DNA Nanomachines

1
DNA Nanomachines

• Presented by Jenna Cameron
• Class CS4462
• DNA Computing
• Date April 3rd 2008

2
Agenda

• Nanomachines of the last 10 years
• Chemistry Introduction
• Detection of Thrombin in Blood Serum
• Background for the Benenson paper
• A molecular computer for gene expression
• A theoretical molecular computer

3
Objectives

• Understand the basic chemistry of redox reactions
• Understand chemistry of electronic aptamer
  beacons
• Understand the importance of MDM2 in the human
  body
• Understand how a molecular computer for gene
  control works
• Understand the basics of cancer and biochemical
  signalling
• Understand the theoretical MEK1/2 inhibitor

4
Nanomachines in the last 10 years

• 1999 Mao et al - First well-structured device
  that turned right-handed B-DNA into left handed
  Z-DNA
• 2001 Research done on molecules that are real
  world molecular machines - myosin and kinesin
• 2002 Mitchell and Yurke - Tweezers joined at
  hinges with short carbon linkers
• 2004 Dittmer et al Bind and release blood
  clotting factor
• 2005 Shu DNA-induced bending of
  microcantilevers
• 2006 Weizmann et al - detection of viral DNA

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ElectroChemistry

• Electrons circle the nucleus
• Not actually on a path, but contained in clouds
• Electrons can jump into other clouds

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Redox Reactions

• Reduction Oxidization reactions occur when
  there is electron transfer
• Reduction Gain of electron
• Oxidation Loss of electron

7
Label-Free Electronic Detection of Thrombin in
Blood Serum by Using an Aptamer-Based SensorYi
Xiao, Arica A. Lubin, Alan J. Heeger and Kevin W.
Plaxco

• Aptamer DNA or RNA sequences that can bind
  specific molecular targets can have two or more
  conformations
• Aptamer Beacons Aptamers that induce large scale
  conformational changes upon binding in order to
  modulate the emission of a fluorphore
• E-AB Electronic aptamer based sensor
• Methylene Blue (MB) Heterocyclic aromatic
  chemical that has a blue colour in solution
• Electrode Chemical conductor
• Self Assembly Monolayer Organized layer of
  amphiphilic molecules where the head group shows
  specific affinity for a substrate

8
Label-Free Electronic Detection of Thrombin in
Blood Serum by Using an Aptamer-Based SensorYi
Xiao, Arica A. Lubin, Alan J. Heeger and Kevin W.
Plaxco

• Aptamer is an oligonucleotide probe with a gold
  electrode at one end and a redox reporter
  molecule at the other
• Thrombin is a blood clotting protein in humans
• Thrombin binding induces conformational change
  influencing electron transfer between reporter
  and electrode
• Developed a reusable sensor the the detection of
  a-thrombin, blood clotting factor enzyme

9
Label-Free Electronic Detection of Thrombin in
Blood Serum by Using an Aptamer-Based SensorYi
Xiao, Arica A. Lubin, Alan J. Heeger and Kevin W.
Plaxco

• Construction
• Covalently attached a MB labelled thrombin
  binding DNA aptamer to a gold electrode by self
  assembly monolayer
• Aptamer is a 32 base strand that is immobilized
  by the gold plate
• Gold plate is a polycrystalline gold disk
  electrode
• Electrodes prepared by polishing with diamond
  and alumina, sonification in water,
  electrochemical cleaning, and application of
  probe DNA
• Aptamer is unfolded in absence of target and can
  move more freely
• Upon binding, aptamer undergoes conformation
  change and is relatively stiff

10
Label-Free Electronic Detection of Thrombin in
Blood Serum by Using an Aptamer-Based SensorYi
Xiao, Arica A. Lubin, Alan J. Heeger and Kevin W.
Plaxco

• Mechanism
• When thrombin is not bound, end that is MB
  labelled can move freely
• Collides with electrode and an electron is
  transfered
• With thrombin, can no longer move and electron
  transfer is inhibited
• Conformational change alters electron tunnelling
  distance and pathway

11
Label-Free Electronic Detection of Thrombin in
Blood Serum by Using an Aptamer-Based SensorYi
Xiao, Arica A. Lubin, Alan J. Heeger and Kevin W.
Plaxco

• Aptamer is in equilibrium
• Goes between unfolded and folded state
• In folded has a G-quartet and is capable of
  binding
• Target molecule pushes equilibrium toward folded
  conformation

