Gamow explaining a point of interest to members of the Junior ...

triteritzyBiotechnology

Dec 14, 2012 (4 years and 8 months ago)

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MAE 291


Biological Nanotechnology


Goals


entrée into
nanoscale

biology for engineers


and engineering point of view for life scientists


learn enough about subject to be able to



recognize where biology is relevant to



engineering and vice versa


learn enough to read current literature yourself


learn to read research papers critically


Philosophy


course should be intellectually challenging


and fun; grading will take into account divergent


backgrounds and effort




Didactic plan



lecture
-
discussion
based on
~
1
-
2 literature papers/week


sources of info: Google, Wikipedia, Philip Nelson’s Biological


Physics: Energy, Information, Life



homework
problems based on
lit.
paper each
week: may do as a



group
, get
any help you want, but present in your own words


mid
-
term, final will resemble homework problems


1 oral or written presentation/critique
of
lit. paper: I’ll suggest good


candidates each week, or pick yourself (but check with me)


grading
: 20%
each class participation, homework, midterm, student


presentation, final; or 25% each if you choose not to take final

Contact


Prof. J. Silver,
jesilver@gwu.edu

Office hours:
TBA


Papers, lecture notes, homework, previous

week’s homework answers, announcements

will be on Blackboard
(along with related papers

that might make good candidates for student

presentations)

Theme of course




biological molecules have rich structure

and functions as
nanomachines


engineering tools help make it possible to learn about

their structure and how they work


with increasing knowledge we can make new

nanostructures/machines with the same components


principles/processes of
nanoscale

biological materials

can inform design of non
-
biological nanotech./engineering

References for class 1


Philip Nelson Biological Physics Ch 2 section 2.2

The Molecular Parts List, pp.45
-
62.


http://www.exploratorium.edu/origins/coldspring

/ideas/printit.html



Watson/Crick paper 1954


http://www.neb.com/nebecomm/tech_reference

Genetic code; amino acid structures; DNA base pairs;

Products


Wikipedia



DNA structure, mechanical properties, etc.

DNA double helix
2nm
3.3nm
10
bp
1
2
4
5
Biological Macromolecules
-

DNA

Base pairing


at edges



holds strands

together


Base stacking


above & below
-

compresses

ds

into helix


Boiling separates

strands

RNA


like DNA, except OH at 2’ position, and
Uridine

for Thymine

5’

5’

3’

3’

N

http://www.google.co
m/images?q=DNA&hl
=en&gbv=2&tbs=isch:
1,simg:CAISEglYWPmr
g53qoyHd2bQ6MaaJl
A,sit:o&iact=hc&vpx=
409&vpy=76&dur=13
73&hovh=232&hovw=
217&tx=73&ty=305&e
i=theBTJiwK4aBlAf_zp
WuDg&oei=theBTJiwK
4aBlAf_zpWuDg&esq=
1&page=1&tbnh=127
&tbnw=118&ved=1t:7
22,r:12,s:0&biw=956&
bih=572

3

2

1

4

5

5

3

5

5

5

5

3

3

3

3

3

5

Cutting at P

-
> one 3’ and

one 5’ end

purines


pyrimidines

(2 rings)



(1 ring)

Watson
-
Crick base pairs


G
-
C 3 hydrogen bonds







A
-
T 2 hydrogen bonds



other hydrogen bond


base pairs are possible


in special circum
-


stances (class 4)

Implications of
double helix
structure


Suggests a replication mechanism based on separating


strands, assembling a new copy strand with



complementary sequence by base pairing



engineering application


pol. chain
rxn
. (
pcr
)


Suggests a sequence
-
specific binding between strands



with complementary sequence


many applications!


Makes
dsDNA

stiffer than
ssDNA
, but how stiff?



Over what length? (
~
50nm, class 5)


Helix suggests rotational motion may be important


e.g.


to unwind, to move along grooves in helix (class 6)

DNA can assume other structures besides the “B
-
form”


Watson
-
Crick described, when single
-
stranded or


when
dsDNA

is subjected to unusual conditions


(dehydration, non
-
physiologic salts, interaction with


intercalating dyes, pulling/twisting forces) e.g.


