2015 化學生物學handout Ch. 3-2

Section 23-6 National Tsing Hua University
CHEM3100
Intramolecular reactions
6-membered ring
formation
Restriction of C-C
bond rotation
5-membered
ring formation
Locked into the

same plane
Locked into the

same plane, with
© 2014 Pearson Education Inc. better orientation
CHEM5980
DNA pairing in organic synthesis

• Exploit the DNA
pairing as
driving force to
limit the
reactants in
close proximity.
• Limited in such
close space,
reaction can
take place
under very
diluted
conditions.
• The orientation
of fragment
may be
restrained that
the selectivity
may be
controlled.
J Am Chem Soc 2010, 132, 11779
CHEM5980
DNA pairing in organic synthesis
• Replace the pairing DNA conjugate

with a longer one to install more
fragments into the target molecule
J Am Chem Soc 2011, 133, 9972

CHEM5980
DNA hairpin structure

• When complementary sequences are available in a DNA strand, it is possible to
the DNA to fold into a hairpin structure.
• When such hairpin structure were formed it resist hybridization with probes.
• DNA hairpin folding can cause some health problems. Huntington’s disease
(neurodegenerative) is caused by CTG repeats that compete with DNA duplex
formation.
CHEM5980
DNA superstructure
• Relaxed B-DNA has 10.5 base pairs per turn
• In the case of circular DNA from cells usually underwinding-fewer turn than it
would expected from B-DNA structure.
CHEM5980
DNA superstructure
• As DNA replication requires unwinding of

duplex, DNA at underwinding state is a way
for cell to save energy
• Underwinding can be stabilized by strand
separation or supercoiling, which is
preferred as it takes less energy.
• The underwind state can only be maintained
when DNA is circular or bound to protein.
Otherwise it would just twist to relax.
• Supercoil reduces the size of DNA that helps
packing the otherwise 2m DNA in total into
nucleus.
CHEM5980
Defining DNA underwinding

• How to define DNA underwinding?
• In mathmatics, topology uses linking number (Lk) to describe interwind strands.
• When 2 circular strands are linked, linking number can be described as the
times one strand pierce the surface formed by the other strand.
• If the DNA strands are arranged in right-handed helix, it is denoted (+) and vice
versa.
• Molecules that differ only at linking numbers are called topoisomers.
• Topoisomers can only be interconverted by cutting DNA strand and rejoining it.
CHEM5980
Changes in DNA conformation

• For a B-DNA with a given length, there is a number of turns required to fulfill
structural stability. If such double helix can be view as a ribbon, there are two
ways to reduce torsional strains from under-or over-winding.
• One way to reduce torsional strain is twisting the ribbon and the other is to
make writhe. Therefore linking number can be break down into these two
components, which do not necessary to be integers:
Lk = Tw + Wr
• DNA with the same Lk (topoisomers) would have many possible ways
of folding in terms of twist and writhe. The actual folding would favor
the least cost in energy.
Cell 2010, 142, 519

CHEM5980
Enzymes that change supercoiled state of DNA
• DNA replication requires opening

up the twisted helix structure.
• Circular DNA cannot change LK
without dissection and/or DNA is
bound to protein that prevent the
twisting of the double strands.
• This dilemma need to be solved
for cell to replicate. A group of
enzymes called topoisomerase
are used for this purpose.
• Topoisomerase binds to DNA,
then cleaves and reconnect DNA
to change linking number.
Cell 2010, 142, 519

CHEM5980
Enzymes that change DNA supercoiled state

• One essential feature of topoisomerase is the formation of the transient
enzyme-DNA covalent adduct to avoid the release the damaged DNA. In all
topoisomerases, this connection is made by nucleophilic tyrosine.
• The topoisomerase family can be divided into two class, type I cleavages only
one strand of DNA, while type II cleavages both strand.
• There are two mechanisms for topoisomerase type I, “strand passage” and
“swiveling”.
Nat Rev Mol Cell Biol 2011, 12, 827

CHEM5980
Enzymes that change DNA supercoiled state

• Topoisomerase type II cleavages both strand of DNA. For the mechanism, the
enzyme make passage for DNA like the type I, but the duplex are used instead of
single strand.
Nat Rev Mol Cell Biol 2011, 12, 827

CHEM5980
Targeting topoisomerase
• Given the essential functions in adjusting DNA superstructure, topoisomerases
are interesting pharmaceutical targets.
• Natural product novobiocin is produced by the actinomycete Streptomyces
niveus. This natural antibiotics binds to bacterial topoisomerase and inhibits its
function. In contrast, ciprofloxacin is synthetic compound for the same purpose.
• Other compounds target human topoisomerase II (TOP 2) and were developed
as anti-cancer drugs. They functions on TOP 2 with several mechanisms, for
example, doxorubicin is a TOP 2 poison that intercalate DNA and traps TOP 2.
Alternatively, ICRF-187 inhibits TOP 2 by trapping it as a closed clamp.
Bacterial topoisomerase II
CHEM5980
Bacterial plasmid
• Bacteria carry circular DNA called plasmid, that is not a part of genome DNA
but carries gene that confers selective advantage.
• Plasmid and genomic have sequence called origin of replication (ORI), which is
the starting point for cellular enzyme to make replicate plasmid. Different ORIs
lead different levels of replication.
• Plasmid map shows the gene encoded in the plasmid.
Tetracycline
resistant gene
CHEM5980
Selection of plasmid
• Plasmid can passes horizontally to other bacteria and provides selection
advantage to its host.
• To collect the bacteria received the engineered plasmid, an efficient selection
method was introduced.
• The antibiotic resistant gene is usually inserted into plasmid with target gene,
so that if the transformation is successful, both antibiotic resistant protein and
target protein will be expressed.
• In the media in which the bacteria grow, the
cognate antibiotic was added. These conditions kill
the bacteria did not receive engineered plasmid
cannot survive that only those successfully
transformed grow into colonies.
• Examples of lactam-based antibiotics

CHEM5980
Packing of eukaryotic DNA

• How to pack 2m long DNA in cell?
• Human genome has about 6 billion base
pairs and has to fit within nucleus.
• Human DNA is packed into several levels:
DNA duplex first wrap around a protein
called histone resulted in nucleosome.
• The bead-on-string format of
nucleosomes further packed into 30 nm
chromatin fiber.
• The 30 nm fiber can further pack into
tighter superstructure depending on the
stage in the cell cycle.
• The stored information stored must be
easy access for gene activation.
• Therefore the DNA must be pack in a very
compact way without compromise its
rapid accessibility.
CHEM5980
Histone and its modification

• Histones are found in the nucleus of
eukaryote cells to pack DNA into compact
size. DNA wrap around histone octamer
1.65 times to give a particle of ~100 Å in
diameter.
• Histones are alkaline protein that have
many positive charges on surface that
generate strong interaction with the
phosphate back bone of DNA. H2A’ H2A
• The positive charges come from the H2B H2B
arginine and lysine residues.
• Histones can be divide into 5 families
including H1/H5 as linker protein, and the
rest H2A, H2B, H3 and H4 family as core. H4 H4’
• Active genes less bind to histone, while
the inactive genes are tightly histone H3 H3’
bound.
Acta Cryst 2005, F61, 541

CHEM5980
Histone and its modification

• Histones use positive charges from Lys and Arg residue to interact with phosphodiesters
of DNA. The positive charge can be alter by acetylation to release DNA into extended
flexible form to allow transcription.
• The state of acetylation can be controlled by the enzymes called histone
acetyltransferases (HATs) and histone deacetylases (HDACs)
• The regulation of nucleosome structure through histone modification provide a powerful
way for cell to control transcription. In addition to acetylation, a number of modification
to histone have been identified including: methylation, phosphorylation, citrullination,
ADP-ribosylation and conjugation of protein such as ubiquitin and SUMO.
• Histone modification is also essential to the field of epigenetics.
Typical sites for modification

