

470 nm

Acceptor Morpholino(TMR)

Donor Morpholino(Alexa 488) and Acceptor(TMR)

Excitation @

470 nmEmission

@ 575 nm

Target DNAAcceptor

Target

DNA

Excitation @ 470 nm

Emission Wavelength

Inte

nsit

y

Donor

600 700

Donor & Acceptor

Emission @

575 nm

Figure2: Fluorescence resonance energy transfer caused by PMO probes binding to target DNA sequence.

Blue line: without target DNA(Control)Red line: with target DNA

0 200 400 600 800 1000

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

R

Target Concentration (pM)

Adjusted R2 = 0.97

Morpholino probes detected as low as 20pM target sequence.

0.0

0.2

0.4

0.6

0.8

1.0

500 550 600 650

-0.2

0.0Flu

ore

scence

Wavelength (nm)PMO probes detection of target DNA doped in human saliva.

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0.25

0.50

0.75

1.00

500 550 600

B. SalivaA. Buffer

filename is Figure3BufferSaliva

Wavelength (nm)

Flu

ore

scence

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DNA Probes

Hight SaltFlu

ore

sce

nce

Wavelength (nm)

C

PMO Probes

Low Salt

B

Wavelength (nm)

PMO Probes

Hight Salt

A

Wavelength (nm)

PM

O+d

sTar

get/T

ES

PM

O+s

sTar

get/T

ES

PM

O+d

sTar

get/T

E

PM

O+s

sTar

get/T

E

DNA+d

sTar

get/T

ES

DNA+s

sTar

get/T

ES

0.0

0.2

0.4 dsTarget

ssTargetD

R

PMO probes detection of hybridized double strand target.

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1.00

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ds target ds target

ss target

DNA Probe Pair

High Salt

Flu

ore

sce

nce

A

D E

ds target

ss targetss target

PMO Probe Pair

Low SaltC

PMO Probe Pair

High SaltB

Wavelength (nm)

0.00 0.05 0.10 0.15 0.20 0.25

filename is Morpholinos/Figure6ModifiedC

high salt

high salt

low salt

PMO probe pair

PMO probe pair

DNA probe pairds target

high salt

low salt

R 0.00 0.05 0.10 0.15 0.20 0.25

high saltss target

R

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1.00

-Urea +Urea

Low

Salt

A

Flu

ore

sce

nce

High

Salt

B

C

Wavelength(nm)

D

Morpholino probes bind to target in denaturing buffer.

500 550 600 650

0.00

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1.00

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1.00

500 550 600 650

0.00

0.25

0.50

0.75

1.00

A

Flu

ore

sce

nce

(a

.u.)

B

High

Salt

Low

Salt

+Urea

C

Wavelength(nm)

-Urea

D

DNA probes bind to target in denaturing buffer.

• Morpholino probes bind to DNA and emit FRET in denaturing

conditions in which DNA probes cannot.

• Morpholino probes provide a rapid and efficient method for

detecting pathogens in human saliva or blood.

Stanek, J. MS, Powell, J. BS, Mata, J. PhD, McQuistan, T. MS, Smythe, C. BS, Summerton, J. PhD, Squier, T. PhD, Xiong, Y. PhD

Authors and affiliations: