HiroshiSugiyamaDepartmentofChemistry,GraduateSchoolofScienceInstituteforIntegratedCell-MaterialSciences(iCeMS)

KyotoUniversity1

100nm

生体分子機能論 2018-2配列決定

Advanced Course in Molecular Biology and Biochemistry

Basics 1 Basic elements of nucleic acids and their synthesis 2 Sequencing of DNA 3 3D structure of DNA 1 4 3D structure of DNA 2 Chemistry 5) DNA alkylation 6) Hydrogen abstraction 1 7) Hydrogen abstraction 2 8) Charge transfer Biology 9) Epigenetics 1 10) Epigenetics 2 11) ATRX

Sequencing of DNA

1)  In 1970s Maxam and Gilbert sequencing Sanger sequencing Slab gel electrophoresis (Nobel Prize Chemistry 1980) 2) In late 1990s to 2000s Capillary gel electrophoresis (Human Genome Project) 3) 2010- Next generation sequencing (NGS)

Maxam-Gilbert Chemical Sequencing

STEP1 Labeling A polynucleotide is labeled at either its 5’- or 3’-end with 32P.

STEP2 Chemical treatment Labeled polynucleotide is treated with a base-specific reagent and then the chemically modified polynucleotide causes strand breakage at each modified site.

STEP3 Electrophoresis Labeled fragments are electrophoresed side by side in denaturing acrylamide gels for size separation.

Maxam-Gilbert Chemical Sequencing

Maxam, A.; Gilbert, W. Methods in enzymology 1980, 65, 499–560.

Maxam-Gilbert Chemical Sequencing

type of modification reagent cleavage

A + G deprination 88 % formic acid (pH 2) 1 M piperidine (90 oC for 30 min)

G methylation dimethyl sulfate 1 M piperidine (90 oC for 30 min)

C base ring-opening hydrazine 1 M piperidine (90 oC for 30 min)

C + T base ring-opening hydrazine (1.5 M NaCl) 1 M piperidine (90 oC for 30 min)

Maxam-Gilbert Chemical Sequencing

A + G specific reaction

G specific reaction

Maxam-Gilbert Chemical SequencingC + T specific reaction

Maxam-Gilbert Chemical Sequencing

Maxam, M.; Gilbert, W. PNAS 1977, 74, 560–564.

10

Prof Teruo Matsuura Feb. 20th,1984

Organic Chemical Approaches to DNA Photochemistry -New Aspects of Thymine Photochemistry-

J. Am. Chem. Soc., 103, 1598 (1981), ibid 105, 698 (1983), ibid 105, 956 (1983)

Sequencing of DNA

1)  In 1970s Maxam and Gilbert sequencing Sanger sequencing Slab gel electrophoresis (Nobel Prize Chemistry 1980) 2) In late 1990s to 2000s Capillary gel electrophoresis (Human Genome Project) 3) 2010- Next generation sequencing (NGS)

Fluorescent labeling ddNTP

The human genome project (HGP)

DNA sequence of the fragment

Sequence

Sequence

Sequence Sequence

Sequence

Sequence

(Bioinformatics)

Check for overlapping sequences

Genomic sequence

The human genome project (HGP)

Historical overview

The human genome project (HGP)

Sample of genomic DNA

Shotgun-method 1. step: Digestion of the genomic DNA

into smaller fragments (enzymes or shear).

2. step: Multiplication (cloning fragments in vector).

