Exam at 10-12 in FYS2Mo12.5

11)" Miniaturized PCR chips for nucleic acid research - Matti Vuento, YN121 10.15-12.00!Tu6.5

10)" Protein motors and force resistance of proteins - Jari YlänneMo28.4

9)""Structure and functions of biological membranes - Kirsi PakkanenFr25.4

8)"""Reaction mechanisms of enzymes - Ulla PentikäinenTu22.4

7)"""Regulation of cellular traffic - Varpu MarjomäkiFr18.4

6)"""Targeted molecular discovery - Olli PentikäinenTu15.4

5)""Viruses as biological self assembling systems - Jaana BamfordFr11.4

4)" Live cell imaging demo - Johanna/Ari/Einari/TeemuTu8.4

3)""Non-viral nano-carriers for molecular therapy - Johanna LaakkonenFr4.4

2) Methods for cellular imaging - Teemu IhalainenTu1.4

1) Viruses, natural nano-carriers for molecular therapy - Maija Vihinen-RantaFr28.3

Biology

SMBS813 Basics of Nanoscience 7 crp

Lectures at 8.15-10.00 in YN121

Bionanotechnology is the intersection of biology and nanotechnology

Bionanotechnology uses biological principles such as recognition and uses biological principles such as recognition and

assembly for assembly for nanotechnological nanotechnological applicationsapplications

In a wider sense, bionanotechnolgy refers to synthetic technology based on

the principles and chemical pathways of living organisms

Examples

• Molecular machines-created by studying the structure and function of the natural nano-machines found in living cells or microbes. Usage of proteins in which enzymatic activity e.g.

energy conversion or self assembly, are integrated to produce linear or rotary motion on a

nanometre scale.

• Nano-electronics and photonics - integration of electrically and optically active biomolecules toproduce devices, networks and sensors

• DNA nanotechnology, which uses self-assembling nucleic acid structures to control matter at

the nanoscale. DNA is a wonderful material for construction on the nanometre-scale

• Molecular therapy - viral and nonviral vectors

Bionanotechnology?

Taken from George Oster!s website (http://www.cnr.

berkeley.edu/~goster/oster)

Motor Proteins - Molecular machines?

Movement ofkinesin motorprotein along

microtubules

Time accelerated!

Inverted Motility Assay

Movie by Emilie Warner, Dep. of Bioengineering, UW

Howard J. et al, Methods in Cell Biology 1993, 39:137

Speed: 800 nm/s at 1 mM ATP

Microtubule

KinesinCasein

Glass

Paralleltracks

Complexpattern

Patterns fabricated by

photolithography /

soft lithography

Directed transport

Movie by Emilie Warner

Dep. of Bioengineering, UW

DNA Trapping With Dielectrophoresis

Tuukkanen et al., (2006)

Nano Letters 6: 1339-

Päivi Törmä´s group

Fluorescent DNA

DNA nanotechnology

The self-assembly properties of the DNA double helix can be exploited to

make a variety of structures.

The aim of studies is to build large, complicated, reasonably rigid 3D

structures that have controllable moving parts.

Prof. Nadrian C. Seeman, Department of Chemistry, New York University New York, NY 10003, USA

Paul W. K. Rothemund 2006. Folding DNA to createnanoscale shapes and patterns Nature 440, 297-302

Delivery

Challenges in molecular therapyChallenges in molecular therapy

DeliveryDelivery DeliveryDelivery

1)1) Packaging of the gene/drug Packaging of the gene/drug

2)2) Protection Protection

3)3) Cellular internalization, nuclear delivery and release in an active form Cellular internalization, nuclear delivery and release in an active form

Carriers/Vectors#Trojan horses! that sneak the gene/drug into the

cell

Strategies of gene therapyStrategies of gene therapy

EX-VIVO GENE TRANSFEREX-VIVO GENE TRANSFER IN-VIVO GENE TRANSFERIN-VIVO GENE TRANSFER

•• Gene repair Gene repair

•• Gene addition) Gene addition)

What is Gene Therapy

• It is a technique for correcting defectivegenes that are responsible for diseasedevelopment

• There are four approaches:1. A normal gene inserted to compensate for a

nonfunctional gene.

2. An abnormal gene traded for a normal gene

3. An abnormal gene repaired through selectivereverse mutation

4. Change the regulation of gene pairs

Viruses, natural nano-carriers formolecular therapy

How viral gene therapy works?