H3K4 H3K9 H3K14 H3K27 H3K79 H3K36 H4K20 H2BK5
CHEM5980
chromatin immunoprecipitation (ChIP) on chip

• Crosslinking histone to DNA, then break DNA into fragments and collect histone-complex.
• Purify and label complex DNA and analyze with whole genome DNA microarray.
• Generally, high levels of histone acetylation in promoter regions of active genes.
http://upload.wikimedia.org/wikipedia/en/8/8d/ChIP-on-chip_wet-lab.png
CHEM5980
Targeting histone modification

• Cell division and DNA replication require unwinding DNA from histone. Therefore
the control of histone modification may intervene such physiological processes.
• Protein families that methylate Lys and Arg are found to involve cancer and
neurological disorder.
• Unlike acetylation,
methylation on amine group
do not change the charge.
But this change can be
recognize by other proteins
to recruit additional enzyme
activities.
Nat Rev Drug Disco 2013, 12, 917

CHEM5980
Targeting histone modification

• As histone modification regulate DNA transcription and replication, it is not
surprising that many histone deacetylase and demethylases are involved in cancer.
• Evidences have shown the expression of several histone demethylase families
increased in tumors and that functional studies have demonstrated the requirement
of specific histone demethylase in tumor growth.
• Some promising target histone demethylases include: LSD1, JMJD2 subfamily,
JARID1B and FBXL10. Development of their inhibitors is an important topic in
pharmaceutical research.
Trapoxin A
HDAC inhibitor
Tranylcypromine
LSD1 inhibitors
GSK-J1
JMJD3 inhibitor Nat Rev Drug Disco 2013, 12, 917
CHEM5980
DNA biosynthesis
• DNA polymerase is the essential enzyme responsible for DNA biosynthesis. Many
families of DNA polymerases have been identified with different functions.
• DNA polymerase can only add new nucleotide in the direction from 5’ to 3’, also
the free 3’-OH must from a nucleotide paired with template strand. Therefore the
existence of a DNA primer strand is necessary before the reaction can begin.
• Pol δ and Pol ε are the DNA polymerases in human cells that responsible for the
replication of genome DNA.
CHEM5980
DNA biosynthesis
• The monomers used by DNA polymerase are 2’deoxynucleotidyl 5’-triphosphates
namely dATP, dCTP, dGTP and dTTP for corresponding bases.
• Mg2+ ion are required in human DNA polymerase. In each incorporation of
nucleotide, magnesium alkoxide was formed to attack triphosphate group.
• The leaving pyrophosphate group is stabilized by another magnesium ion.
CHEM5980
Fidelity in DNA biosynthesis

• DNA is a major media to store genetic information that mutation in DNA sequence may
cause change in protein expression, which usually gives adverse effect.
• Mutations in genomic DNA will also pass to future generations.
• The fidelity of DNA replication/transcription is essential for the survival of species.
• DNA polymerase grips growing DNA like holding a rope in palm to sense the base pairing.
• To aid the fidelity in DNA biosynthesis, DNA polymerase is equipped with an exonuclease
domain that removes mismatch nucleotide in the growing strand.
• With this proofreading mechanism, the error rate for DNA polymerase is about 1 out of
100,000,000.
Growing
strand
Exonuclease Templat
domain e strand
Nat Rev Genetics 2008, 9, 594

CHEM3100
DNA replication
• For DNA to replicate, the double strand
structure unwinds and each of the parent
strand is used as template to synthesize a
new daughter strand, which readily forms
new double helix with a parent strand
• The replication proceeds toward the
direction od parent double helix and
forms a fork-like shape
Issues regarding DNA replication:
• How does double strands unwind? How

does this process impact the entire
structure?
• DNA is synthesized in 5’ to 3’ direction.
How is this DNA synthesis achieved for
the both daughter strands?
• What is the error-proof mechanism for
DNA replication?
© 2014 Pearson Education Inc.
CHEM3100
DNA replication fork