3. step: Sequencing (Sanger, dye terminator)

Laser induced fluorescence. Readout in capillary electrophoresis

The instrument: capillary electrophoresis

H3CO2C OH

NNN

N

NH2

O

NH

NO

H3C

CGTATpO

OpCG

OHO

NH

OCH3

OCH3OCH3

90 °C

(Tetrahedron Lett. 1990, 31, 7197)(Tetrahedron Lett. 1993, 34, 2179)

5 min

90 °C

DNA Cleavage

+

20 min(Chem. Res. Toxicol. 1994, 7, 677)

C A T A A A G

T G T A A A G

G G C A A A C

C A T T A A T H3CO2C

ON

NH

NNN

NHN

OHH3C

OCGTATpO

OpCG

O

NH

OCH3

OCH3OCH3

Duocarmycin A Alkylates Adenine N3 at the 3' End of AT Rich Sequences

5'-d(CGTATACG)-3'3'-d(GCATATGC)-5' 5'-d(CGTATACG)-3'

3'-d(GCATATGC)-5'

5'-d(CGTATACG)-3'

3'-d(GCATATGC)-5'

CO2CH3O

N

NHO

ONH

CH3O

CH3OOCH3

CH3

NO

HN

NO

NH

O

NH

N

N

O

HN

N

HN

O

N

N

O

NH

NO

NH

NO

NH

O

HN

O

NH

O N

OH

Cl

R

R = H (16S) , F (17) , Me (18), Br (19)

Figure 2. (a) Thermally induced strand cleavages of the 5'-Texas Red-labeled 208-bp DNA

fragment (6 nM) by conjugates 16S and 16–19 incubated for 1 h at 23 °C: lane 1 = DNA control;

lanes 2–5, 2.5, 5, 10, 20 nM of 16S; lanes 6–9, 2.5, 5, 10, 20 nM of 17; lanes 10–13, 2.5, 5, 10, 20

nM of 18; lanes 14–17, 2.5, 5, 10, 20 nM of 19; lanes 18–21, 2.5, 5, 10, 20 nM of 16. (b) A

schematic representation of sequence-specific alkylation by conjugates 16–19. Arrows indicate the

sites of adenine N3 alkylation. The alkylating base is shown in red bold.

Conformational analysis of CBI-vinyl moiety

To gain insight into the basis of the different reactivity of conjugates 16-19, we carried out

conformational analysis of CBI-vinyl moiety. Optimized structures using density functional theory

B3LYP calculation at the 6-31G (d,p) level (Gaussian 09 W) 21 are shown in Figure 4c. The results

clearly indicated that analogues of 16 and 17 mostly keep planer conformation. On the contrary,

analogues of 18 and 19 distort their conformation at the position of C2-C3 bond and cyclopropane

subunit far from the adenine N3, thus reduce their DNA alkylating activity. These results well

explain with the PAGE analysis for 16-19.

Figure 4. ������Figure 4 c, d Figure 5 a, b

Energy minimized structure of the d(CGCTTTGTCACGC) ODN1/ d (GCGTGACAAAGCG)

ODN2-16S′ complex. Vinyl linker is drawn in brown, cyclopropane unit of CBI is drawn in yellow

and ODN1 is drawn in purple, especially a reacting adenine is drawn in bold purple. (a) Overall

structure of conjugate 16 and ODN1/ODN2 complex. (b)Position and distance between the

CBI-vinyl moiety and adenine N3. (c)Chemical structures of AcPL(R)CBI moiety and dihedral

5'-CGCTTTGTCACGC-3'

3'-GCGAAACAGTCGC-5'

-β--β-

DNA alkylation at N3 of A can be evaluated by thermal DNA strand cleavageJ.Am.Chem.Soc.134,13074(2012)

PIPolyamideseco-CBIConjugateswithaVinylLinker

5'

5'

3'

3'

5'

3' 5'

3'

5' 3'

ｐUC18

378-827

Photoreaction of 5-Halouracil-Containing DNA Fragment

dXUTP ( X = Br or I )

