ĐẠI HỌC NÔNG LÂM TP.

HCM
KHOA CHĂN NUÔI THÚ Y
Bộ môn Khoa học Sinh học Thú Y

CASE STUDY 2

Duy Ba NGO, DVM., Ph.D. Biotech.,

E: duy.ngoba@hcmuaf.edu.vn M: + 84 9043 8273 7
Web: https://vet.nlu.edu.vn/profile/ngobaduy

NL, 03/2024 1
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Chem. Sci., 2015, 6, 6777-6782, doi: 10.1039/c5sc02784e
OUTLINE 2
Introduction of target fueled DNA walker
1

Literature review (DNA nano-structure, MiRNA, Fluorescence

2 quenching in probe, RCA DNA, & DNA displacement)

Methodology
3

Result & Discussion

4

Conclusion
5
3
1. INTRODUCTION 3
Target-fueled DNA walker
DNA nanotechnology,
DNA nanotechnology 1982
Discovery

MiRNA, 1989

Fluorescence Quenching
in Probes, 2002

DNA walker, 2004

TARGET-FUELED DNA WALKER FOR HIGHLY SELECTIVE

MiRNA DETECTION DNA displacement, 2009 Synthetic DNA RCA,
2008

4
Cell Vol. 57, 1, p49–57, 1989, doi: http://dx.doi.org/10.1016/0092-8674(89)90171-2; J. theor. Biol. (1982) 99,237-247, doi: https://doi.org/10.1016/0022-5193(82)90002-9; Angew. Chem. Int. Ed.2008, 47, 6330 – 6337, doi: 10.1002/anie.200705982; J. Am.
Chem. Soc., 2004,126, 10834–10835, doi: 10.1021/ja047543j
2. Literature review 4
2.1. DNA nanostructure
A B The DNA formation and structure (adapted
Sinden, 1994)

- A: The binding model of base pairs (C.G) by

three hydrogen bonds;

- B: The binding model of base pairs (A.T) by

two hydrogen bonds;
C D

- C: The elongate model of the nucleotides;

- D: The helix structure DNA with

determining the nano-scale number

A,B, C: Watson-Ricks D: Nano scale size

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2. Literature review 5
2.1. DNA nanostructure

DNA duplex

DNA:RNA hetero-duplex

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Anosova et al., 2016. Nucleic Acids Research, Vol. 44, No. 3 1007–1021. Doi: 10.1093/nar/gkv1472; wikispaces.psu.edu/; Barone et al,. 2000. Biophysical Chemistry (86): 37-47
2. Literature review 6

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2. Literature review 7
The miRNA:
• MicroRNA are small and short RNA around 19-30
nucleotides at mature (1-3kb length for pri-miRNA)
• Regulators of gene expression
• Amount of miRNA depend on organism
• Encoded by endogenous genes
• Single strand RNA with stem-loop structure
• Partial complement to the 3’UTR of target genes
The pre-miRNA (black) and mature miRNA (red)
• Recognize multiple targets at Worm and human

The miRNA: Cell, Bacteria, Virus, Worm, plant, human

Conventional method miRNA detection: nothern blot, microarray, and qRT-PCR

that are more material, less sensitivity and selectivity

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Hausser et al., 2015. Wiley VCH: 833-859; Abdurakhmonov 2016. Doi: 10.5772/62038; https://en.wikipedia.org/wiki/MicroRNA
2. Literature review 8
Virus family Name Host No. pre-miRNA

Herpesvirus HSV-1 Human 6

MDV-1 Avian 14
mCMV Murine 18
rLCV Simian 16

Polyomavirus SV40 Simian 1

BKV Human 1
mPy Murine 1

Adenovirus hAv Human 1

Ascovirus HvAV Lepidopteran 1

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2. Literature review 9

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Chem. Rev. , 2013, 113 (8), pp 6207–6233. DOI: 10.1021/cr300362f
2. Literature review 10

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Frontiers in Oncology | Gastrointestinal Cancers, doi: 10.3389/fonc.2015.00068
2. Literature review 11
9
Introduction of conventional method of miRNA detection
Northern blot and Microarry for miRNA detection

Microarray
Northern blot
Graybill et al., 2015. Anal. Chem.: 1-63
2. Literature review 10
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Introduction of conventional method of miRNA detection (cont.)
qRT-PCR for miRNA detection