DNA DNA
polymerase primase
Lagging 3’
Topoisomerase
strand
5’ RNA
DNA Okazaki primer Helicase 5’
ligase fragment
5’ 3’
Leading
strand
3’
DNA Single strand
polymerase binding protein
• In the leading strand, one RNA primer was used and the daughter strand is
synthesized by DNA polymerase in a continuous manner
• In the lagging strand, the daughter is synthesized in fragments, in which RNA primer
is required for each on them. Then the RNA portion is removed by RNase and the
vacancy is DNA by the action of another DNA polymerase. The nicks are sealed by
DNA ligase
CHEM5980
Reverse transcriptase
• The common flow of genetic information is from DNA to RNA, however some
RNA virus have genome composed of RNA.
• Reverse transcriptase is a type of DNA polymerase that uses RNA as template.
• DNA polymerase requires primer to initiate reaction, in case of reverse
transcriptase the primer is as simple as human transfer RNA (tRNA).
• The unique properties of reverse transcriptase has made it as useful tool in
molecular biology. Other than to synthesize one strand of DNA from RNA
template, reverse transcriptase can destroy RNA template.
• The RNA cleaving property of reverse transcriptase allows its subunit, RNase H,
to be used to cleavage RNA in research.
HIV Life cycle
HIV Virion
Co-receptor Golgi
CD4 Glycosylation
dsDNA
ER Translation
Reverse
transcription
Integration
ssRNA Host DNA

T cell Transcription ssRNA
Nucleus
• Error-prone reverse transcription result in high mutation rate

• Envelope protein heavily modified with carbohydrate
CHEM5980
Drugs targeting HIV reverse transcriptase

• Reverse transcriptase in HIV
infection is encode by HIV and
packed in virion.
• Therefore it has become a obvious
pharmaceutical target. A number of
inhibitors were approved and widely
use to prolong the progression to
AIDS.
• Highly active antiretroviral therapy
(HAART), also known as a cocktail
therapy, is current strategy to treat
HIV infection. Inhibitors with
different mechanisms used
concurrently.
Nat Rev Drug Disco 2002, 1, 13

CHEM5980
Modified substrate for DNA polymerase

• DNA polymerase is efficient and reliable enzyme for DNA synthesis that it
recognize natural nucleotide pairing with high fidelity, but does it take artificial
substrates?
• If artificial substrate can be exploited by DNA polymerase, unnatural nucleic base
can be incorporated into DNA.
• Modified thymidylate residues with compatible Watson-Crick pairing pattern have
been accepted by DNA polymerase to inserted into DNA.
• Unnatural base pair in DNA could expand the genetic code and aid development
in DNA nanotechnology.
CHEM3100
Polymerase chain reaction
• The amount of gene copies in cell is too little

to handle in vitro and the entire DNA is too
long for the gene of interest: Need for a
method to amplify gene of interest
• DNA polymerase is an efficient enzyme with
high fidelity in DNA replication
• Specific DNA sequences can be amplified by
repeated replication cycles
• Replication with DNA polymerase requires
the separation of 2 DNA strands, which is
achieved by heating to break hydrogen
bonds
© 2014 Pearson Education Inc.