PCR

302 nm　

Sequencing Gel

top strand

bottom strand450 bp

TexasRed labeled primer

reverse primerXU

Reaction

Specific Cleavage at (G/C)AAXUXU and (G/C)AXUXU top strand

G C

T A

X =Br X = I reaction time

(sec) 0 15

30 45

60 0 45

90 135

180 G C

T A

X =Br X = I

0 15

30 45

60 0 45

90 135

180

site 1

site 2

site 3

site 4

site 5

site 6

site 7

site 8

site 13

site 12

site 11

site 10

site 9

bottom strand 5’-N80 TCGAATTCGT AATCATGGTC ATAGCTGTTT 　 3’-N80 AGCTTAAGCA TTAGTACCAG TATCGACAAA CCTGTGTGAA　ATTGTTATCC GCTCACAATT GGACACACTT　TAACAATAGG CGAGTGTTAA CCACACAACA　TACGAGCCGG AAGCATAAAG GGTGTGTTGT　ATGCTCGGCC TTCGTATTTC 　 　 TGTAAAGCCT GGGGTGCCTA　ATGAGTGAGC ACATTTCGGA CCCCACGGAT TACTCACTCG TAACTCACAT TAATTGCGTT GCGCTCACTG ATTGAGTGTA ATTAACGCAA CGCGAGTGAC CCCGCTTTCC AGTCGGGAAA CCTGTCGTGC GGGCGAAAGG TCAGCCCTTT GGACAGCACG CAGCTGCATT AATGAATCGG CCAACGCGCG GTCGACGTAA TTACTTAGCC GGTTGCGCGC GGGAGAGGCG GTTTGCGAAT　TGGGCGCTCT N140-3’ CCCTCTCCGC CAAACGCTTA　ACCCGCGAGA N140-5’

site 1

site 2 site 3

site 4

site 5

site 6 site 7

site 8

site 13

site 12

site 11

site 10

site 9

top strand

bottom strand

(G/C)AAXUXU or (G/C)AXUXU

T = XU

Sequencing of DNA

1)  In 1970s Maxam and Gilbert sequencing Sanger sequencing Slab gel electrophoresis (Nobel Prize Chemistry 1980) 2) In late 1990s to 2000s Capillary gel electrophoresis (Human Genome Project) 3) 2010- Next generation sequencing (NGS)

Sanger sequencing Next generation sequencing

PCR vs emulsion PCR

DNA sequencing by Ion Torrent Personal Genome Machine

Genomic Fragment

Adapters

DNA sequencing by Ion Torrent Personal Genome Machine

Genomic Fragment

Barcode

DNA sequencing by Ion Torrent Personal Genome Machine

DNA sequencing by Ion Torrent Personal Genome Machine

Bead/ISP

Adapter Complement Sequences

The idea is that each bead should be amplified all over with a SINGLE library fragment.

DNA sequencing by Ion Torrent Personal Genome Machine

Problem: How do I do PCR to amplify the fragments without having to use 1 tube for each reaction?

DNA sequencing by Ion Torrent Personal Genome Machine

DNA sequencing by Ion Torrent Personal Genome Machine

Shotgun sequencing by PGM/454

DNA sequencing by Ion Torrent Personal Genome Machine

DNA sequencing by Ion Torrent Personal Genome Machine

DNA sequencing by Ion Torrent Personal Genome Machine

DNA sequencing by Ion Torrent Personal Genome Machine

DNA sequencing by Ion Torrent Personal Genome Machine

DNA sequencing by Ion Torrent Personal Genome Machine

Shotgun sequencing by PGM/454

DNA sequencing by Ion Torrent Personal Genome Machine

~3.5 µm for Ion Torrent, ~30 µm for 454

DNA sequencing by Ion Torrent Personal Genome Machine

JM Rothberg et al. Nature 475, 348-352 (2011)

Sensor, well and chip architecture.

Data collection and base calling.

A C G C G C C G G G T C A G A A C C C G A T C G C G 5’3’

5’T G C G C G G C C C A

Primer

Only give polymerase one nucleotide at a time:

If that nucleotide is incorporated, enzymes turn by-products into light:

T C A G T C A G T C A G

1 2 3 4 5

T T T

T T

DNA sequencing by Ion Torrent Personal Genome Machine

A C G C G C C G G G T C A G A A C C C G A T C G C G 5’3’

5’T G C G C G G C C C A

Primer

Only give polymerase one nucleotide at a time:

If that nucleotide is incorporated, enzymes turn by-products into light:

