advanced course in molecular biology and...

HiroshiSugiyamaDepartmentofChemistry,GraduateSchoolofScienceInstituteforIntegratedCell-MaterialSciences(iCeMS)

KyotoUniversity1

100nm

生体分子機能論 2018-1化学構造

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

Basic elements of nucleic acids and their synthesis

1)  Phosphodiester bond

2)  Watson Crick base pair

3)  syn and anti, α and β, C2’ endo and C3’ endo

4)  Phosphoramidite method and PCR

5)  Aptamer, nucleic acid-based drug

Nucleic Acids

In nucleic acid, the phosphate group is phosphodiester

The Bases in Nucleic Acids

Adenine, guanine, cytosine, and thymine are found in DNA

Adenine, guanine, cytosine, and uracil are found in RNA

Why does DNA contain thymine instead of uracil?

The synthesis of thymine is energetically expensive

DNA: G C T A RNA: G C U A

Nucleosides = Base + Sugar

Nucleotides = Base + Sugar + Phosphate

The sugar–phosphate is on the outside, and the bases are on the inside

Complementary Base Pairing in DNA

Major groove

Minor groove

Groove width (Å)

Groove depth (Å)

5.7

11.7

7.5

8.8

Structure sense: Right-handed Residues per turn: 10 C2'-endo

χ: antiγ: g+

A

B-DNA�

Nucleic Acids

Phosphoramidite method Laboratory Synthesis of DNA

Phosphoramidite method 1. Detritylation 2. Activation and Coupling 3. Capping 4. Oxidation 5. Detritylation

1.  Detritylation

2. Activation and Coupling

4. Oxidation

Phosphoramidite method 1. Detritylation 2. Activation and Coupling 3. Capping 4. Oxidation 5. Detritylation

DNA Synthesis

Cycle number

The amino groups of the bases must be protected

HN

N N

N

O

H2N

H3C

H3C

O

O

O CH3CH3 HN

N N

N

O

NH

H3C

H3C

O

guanine

HO

O CH3CH3

+

base-PrO

OPOO

O

Obase-Pr

O

O

CN

base-PrO

OPO

HO

OCN

O

HNO

solid support

base-PrO

OP-OO

O

Obase-Pr

O

OH

base-PrO

OP-O

HO

ONH4OH

rt, 1 h

baseO

OP-OO

O

Obase

O

OH

baseO

OP-O

HO

ONH4OH

55°C, 12 h

Capping

PCR in vitro selection (aptamer) DNA micro array

PCR(polymerase chain reaction)

PCR

Principle

denature annealing elongation

Temperature （℃）

95℃

72℃

55℃

amplification

1 cycle

The polymerase chain reaction (PCR) is a biochemical technology in molecular biology to amplify a single or a few copies of a piece of

DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.

in vitro selection

Louis C. Bock, Linda C. Griffin, John A. Lathan, Eric H. Vermaas & John J. Toole Nature. 1992, 355, 564-566

~1013 96mer DNA Pool

thrombin aptamer

in vitro selection

result

GGNTGGN2-5GGGNTGG


in vitro selection

result


Macugen : VEGF Aptamer (vascular endothelial growth factor) age-related macular degeneration (AMD) Pegaptanib is a pegylated anti-VEGF aptamer, a single strand of nucleic acid that binds with specificity to a particular target. Pegaptanib specifically binds to VEGF 165, a protein that plays a critical role in angiogenesis (the formation of new blood vessels) and increased permeability (leakage from blood vessels), two of the primary pathological processes responsible for the vision loss associated with neovascular AMD.

A)Vitraveneisanantiviraldrug.Itisusedinthetreatmentofcytomegalovirusretinitis(CMV)inimmunocompromisedpatients,includingthosewithAIDS.ItwaslicensedbytheFDAforCMVin1998.

C)Kynamro:isacholesterol-reducingdrug.ItisanantisensetherapeuticthattargetsthemessengerRNAforapolipoproteinB.Itisadministeredasaweeklyinjectionforfamilialhypercholesterolemia.ItwasapprovedFDAin2013.