CHEM3100
Conditions for PCR

To use DNA polymerase in vitro:
• How to separate 2 strands of DNA?
• How to initiate the action of DNA polymerase
• How to assign the target DNA region to be amplified?
Region of target DNA
to be amplified How to improve the tedious process?
• Avoid addition of reagents in

Heat to separate each cycle?
① double strands 95˚C
3’ 5’ Use excess reagents
5’ 3’ • Avoid addition of enzyme in
Cool and addition Repeat each cycle?
② of primers 54˚C ①~③ steps Use heat stable enzymes--
3’ 5’ DNA polymerase from
5’
5’ thermophilic bacteria
5’ 3’
Addition of reagents
③ and DNA polymerase 72 ˚C Only control
reaction
3’ 5’ temperature Target DNA
5’ 3’ amplification
3’ 5’
5’ 3’ with ease
CHEM3100
Outcome of PCR
• The amount of gene copies are amplified exponentially
• Target DNA region predominates after several cycles
2F
3’ 5’
①②
3’ 5’ 2F
5’
5’ 5’ 3’ 6H
5’ 3’
8T
③ 3’ 5’
3’ 5’
2F 5’
5’ 3’
2H 5’
5’ 3’ 95˚C then 54˚C
95˚C then 54˚C then 72˚C
3’ 5’
3’ 5’
5’ 5’
2F
3’ 3’
72˚C 4H
3’ 5’ 3’ 5’
2T
5’ 3’ 5’ 3’
CHEM5980
Quantitative polymerase chain reaction

• Quantitative polymerase chain reaction (qPCR) monitors the
amplification results from each thermo cycle.
• This process quantifies the dynamic change of DNA
concentration with time and therefore also know as real-time
PCR. In this regard, the traditional PCR that only amplifies target
DNA can be called end-point PCR.
ex: λmax = 488 nm
• To quantify the amount of double strand DNA, SYPR Green is em: λmax = 522 nm
used. As this dye becomes more fluorescent when intercalate
with double strand DNA.
• This analysis requires a qPCR instrument that reads fluorescence.
• The application includes: Provide quantitative measurements of
gene transcription; rapid diagnosis of disease; Clinical
quantification and genotyping of virus
http://www.bio-rad.com/en-us/applications-
technologies/qpcr-real-time-pcr
Anal Bioanal Chem 2009, 393, 1629
CHEM5980
Reverse transcription polymerase chain reaction

• Reverse transcription PCR (RT-PCR)
should not be confused with real-time
PCR (qPCR).
• Use reverse transcription to generate
cDNA then follow PCR procedures.
• Allows analysis of RNA concentration
to monitor gene activity.
http://rna-dna.com/wp-content/uploads/2014/05/RT-PCR.jpg
CHEM5980
From gene to protein

• Understanding from gene to protein relies on knowing the genetic code on
DNA. However the sequencing technology was developed much later after the
discovery of DNA structure. Although the composition of DNA can be analyzed
to show the percentage of each nucleic base, this information is not very
useful.
• Theories have been proposed to correlate genetic codes and protein sequence.
One theory, called diamond code hypothesis, suggests the minor groove of
DNA duplex fits complementary amino acid side chains.
Minor groove
Amino acid fits here
• Chemical synthesis of DNA had become a strategy to help this situation

because the sequence can be controlled in stepwise synthesis by the order of
reagent used.
CHEM5980
From gene to protein

• The connection between genetic and
protein sequences was first investigated
by enzymatic synthesized RNAs and
their protein products.
• With RNA consisted with single or
randomly mixed nucleic bases. It can be
concluded that triplet nucleic bases
(codon) are responsible for determining
amino acids.
• 50 out of 64 combinations but not the
order were identified from three-
nucleotide set.
• Chemical synthesis of DNA had become
a strategy to help this situation because
the sequence can be controlled in
stepwise synthesis by the order of
reagent used.
CHEM5980
DNA synthesis from phosphate coupling

• Oligonucleotide are connected by phosphodiester bonds, therefore it is
straightforward to extend such linkage step by step.
• Khorana method uses acetyl group to protect 3’ OH to avoid building block “self-
polymerization”. The protecting group is removed after each coupling to expose
the 3’ OH for the next step.
• The major problem to this strategy is the lower yield, generally lower than 80%,
which also increase the difficulty in purification.
CHEM5980
Phosphite and phosphate chemistry
• To solve the low yield problem from

phosphate-based synthesis, phosphite ester
was exploited.
• Phosphite are more reactive than phosphate
due to the lone pair on the oxygen, which
donates electron into phosphorus empty
orbital and prevent nucleophile from
incoming.
• The relative reaction rate of phosphite,
comparing to phosphate, is especially
overwhelming at acidic conditions.
CHEM3100
Protecting groups in oligonucleotide synthesis