Reverse transcription quantitative polymerase reaction

Graybill et al., 2015. Anal. Chem.: 1-63
2. Literature review 13

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http://www.biologydiscussion.com/biochemistry/electrochemical-techniques/top-10-types-of-electrophoretic-techniques-used-in-biochemistry/12669
2. Literature review
MiRNA are of interest in use for pathological & diseased detection
Years Article
2017 An electrochemical biosensor for highly sensitive
detection of microRNA-377 based on strand
displacement amplification coupled with three-way
junction
2016 Ultrasensitive and multiple disease-related
microRNA detection based on tetrahedral DNA
nanostructures and duplex-specific nuclease-
assisted signal amplification
2015 Electrochemical DNA Biosensor Based on a
Tetrahedral Nanostructure Probe for the Detection
of Avian Influenza A (H7N9) Virus
2014 Hybridization Chain Reaction Amplification of
MicroRNA Detection with a Tetrahedral DNA
Nanostructure-Based Electrochemical Biosensor
2013 Bio-barcode gel assay for microRNA
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Journal
J. ELECTROANALYTICAL CHEMISTRY
DOI: 10.1016/jjelechem.2017.02.038
ACS Appl. Mater. Interfaces
DOI: 10.1021/acsami.6b12214
ACS Appl. Mater. Interfaces
DOI: 10.1021/acsami.5b01438
Anal. Chem
Doi: dx.doi.org/10.1021/ac4037262
Nature Communications
doi:10.1038/ncomms4367
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2. Literature review 15
The miRNA discovery in human cancer and diseases

Cellular miRNAs known to interact with RNA viruses

In thhis miRNA target: let-7a
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Andrew et al., 2013. Nat. Review Immunology: 1-11; Lautner et al., 2014. Electroanalysis (26): 1 – 12; Umbach et al., 2009. Genes & Development 23: 1151-1164; Front. Microbiol., 13 October 2010 | https://doi.org/10.3389/fmicb.2010.00108
2. Literature review 16

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2. Literature review 17

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https://www.biocat.com/products/RA610A-1-SBI
2. Literature review 18
Fluorescence quenching in dual-Labeled oligonucleotide probes: There are two Fluorescence quenching
Static Quenching (also known as contact quenching) and FRET (Förster Resonance Energy Transfer) Quenching

Distinctions between Static Quenching and FRET

In this paper used Static Quenching

Strong coupling Weak coupling

- Physical contact between reporter & quencher - Reporter and quencher interact through space
- Temperature and solvent dependent - Not very temperature and solvent dependent
Non quenching references - Absorption spectrum of reporter distorted - Absorption spectrum unchanged
* Quenching in a dual-labeled probe can occur through a combination of static and FRET
quenching

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J. AM. CHEM. SOC. 2002,124, 6950-6956, doi: 10.1021/ja025678o
2. Literature review 19
DNA Displacement: based on Watson – Rick principle as G.C; A.T (A.U)

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Nature Chemistry 3, 103–113 (2011), doi:10.1038/nchem.957
2. Literature review 20

The diseases and pathogen are complicated day by day,

a new trend of method for detection is necessary

According to WHO, the new method could be “ASSURED” ceteria

(affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable to end users)

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www.who.int/std_diagnostics
3. Methodology 22
Objective

To develop the DNA walker platform for ultra-sensitive and specific miRNA detection

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Chem. Sci., 2015, 6, 6777-6782, doi: 10.1039/c5sc02784e
3. Methodology 23
Platform method Finish steps
let-7a

DNA walker structure Activated

Target-fuels steps steps
activated

Fluorescences report DNA RCA Products Complete circles

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Chem. Sci., 2015, 6, 6777-6782, doi: 10.1039/c5sc02784e
3. Methodology 24
Experiments
Fabrication of To mix 100 nM (H1, H2, H3, H4, H5, C1, & C2) in reaction buffer, pH= 7.9 at 370C. And, incubated
DNA walking
nanostructure 900C, 3 min. Then, a gradient cool 650C, 450C, 370C, 25 0C take 30 min each of step

MiRNA active
steps & The C1 & C2 DNA steps activated by MiRNA at 250C, 2 h. RCA ligation optimize with T4
circularization ligase 0.1 - 10 units and 0 - 50 min at 250C, ligase buffer
of DNA

Rolling circle After ligation quickly, to add 1 ul RCA primer for C2 DNA, 5 ul dNTPs, and 1 ul phi 29
amplification
(RCA) bioassay polymerase, react at 370C for specific time, and stop reaction 900C for 10 min.