S-oligo : 5'-GMeCMeCMeUMeC AGTMeCTGMeCTTMeC GMeCAMeCMeC- 3'

OPO

Na+S-

O

BaseOO

OCH2CH2OCH3

2'

5' MeC : MeU :

OPO

Na+S-

O

BaseOO

H2'

N

N

NH2

O

5

R

N

NH

O

O

5

R

3'

S-oligo : 5'-GCGTTTGCTCTTCTTCTTGCG-3'

OPO

Na+S-

O

BaseOO

2'

5'

3'

B)Macugenisananti-angiogenicmedicineforthetreatmentofneovascular(wet)age-relatedmaculardegeneration(AMD).ApprovalwasgrantedbytheFDAin2004.

G G C

AA C

AC UA

CGU

A A O5'

3'

NH5

O

HN

NH

O

4O

O450

O

O

O

450

PEG

O

P O

O

-O

T

O

OH5'3'

U U A U

A G U G

U C C G

OPO

OO-

BaseOO

OCH3

A, G

2'

OPO

OO-

BaseOO

F

U, C

2'

OPO

OO-

BaseOO

OH2'

A

Nusinersen, marketed as Spinraza,[3] is a medication used in treating spinal muscular atrophy (SMA), a rare neuromuscular disorder. In December 2016, it became the first approved drug used in treating this disorder. Spinraza list price is US$125,000 per injection which puts the treatment cost at US$750,000 in the first year and US$375,000 annually after that. According to the New York Times, this places Spinraza "among the most expensive drugs in the world".

Eteplirsen (Exondys 51, Sarepta Therapeutics Inc.), also called AVI-4658, is a drug designed for treatment, but not a cure, of some mutations that cause Duchenne muscular dystrophy (DMD), a genetic degenerative muscle disease.

A year's worth of treatment with Eteplirsen is expected to cost approximately $300,000.

Alnylam Announces First-Ever FDA Approval of an RNAi Therapeutic, ONPATTRO™ (patisiran) for the Treatment of the Polyneuropathy of Hereditary Transthyretin-Mediated Amyloidosis in Adults Aug 10,2018 − First and Only FDA-approved Treatment Available in the United States for this Indication – − ONPATTRO Shown to Improve Polyneuropathy Relative to Placebo, with Reversal of Neuropathy Impairment Compared to Baseline in Majority of Patients – − Improvement in Specified Measures of Quality of Life and Disease Burden Demonstrated Across Diverse, Global Patient Population – − Alnylam to Host Conference Call Today at 3:00 p.m. ET. − CAMBRIDGE, Mass.--(BUSINESS WIRE)--Aug. 10, 2018-- Alnylam Pharmaceuticals, Inc. (Nasdaq: ALNY), the leading RNAi therapeutics company, announced today that the United States Food and Drug Administration (FDA) approved ONPATTRO™ (patisiran) lipid complex injection, a first-of-its-kind RNA interference (RNAi) therapeutic, for the treatment of the polyneuropathy of hereditary transthyretin-mediated (hATTR) amyloidosis in adults. ONPATTRO is the first and only FDA-approved treatment for this indication. hATTR amyloidosis is a rare, inherited, rapidly progressive and life-threatening disease with a constellation of manifestations.

It was approved by the FDA in August 2018 and is expected to cost around $345,000 to $450,000 per year.

Gregory Verdine, Ph.D.Chairman, WAVE Life Sciences & President and CEO, FOG Pharmaceuticals Paul Bolno, M.D., MBAPresident and Chief Executive Officer, WAVE Life Sciences Peter Kolchinsky, Ph.D.Managing General Partner, RA Capital Management Koji MiuraManaging Director, Miura & Associates Management Consultants Pte. Ltd. Ken Takanashi, MBA, CPASenior Managing Director, Shin Nippon Biomedical Laboratories Ltd. Masaharu TanakaPresident, Kagoshima Development Co. Ltd. Takeshi Wada, Ph.D.Professor, Tokyo University of Science

DNA microarray

advanced course in molecular biology and...

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