Without Thrombin MB can contact gold
electrode With Thrombin Molecule is in a stiff
conformation and no electron transfer occurs
5'-HS-(CH2)6-TAAGTTCATCTCCCCGGTTCCTGTGGTTGGT-(CH2)
2-MB-3' 5'-HS-(CH2)6-TAAGTTCATCTCCCCGGTGGTGGTTGTGG
TT-(CH2)2-MB-3' 5'-HS-(CH2)6-GCGAGGTAAAACGACGGCCAG
TCTCGC-(CH2)2-MB-3'
12
Label-Free Electronic Detection of Thrombin in
Blood Serum by Using an Aptamer-Based SensorYi
Xiao, Arica A. Lubin, Alan J. Heeger and Kevin W.
Plaxco
G-Quartet Four guanine bases are arranged in a
square planner array in a cyclical hydrogen
bonding pattern. Forms a very stable structure.
13
Label-Free Electronic Detection of Thrombin in
Blood Serum by Using an Aptamer-Based SensorYi
Xiao, Arica A. Lubin, Alan J. Heeger and Kevin W.
Plaxco
Left Decrease in signal in presence of thrombin
Regeneration Right Time dependence of sensor
response after 3 hours of incubation
14
Label-Free Electronic Detection of Thrombin in
Blood Serum by Using an Aptamer-Based SensorYi
Xiao, Arica A. Lubin, Alan J. Heeger and Kevin W.
Plaxco
Left Detection of signal in fetal calf
serum Right Lack of signal with thrombin present
in fetal calf serum
15
Label-Free Electronic Detection of Thrombin in
Blood Serum by Using an Aptamer-Based SensorYi
Xiao, Arica A. Lubin, Alan J. Heeger and Kevin W.
Plaxco

• Conclusions
• Successful creation of a novel electronic method
  for detection of blood clotting factor thrombin
• Method is specific and reusable
• Method performs even in the presence of other
  materials
• Limited to targets that have available DNA
  aptamers that undergo large scale conformational
  changes

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An autonomous molecular computer for logical
control of gene expressionYaakov Benenson,
Binyamin Gil, Uri Ben-Don, Rivka Adar, Ehun
Shapiro

• Creation of an autonomous biomolecular computer
  that analyzes levels of messenger RNA and
  'diagnoses' a disease, in turn releasing a drug
  or a drug suppressor
• Computer has 3 parts
• Computational Module a stochastic molecular
  automaton
• Input Module specific mRNA levels regulate
  software molecules, molecular concentrations, and
  the automaton
• Output Module Short ssDNA molecule corresponding
  to an anticancer drug or its suppressor

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An autonomous molecular computer for logical
control of gene expressionYaakov Benenson,
Binyamin Gil, Uri Ben-Don, Rivka Adar, Ehun
Shapiro

• 'Diagnosis' Identification of a combination of
  mRNA molecules at specific levels
• 'Therapy' Production of a biologically active
  molecule, a drug like ssDNA with known anticancer
  activity
• 'Diagnostic Rule' Encodes medical knowledge
• Left hand side consists of a list of molecular
  indicators for a specific disease, right hand
  side is molecule to be released

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An autonomous molecular computer for logical
control of gene expressionYaakov Benenson,
Binyamin Gil, Uri Ben-Don, Rivka Adar, Ehun
Shapiro

• System designed for the detection and treatment
  of two kinds of cancer small cell lung cancer
  and prostate cancer
• In prostate cancer, if PPAP2B and GSTPI levels
  are decreased, and PIM1 and HPN levels are
  increased, the computer will administer
  GTTGGTATTGGACATG which binds the mRNA for MDM2
  and inhibits its production
• Any short oligonucleotide (less than 22nu) can be
  administered in this system

19
An autonomous molecular computer for logical
control of gene expressionYaakov Benenson,
Binyamin Gil, Uri Ben-Don, Rivka Adar, Ehun
Shapiro

• MDM2
• Negative regulator of the tumour suppressor
  protein p53
• P53 is the guardian of the genome
• Functions to sense DNA damage and halt cell
  division
• MDM2 protein recognizes the N-terminal
  transactivation domain of p53 and acts on it as a
  E3 ubiquitin ligase
• E3 ubiquitin ligase is a type of protein that
  can attach ubiquitin to a lysine residue on a
  target protein
• Polyubiquitination marks a protein for
  degradation

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An autonomous molecular computer for logical
control of gene expressionYaakov Benenson,
Binyamin Gil, Uri Ben-Don, Rivka Adar, Ehun
Shapiro

• The left hand side of the rule is a string of
  symbols
• Each symbol can have 3 transitions Yes--Yes,
  Yes--No, No--No
• Automaton is stochastic it has 2 competing
  transitions for each symbol
• The probability of a positive transition is
  regulated by the corresponding molecular
  indicator
• The probability of a positive diagnosis is the
  product of the probabilities of the positive
  transition at each symbol
• Can change the ratio between the positive and
  negative transitions for a particular indicator
  and have fine control of the sensitivity

21
An autonomous molecular computer for logical
control of gene expressionYaakov Benenson,
Binyamin Gil, Uri Ben-Don, Rivka Adar, Ehun
Shapiro

• There are two automata
• There are transition molecules that realize
  automaton transitions and are regulated by
  molecular indicators
• Hardware is the restriction enzyme FokI
• Diagnosis moiety recognizes each symbol by a
  unique dsDNA sequence that is 7bp long
• The first four bases represent the symbol
  combined with the positive state
• The last five represent the symbol and the
  negative state