T C A G T C A G T C A G

1 2 3 4 5

A A A

A A

DNA sequencing by Ion Torrent Personal Genome Machine

A C G C G C C G G G T C A G A A C C C G A T C G C G 5’3’

5’T G C G C G G C C C A

Primer

Only give polymerase one nucleotide at a time:

If that nucleotide is incorporated, enzymes turn by-products into light:

T C A G T C A G T C A G

1 2 3 4 5

G G G

G G G

DNA sequencing by Ion Torrent Personal Genome Machine

A C G C G C C G G G T C A G A A C C C G A T C G C G 5’3’

5’T G C G C G G C C C A

Primer

Only give polymerase one nucleotide at a time:

If that nucleotide is incorporated, enzymes turn by-products into light:

T C A G T C A G T C A G

1 2 3 4 5

G

T T T

T T T

DNA sequencing by Ion Torrent Personal Genome Machine

A C G C G C C G G G T C A G A A C C C G A T C G C G 5’3’

5’T G C G C G G C C C A

Primer

Only give polymerase one nucleotide at a time:

If that nucleotide is incorporated, enzymes turn by-products into light:

T C A G T C A G T C A G

1 2 3 4 5

G T

C C C

C C C

DNA sequencing by Ion Torrent Personal Genome Machine

A C G C G C C G G G T C A G A A C C C G A T C G C G 5’3’

5’T G C G C G G C C C A

Primer

Only give polymerase one nucleotide at a time:

If that nucleotide is incorporated, enzymes turn by-products into light:

T C A G T C A G T C A G

1 2 3 4 5

G T C

A A A

A A

DNA sequencing by Ion Torrent Personal Genome Machine

A C G C G C C G G G T C A G A A C C C G A T C G C G 5’3’

5’T G C G C G G C C C A

Primer

Only give polymerase one nucleotide at a time:

If that nucleotide is incorporated, enzymes turn by-products into light:

T C A G T C A G T C A G

1 2 3 4 5

G T C

T T T

T T T T

DNA sequencing by Ion Torrent Personal Genome Machine

A C G C G C C G G G T C A G A A C C C G A T C G C G 5’3’

5’T G C G C G G C C C A

Primer

Only give polymerase one nucleotide at a time:

If that nucleotide is incorporated, enzymes turn by-products into light:

T C A G T C A G T C A G

1 2 3 4 5

G T C T T

G G G

G G G G G

The real power of this method is that it can take place in millions of tiny wells in a single plate at once.

DNA sequencing by Ion Torrent Personal Genome Machine

Nat. Rev.Genet. 2010, 11, 31–46

Nature 2005, 437, 376–380

Fluor

Science 2009, 323, 133-138

Future: Nanopore sequencing

Chromatin Immunoprecipitation (ChIP) is a type of immunoprecipitation experimental technique used to investigate the interaction between proteins and DNA in the cell. It aims to determine whether specific proteins are associated with specific genomic regions, such as transcription factors on promoters or other DNA binding sites, and possibly defining cistromes. ChIP also aims to determine the specific location in the genome that various histone modifications are associated with, indicating the target of the histone modifiers.

Wikipedia, Chromatin Immunoprecipitation

RNA-seq analysis of small molecule-regulated RNA.

Sequencing

RNA seq

SPI-seq

Cross linking IP-seq

ChIP-seq TAmC-seq

Mapping of non-B DNA

DNA strand breakage mapping

Protein-DNA interaction study

Transcriptome profiling

DNA epigenetic modification

NGS

Applications of high-throughput sequencing technologies in PIP design

Dervan et al. J. Am. Chem. Soc. 2012, 134, 17814–17822 Dervan et al. J. Am. Chem. Soc. 2014, 136, 3687-3694

ChemBioChem 2014, 15, 2647-2651

IdentificationoftargetsiteforalkylatingPIPinthehumangenome

Genome-wide alkylating PIP’s high affinity binding site

ParallelSequencing

Nucleus

Micrococcalnuclease(MNase)digestion

AffinityPurification

NAR 2016, 44, 4014