A



B



Z











some
alterna
-








tive

forms will








be considered








in classes 4
-

6




Enzymes that act on DNA or RNA


DNA
polymerase
N









strands are







“anti
-
parallel”
















extension







adds to 3’
-
end








pol

requires primer to start synthesis

primer

template

Can chemically
synthesize
N

(buy) short pieces of DNA



(“
oligo”nucleotides

~
1
-
100 bases) of any sequence



(
~

$1/base for
m
mol

= 6x10
17

molecules)


So can “prime” synthesis at any chosen location on template




-
> idea for
exponential

synthesis of segment of DNA


using 2 primers that hybridize to opposite strands


with appropriate 5’
-
>3’ orientations





Polymerase chain
reaction
N

(
pcr
) amplification of DNA

w/
thermostable

DNA polymerase (e.g.
Taq

pol
)

forward

primer

template

strand (A)

copy strand (B)

Taq

pol

Melt DNA (94
o
C), cool (60
o
C) to anneal primers, extend (72
o
C)

reverse primer

new strand A

Old and new templates are not destroyed by melting,


so repeated cycles of melting and polymerization
-
>


1
-
> 2
-
> 4
-
> 8
-
> …
-
> 2
n

copies of DNA region lying

b
etween 2 primer
-
annealing sites on initial template


Polymerase copies
~
1000b/m =>

~
1 hour for 2
30

=10
10
-
fold amp. of a kb piece of DNA



http://www.youtube.com/watch?v=_YgXcJ4n
-
kQ


http://www.dnalc.org/ddnalc/resources/animations.html


1
gggcggcgac

ctcgcgggtt

ttcgctattt

atgaaaattt

tccggtttaa

ggcgtttccg

61


ttcttcttcg

tcataactta

atgtttttat

ttaaaatacc

ctctgaaaag

aaaggaaacg

121


acaggtgctg

aaagcgaggc

tttttggcct

ctgtcgtttc

ctttctctgt

ttttgtccgt

181


ggaatgaaca

atggaagtca

acaaaaagca

gctggctgac

attttcggtg

cgagtatccg

241


taccattcag

aactggcagg

aacagggaat

gcccgttctg

cgaggcggtg

gcaagggtaa

301

Portion of sequence of lambda phage DNA


reads 5’
-
>3’, only one strand of
dsDNA

shown

Could you write sequence of opposite strand?


Could you specify sequence of two 20 base primers to


amplify segment consisting of bases 11
-
290?




More enzymes that work on DNA



Reverse transcriptase


copies
ss

RNA into DNA




DNA primer

RNA template









(can also use
ssDNA

as template)

Reverse
transcriptase
N

Other enzymes that act on DNA


restriction
enzymes
N


Cut DNA backbone at

specific short sequence;

may leave
ss

overhangs

that can be used to direct

assembly of DNA pieces

with complementary

overhangs

EcoRI


5’
--
GAATTC
--



3’
--
CTTAAG
--


NheI

5’
--
GCTAGC
--



3’
--
CGATCG
--


AvrII

5’
--
CCTAGG
--



3’
--
GGATCC
--



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

http://www.dnalc.org/resources/animations/restriction.html

What would

cutting with

EcoRI

produce?


Cutting with

EcoRI

+
PstI
?


Cutting with

SpeI

+
SacI
?