Fluorescence To mix 50 ul 200 nM MB and 10 unit of Nb.Mva1269I in 96 well measure after

assay incubated at 370C, 45 min with the excitation 495 nm and emission 520 nm

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Chem. Sci., 2015, 6, 6777-6782, doi: 10.1039/c5sc02784e
3. Result & Discussion 25
Fabricated DNA Walker
H1 5'‐ AAC TAT ACA ACC TAC TAC CTC ACT TTT ACG ACA CGG GTC
AAG TCT AG ‐3'
H2 5'‐ CTA GAC TTG ACC CGT GTC GTG AGT GTG TGC AAT AGT TAG
CTA TGA TGT CGT TAA CAA CCT ACT ACA
GCA GCG AAT ‐3'
H3 5'‐ TAC TAC ACA TTC GTA CTT TGA CAG GAT CTA GTC TTG CCA TTA
TGA CCA TCG ACA TCA TAG CTA ACT
ATT GCA CAC ACT C ‐3'
H4 5'‐ ATG GTC ATA ATG GCA AGA CTA GAT CCT GTC CAT GCA ACT
CGT AGC ‐3'
H5 5'‐ GCT ACG AGT TGC ATG ‐3'
C1 5'‐ p GTA GTA GGT TGT ATA GTT CAT CTT TAT TAG AGG TGC ATT DNA Walker structure
CAT CAG TCC TTA GAT TCG CTG CT ‐3'
C2 5'‐ p GTA GTA GGT TAC TTT ATA TTA GAG GTG CAT TCA TCA GAT
CTT AGG TAC GAA TGT ‐3'

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Chem. Sci., 2015, 6, 6777-6782, doi: 10.1039/c5sc02784e
3. Result & Discussion 26
Selective and specific of platform
Finish steps
let-7a

Activated steps
DNA walker structure

Fluorescences report DNA RCA Products Complete circles

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Chem. Sci., 2015, 6, 6777-6782, doi: 10.1039/c5sc02784e
3. Result & Discussion 27
The result of cross-linking check DNA walker with let-7a (miRNA, 10 nM)
1 2 3 Marker
let-7a + C1 + C2

let-7a + C1

C1 + C2

C1

RCA products checking (agarose gel 1%)

1: let-7a activated C1 only;
2: let-7a activated C1 + C2;
3: Not let-7a activated 27
Chem. Sci., 2015, 6, 6777-6782, doi: 10.1039/c5sc02784e
3. Result & Discussion 28
The result of sensitivity and calibration curve are relationship on let-7a detection

10 nM let-7a

10 fold up each one

100 fM let-7a

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Chem. Sci., 2015, 6, 6777-6782, doi: 10.1039/c5sc02784e
3. Result & Discussion 29
The result of specific DNA walker detect let-7a (miRNA)

Control

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Chem. Sci., 2015, 6, 6777-6782, doi: 10.1039/c5sc02784e
3. Result & Discussion 30
Characterization of fabricated DNA Walker (Agarose)

H1 (47 bp)

H1+ H2 (75 bp)

H1+ H2 + H3 (79 bp)

H1+ H2 + H3 + H4 (45 bp)

H1 + H2 + H3 + H4 + H5 (15 bp)

H1 + H2 + H3 + H4 + H5 + C1 (62 bp)

H1 + H2 + H3 + H4 + H5 + C1 + C2 (54 bp)

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Chem. Sci., 2015, 6, 6777-6782, doi: 10.1039/c5sc02784e
3. Result & Discussion 31
T4 DNA ligase optimizing

Phi 29 polymerase optimizing

Nicking enzyme optimizing

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Chem. Sci., 2015, 6, 6777-6782, doi: 10.1039/c5sc02784e
In conclusion 32

The platform of DNA nanostructrure show:

The DNA can walk by target-fueled
Detection limit of 58 fM of let-7a (miRNA)
Only one step of DNA walker gave a signal significantly
This DNA nano-structure is ultraselective and ultrasensitive sensor for point of care in the future

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 CẢM ƠN
THANK YOU
KHAP KHUN KHRAP
GRACIAS MUCHAS

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