22
An autonomous molecular computer for logical
control of gene expressionYaakov Benenson,
Binyamin Gil, Uri Ben-Don, Rivka Adar, Ehun
Shapiro
23
An autonomous molecular computer for logical
control of gene expressionYaakov Benenson,
Binyamin Gil, Uri Ben-Don, Rivka Adar, Ehun
Shapiro

• Both drugs have a double stranded stem protecting
  the looped form of the drug
• Stem length is 3 bp
• After positive diagnosis the drug is released as
  the stem is cleaved by the yes-verification
  transitions
• Presence of a molecular indicator entails
  increased concentration of the positive
  transition molecule and low concentration of its
  competing negative transition molecule

24
An autonomous molecular computer for logical
control of gene expressionYaakov Benenson,
Binyamin Gil, Uri Ben-Don, Rivka Adar, Ehun
Shapiro
25
An autonomous molecular computer for logical
control of gene expressionYaakov Benenson,
Binyamin Gil, Uri Ben-Don, Rivka Adar, Ehun
Shapiro
26
An autonomous molecular computer for logical
control of gene expressionYaakov Benenson,
Binyamin Gil, Uri Ben-Don, Rivka Adar, Ehun
Shapiro
27
An autonomous molecular computer for logical
control of gene expressionYaakov Benenson,
Binyamin Gil, Uri Ben-Don, Rivka Adar, Ehun
Shapiro
28
An autonomous molecular computer for logical
control of gene expressionYaakov Benenson,
Binyamin Gil, Uri Ben-Don, Rivka Adar, Ehun
Shapiro
29
An autonomous molecular computer for logical
control of gene expressionYaakov Benenson,
Binyamin Gil, Uri Ben-Don, Rivka Adar, Ehun
Shapiro
Conclusions Molecular computer is capable of
diagnosis and treatment Releases a
currently known anti-cancer drug Has a built-in
fail safe mechanism for false positives Is
highly confident in its diagnosis
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A theoretical approach to cancer treatment by
targeting MEK1/2 Jenna Cameron

• Cancer can be broken down to six common problems
• When one or more of these are present, cells can
  proliferate unchecked and lead to tumours
• Many of these are the site of cancer treatment
  ideas

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A theoretical approach to cancer treatment by
targeting MEK1/2 Jenna Cameron

• Biochemical Introduction
• The cell has a membrane that receives signals
  from the outside world
• These signals activate or inactivate proteins
  inside the cell
• Proteins can be activated by phosphorylation
• Phosphorylation is the process of adding a
  phosphate (a small molecule that is the backbone
  of DNA) onto a protein
• Many proteins are only active once they have a
  phosphate on them
• If a protein is inappropriately phosphorylated
  or always phosphorylated it can behave improperly

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A theoretical approach to cancer treatment by
targeting MEK1/2 Jenna Cameron

• Proteins are made from DNA that is translated in
  the nucleus
• DNA is not always being translated
• It needs to be told to translate and create
  proteins
• Proteins termed transcription factors bind the
  DNA and recruit proteins to translate the DNA and
  make other proteins
• Many transcription factors are tumour suppressors
  or oncogenes

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A theoretical approach to cancer treatment by
targeting MEK1/2 Jenna Cameron
34
A theoretical approach to cancer treatment by
targeting MEK1/2 Jenna Cameron
http//www.arraybiopharma.com/ProductPipeline/Canc
er/MEK.asp
35
A theoretical approach to cancer treatment by
targeting MEK1/2 Jenna Cameron

• Structure of MEK1
• Arrow is pointing to activation loop
• This is the site of phosphorylation
• When phosphorylated, there is a conformation
  change and there is room for ATP to bind
• This makes it active
• If always active, activates ERK and causes cancer

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A theoretical approach to cancer treatment by
targeting MEK1/2 Jenna Cameron

• Currently there are drugs that can bind in the
  groove and block other proteins
• I propose a DNA computer that contains the
  binding drug in loop form (as in the Benenson
  paper) with a phosphorylation sensor
• When phosphorylation is sensed, the drug will be
  excised and can bind in the loop
• Simultaneously there should be dephosphorylation

37
A theoretical approach to cancer treatment by
targeting MEK1/2 Jenna Cameron

• There are currently known proteins that can sense
  if a protein is phosphorylated
• When it is, there should be a conformational
  change that opens up the protecting sequence to a
  restriction enzyme
• There will be a known RE site located on the
  protecting sequence
• This will allow the drug to be released
• Simultaneously, the cleavage product that is not
  the drug will be a protein phosphatase which can
  remove a phosphate from the protein making it
  inactive

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Conclusions

• Molecular computers and nanomachines are
  currently small scale
• The idea of using existing drugs is a good place
  to start as then only the mechanism of delivery
  is being tested
• More complex systems will be more beneficial
• This is a very promising area