Map of enzymes that cut once

http://www.neb.com/nebecomm/tech_reference/restr
iction_enzymes/dna_sequences_maps.asp


Other enzymes that act on DNA


Ligases



reform
phosphodiester

bonds


join pieces of DNA


reverse effects of restriction enzymes


may be guided by annealing of complementary overhangs



fragments A



fragments B

GATC
--
atc
--
-


+

GATC
-
gcc
--


--
tag
---
CTAG



-
cgg
--
TTAA


note multiple possible ligation products:

AA, A , AB, B, AAA…, A ,
gatc
B

B
ttaa
,…

Can you get BA, BBB?

gatc
B

B
ttaa

A

A

Some
ligated

fragments can be
recut
:


EcoRI

(GAATTC)
EcoRI


product restores
EcoRI

site

5’
--
G AATTC
--

3’

5’
--
GAATTC
--

3’
--
CTTAA G
--

5’

3’
--
CTTAAG
--




Others cannot:

NheI

(GCTAGC)

AvrII

(CCTAGG) product =
NheI

or
AvrII

5’
--
G


CTAGG
--
3’


5’
--
GCTAGG
--

3’
--
CGATC


C
--
5’


3’
--
CGATCC
--

\


More enzymes that act on DNA



Gyrases



unwind
ds

DNAs


Topoisomerases



cut and
religate


DNA strands, allowing one DNA

segment to pass through another,

r
elieving
torsional

strain or

u
ntangling entwined
dsDNA

circles

(class 7)


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

RNA
polymerase
N



partially melts
dsDNA

template





and makes
ssRNA

copy






Some RNA
pol’s

can use





ssDNA

as template






(RNA is same as DNA except for





OH group instead of H at pos. 2





on sugar and base U instead of T;





RNA can be
ss

or
ds
, can





base
-
pair with “complementary”





DNA or RNA)

Protein = linear polymer of amino acids (
aa
)

Chains from a few to


~ 1000
aa

long


Order of
aa’s

determine

protein structure,

interacting surfaces,

properties, function;

m
ost enzymes are
prot
.


aa

order encoded in

order of bases in RNA

Ribosome (protein
-
RNA complex) “reads”
ssRNA

sequence and assembles corresponding protein

Coding conundrum

How can 4 bases encode
20

aa
?

Pairs of bases could only encode 16
aa

Code must involve at least triplets…


but then 64
codons
. Why so many?

Another problem for triplet code:


Which is correct reading frame?


…ACGTGCCTGATT…


…ACG
-
TGC
-
CTG
-
ATT… or


..A
-
CGT
-
GCC
-
TGA
-
TT… or


.AC
-
GTG
-
CCT
-
GAT
-
T…?

(
Photo: Gamow explaining a point of
interest to members of the Junior
Academy of Scientists (GW
Lisner

Auditorium, 1952
))

Maybe Nature doesn’t

use
codons

that allow

reading frame ambiguity


A
AA
-
A
AA
-
>
AAA

(
-
4)

A
AN
-
A
NN
-
>
ANA

(
-
12)



-
>


miraculously, only 20
codons

left

George Gamow (GW physics
prof
) proposed elegant solution

Unfortunately for Gamow, nature preferred degeneracy!

Several
codons

encode particular single amino acids;

Other mechanisms control choice of correct “reading frame”


And 3 “stop”
codons

(UAA, UAG, UGA) direct the ribosome


to release template RNA and nascent protein


Genetic
code
N

RNA
pol

ribosome

DNA
pol
,
pcr

Central Biological Dogma: DNA <
--
> RNA
-
> protein

(reverse)

The enzymes involved act

as
nanoscale

motors/

machines; we will see

how engineering has been

used to study them in

exquisite, single
-

m
olecule detail; how

e
ngineering is going

b
eyond biology to make

n
ew materials and

a
ssemble devices on nm

s
cale using biological

components and principles

Next week


DNA as
nanoscale

building material


2 basic ideas


use sequence
complementarity

as “glue” to stick


pieces of
ssDNA

together in precise way


flexibility of
ssDNA

allows strands to “switch”


double helix partners
-
> branched structures

Reading for next week (heavier than usual but don’t have


to read critically


just try to understand what they did):


1
st

half of
Seeman

review (through static structures; we’ll


consider dynamic structures the following week)


He et al, JACS


first look at fig 1 in SOM
to understand


how they made 2
-
d assemblies


He et al, Science


shows follow
-
up 3
-
d
polyhedra


Yin et al, Science


man
-
made
tubes from DNA