• Two orthogonal protecting groups are require for phosphite-based oligonucleotide
synthesis. The 5’ OH protecting group needs to be removed after every coupling,
while the one on phosphite can be removed after the entire oligonucleotide
completed.
• Trityl group is used to 5’ OH and can be removed by acid.
• Cyanoethanol group protects phosphite and can be removed by ammonia
O O
O
H+ H
Deprotection of O R + R OH
O R
trityl group
O O
O
Dimethoxytrityl ether Orange colored
(DMTr-OR)
Deprotection of R1O OR2

N R1O OR2
cyanoethanol group C
O
P
O
N
C + P
NH3 HO O
H H
CHEM5980
DNA synthesis from phosphite coupling

• In addition to the trityl and cyanoethanol
protecting groups, Caruthers method use
stable phosphoramide as building blocks.
• Three out of four nucleic bases have free
amine groups that need to be protected.
Adenine and cytosine are protected by
benzoyl groups, while guanine is protected as
dimethylformamidine.
• Under acidic conditions, protonated
diisopropylamine becomes a good leaving
group and facilitate the substitution reaction.
CHEM5980
Solid-phase strategy for DNA synthesis

• The concept of solid-phase synthesis was initially used on peptide synthesis. The
idea allows quick purification and is extremely beneficial for biopolymer synthesis.
• For the ease of purification, the reagent can be used in large excess to increase
reaction rate and to drive the reaction to completion.
• In the case of nucleic acid synthesis, porous glass bead were used as solid support.
• 3-aminopropyl triethoxysilane (APTES) is usually used to functionalize the glass
surface for loading compounds.
wash
Porous glass bead under

electron microscope
CHEM3100
Solid phase oligonucleotide synthesis

• Solid phase synthesis avoids tedious separation steps for oligonucleotide synthesis
and allows automated production
• Current solid phase synthesis allows up to 200 nucleotide residues
DMTr O
Base HO
Base
O
O
CN
DMTr O O DMTr O
CN
HO Base HO Base O
Base H O Base O P O O
H O P O
O N CN O Base N CN
+
N O N O Base
CN O O
N N O P N(i -Pr)2 CN N N O P N(i -Pr)2 O
O NH4OH, heat
oligonucleotide
O CN
O CN
O P O O P O
O O
Base
O
Base O P O
Base
coupling O
O P O
O Base
trace O O O O
unreacted
O O
O
chain O
Ac2O
DMTr HA
capping deprotection
DMTr O
DMTr O Base
Base O
O
CN
AcO CN O
Base O
O O P O
O P O Base
Base O O
CN
O O I2
CN
CN
oxidation
O P O O
Base O
O O O P O
O P O Base
capped O O O
Base O O
chain O
O
CHEM5980
The role of tetrazole

• Tetrazole act as a catalyst in the substitution reaction. It replaces diisopropylamine
and has pKa similar to acetic acid that make it a good leaving group.
• Explosive, now sold in solution.
CHEM5980
Capping process
• The 1% difference in yield for a single-step reaction may
not seem important, but to make a long oligomer in
stepwise manner, the overall yield decay exponentially.
• In addition to the overall yield, the final purification will
be affected by the accumulation of impurity from
lengthy steps.
• One way to aid final purification is capping process. This
reaction will terminate the free functional groups that
failed to couple. Termination cause the faulty oligomer
to be very different to product in length and therefore
easier to separate them.
• Acetylation on free OH is the capping method in solid-
phase DNA synthesis.
CHEM5980
Oxidation to phosphate
• Phosphites are known for the activity in the substitution reactions and
therefore not stable. In contrast, phosphates are much more stable.
• To avoid side reactions, the phosphites are oxidized to phosphate after
each coupling steps.
• Molecular iodine is used as very mild oxidant.
CHEM5980
Deprotection of cyanoethanol and cleavage