A vector delivers the therapeutic gene into apatient!s target cell

Phases

• The target cells become infected with the viralvector

• The vector!s genetic material is inserted into thetarget cell

• Functional proteins are created from thetherapeutic gene causing the cell to return to anormal state

Virus-mediated gene therapy

http://encarta.msn.com/media_461561269/Gene_Therapy.html

Viruses as gene transfer vehicles -why?

Viruses are among the most efficient and economical of lifeforms that serve its purpose to perfection

A virus particle is astructure that hasevolved to transfernucleic acid from onecell to another.

•Extensively studied

• genome structures known

• replication cycles known

Viral structures

Helical viruses spherical Complex

viruses viruses

vesicular stomatitis virus Adenovirus T4-fagi

Structures of viruses

Brock & Madican, Biology of microorganisms,

Pearson Prentice hall

How to make a virus vector?

• Remove viral genes, replace with foreign genetic

material

• Tame the virus

- less toxic protein products

- milder replication

• Make room for a transgene cassette

• Change tropism

Virus entry in human body

Adenovirus-mediated gene therapy

http://en.wikipedia.org/wiki/Gene_therapy

1. tumor surrounded by

healthy tissue

Introduce

tumor-selectivevirus 2. virus replication in

malignant cells

3. tumor destruction

Viral cancer gene therapy

Virus-like particles (VLPs)

- hollow VLPs lack the viral DNA

- VLPs are potential vectors for gene therapy

- synthetic therapeutic nano-containerscan be functionalized with

viral ligands

Polyoma virus-like particles (green), are taken up by cells and are often found

associated with actin fibres (red). The nuclei are stained blue (Kenny Linton, MRCClinical Sciences Centre, Faculty of Medicine, Imperial college , London UK.

Animal Cell

Steps of viral infectionIn animal cell

Virus attachment

Virus entry

Nuclear import

Replication and gene expression

Assembly

Exit (Egress)

Binding to cell surface

• The first event in any virus life-cycle - often limits infection

to the “correct” cell

• Tissue tropism - e.g. measles (skin cells) vs. mumps

(salivary gland)

• Binding to cell surface receptor - initially electrostatic,

followed by local hydrophobic interactions

Atomic force

microscopy imge of

parvoviruses (26 nm)

with receptors(TO Ihalainen)

Apical

domain of

Transferrin

receptor

CPV capsid

Viral entry into cell - pathways

Marsh M, and Helenius A (2006). Virus Entry: Open Sesame. Cell 124: 729-740

Steps in the endocyticentry of a typicalanimal virus

Marsh M, and Helenius A (2006).

Virus Entry: Open Sesame

Cell 124: 729-740

Uncoating

• Uncoating means that the stable virus stucture mustbecome unstable

• transition from extracellular (chemical) form to intracellular(biological) form

• There must be some sort of “trigger” or regulateddisassembly process

Uncoating

From Principles of Virology, Flint et al, ASM Press

Transport inside the cell cytoplasm

Microtubules and virus entry

To facilitate transport viruses often bind to the cytoskeleton

and use microtubule-mediated motor proteins for transport,

i.e. dynein

VSV/Rabies,

influenza

Adenovirus

Herpesvirus

From

Sodeik,TrendsMicrobiol8: 465

Herpesviruses

Microtubules

From Principles of Virology

Flint et al, ASM Press

Cytoplasmic capsids:

• binds microtubules via dynein

• is transported as far as the pore

• docks to the nuclear pore and then undergoes

final disassembly, and the DNA is “injected” into the nucleus

Nuclear import

Shahin V (2006). The nuclear barrier is structurally and functionally highly responsive to glucocorticoids,BioEssays 28:935-42.Kastrup L, Oberleithner H, Ludwig Y, Schafer C, Shahin V (2006). Nuclear envelope barrier leak induced bydexamethasone. J Cell Physiol, 206:428-34.

The nuclear envelope is final transport barrier

- Nuclear pore complex is the gate into the nucleus

AFM images of the cytoplasmic face of the nuclear envelope

Complexes that havenuclear localization signalare transported into thenucleus

Cytoplasm

Nucleoplasm

http://sspatel.googlepages.com/nuclearporecomplex

Classical nuclear localization signals of proteins- contains basic amino acids

Viruses, natural nano-carriers for

therapy

Advantages

• Efficiency through evolution

• Viruses are designed to penetrate the cell membrane and deliver their

medicinal payload

Problems• Immune Response. Patients' immune systems often see these viral

delivery agents as invaders rather than ambulances

• Short lived

• May induce a tumor