DNA Nanomachines - PowerPoint PPT Presentation

Loading...

PPT – DNA Nanomachines PowerPoint presentation | free to view - id: 14e78-Nzg5M



Loading


The Adobe Flash plugin is needed to view this content

Get the plugin now

View by Category
About This Presentation
Title:

DNA Nanomachines

Description:

Jenna Cameron. Proteins are made from DNA that is ... Jenna Cameron. There are currently known proteins that can sense if a protein is phosphorylated ... – PowerPoint PPT presentation

Number of Views:700
Avg rating:3.0/5.0
Slides: 39
Provided by: jennac
Category:

less

Write a Comment
User Comments (0)
Transcript and Presenter's Notes

Title: 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

5
ElectroChemistry
  • Electrons circle the nucleus
  • Not actually on a path, but contained in clouds
  • Electrons can jump into other clouds

6
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

16
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

17
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

18
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

20
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
30
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

31
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

32
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

33
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

36
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

38
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
About PowerShow.com