• After the entire coupling of DNA is complete, the cyanoethanol protecting
group can be removed under heated ammonia.
• The basic condition also cleaves the deprotected DNA from porous glass
beads.
• Under basic conditions, the ester bond that links DNA to glass bead will be
cleavaed to release DNA
CHEM5980
Automated and custom DNA synthesis

• The solid-phase synthesis of DNA allows the Exemplary pricing in US$
production of DNA to be automated.
• This technology allowed regular laboratory
to make specific DNAs for their investigation.
• For the laboratories that do not want to
invest the instrument, custom service are
always available. This is extremely useful for
biological related laboratory, which are not
specialists in chemical synthesis.
CHEM5980
Synthetic life
• Given that longer nucleic acid can be synthesized with automation, is it possible to
chemically synthesize the genome of an organism?
• Do the chromosomes contain the entire genome information? If the genome of an
organism can be synthesized, can such organism survive?
• Modification of genome may create new life forms.
• To test this idea and to develop the technology, it took more than 10 years with an
estimated cost of US$ 40 million.
Science 2010, 329, 52

CHEM5980
Synthetic life
• Due to small genome size at about 1 million base pairs, Mycoplasma mycoides was
chosen for the project.
• The genome can be break into about 100 of 1000 base pair fragment that can be
chemically synthesized. Then the fragments were connected and transplant to
recipient cells.
Science 2010, 329, 52

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Section 23-6 National Tsing Hua University

Locked into the

Locked into the

DNA pairing in organic synthesis

DNA pairing in organic synthesis

• Replace the pairing DNA conjugate

J Am Chem Soc 2011, 133, 9972

DNA hairpin structure

• As DNA replication requires unwinding of

Defining DNA underwinding

Changes in DNA conformation

Cell 2010, 142, 519

Enzymes that change supercoiled state of DNA

• DNA replication requires opening

Cell 2010, 142, 519

Enzymes that change DNA supercoiled state

Nat Rev Mol Cell Biol 2011, 12, 827

Enzymes that change DNA supercoiled state

Nat Rev Mol Cell Biol 2011, 12, 827

• Examples of lactam-based antibiotics

Packing of eukaryotic DNA

Histone and its modification

Acta Cryst 2005, F61, 541

Histone and its modification

Typical sites for modification

chromatin immunoprecipitation (ChIP) on chip

Targeting histone modification

Nat Rev Drug Disco 2013, 12, 917

Targeting histone modification

Fidelity in DNA biosynthesis

Nat Rev Genetics 2008, 9, 594

Issues regarding DNA replication:

• How does double strands unwind? How

DNA replication fork

ssRNA Host DNA

• Error-prone reverse transcription result in high mutation rate

Drugs targeting HIV reverse transcriptase

Nat Rev Drug Disco 2002, 1, 13

Modified substrate for DNA polymerase

Polymerase chain reaction

• The amount of gene copies in cell is too little

© 2014 Pearson Education Inc.

Conditions for PCR

• Avoid addition of reagents in

Quantitative polymerase chain reaction

Reverse transcription polymerase chain reaction

From gene to protein

Amino acid fits here

• Chemical synthesis of DNA had become a strategy to help this situation

From gene to protein

DNA synthesis from phosphate coupling

Phosphite and phosphate chemistry

• To solve the low yield problem from

Protecting groups in oligonucleotide synthesis

Deprotection of R1O OR2

DNA synthesis from phosphite coupling

Solid-phase strategy for DNA synthesis

Porous glass bead under

Solid phase oligonucleotide synthesis

The role of tetrazole

Deprotection of cyanoethanol and cleavage

Automated and custom DNA synthesis

Science 2010, 329, 52

Science 2010, 329, 52