Encapsulated in vitro selection of a DNA ligase ribozyme

1
Encapsulated in vitro selection of a DNA ligase
ribozyme
H. Alexander Ebhardt and Peter J. UnrauDept. of
Molecular Biology and BiochemistrySimon Fraser
University
Selection scheme
Motivation
In vitro selection is a powerful tool used to
explore the range of chemistry a ribozyme can
catalyze. In practice, a substrate for the
ribozyme reaction is tethered to the 5' or 3' end
of the RNA pool molecules. Active sequences able
to react with a second tagged substrate can be
purfied away from the inactive molecules in the
pool. These ribozymes act in cis. To enable
ribozymes to act in trans, we propose to use
man-made cell-like vesicles in which the ribozyme
acts on its own genome. This would allow the
direct selection of trans acting ribozymes which
has not been done so far.

• These genomes are amplified using asymmetric
  PCR. The bottom strand is isolated by denaturing
  PAGE.
• The selected genomes are prepared for next
  round by adding splint and biotin tagged DNA.

• After encapsulation, T7 RNA polymerase
  transcribes single-stranded DNA into RNA.
• A catalytic active RNA fold will ligate the
  nicked DNA which has a biotin tag. The tagged DNA
  is now attached to the genome of the catalytic
  active RNA fold.

• After incubation, vesicles are harvested. Through
  denaturation of dsDNA, only successfully repaired
  nicks will bind to streptavidin columns through a
  biotin tag.
• The purified genomes are amplified directly
  from magnetic beads.

B
p
RNAp
RNAp
B
ppA HO
genome DNA
biotin
magnetic bead
strept- avidin
B
ppA HO
pA
primer 1
B
p
B
primer 2
p
genome DNA
RNAp
RNAp

• These genomes will be encapsulated again (see
  step 1).

Adenylylation of DNA
Encapsulation
Encapsulated transcription
To assist the ribozyme, the biotin tagged DNA
will be 5' adenylylated. This adenylylation
mimics the first step of the ligation mechanism
of T4 DNA ligase.
For the first round, I expect to encapsulate 100
mL of transcription mix in 2 L of mineral
oil-tween-span mix. These large volumes are
necessary to encapsulate the initial pool which
is comprised of 6x1014 different DNA molecules.
We expect to have 30 genomes per vesicle.
Each vesicle contains a homogenous aqueous
mixture of transcription buffer, NTPs, DNA
template and T7 RNA polymerase. Once
encapsulated, transcription takes place and has
been shown to be stable for over 48 hours.
To track the location of radiolabelled RNA, all
fractions underwent scintillation counting after
incubation.
Enzymatic 5' adenylylation (T4 DNA ligase)
A B C D E
F
0h 1.5h 3.5h 20h
p
8 denaturing PAGE A transcription reaction
terminated after 5 min. B in vitro transcription
after 2 hours C encapsulated transcription after
2 hours D transcription reaction terminated
after 5 min. E encapsulated transcription 24
hour time point F encapsulated transcription 48
hour time point
3'
App
Vesicles 90 mineral oil 5.0 aqueous 4.5
span80 0.5 tween80 Each vesicle holds 35 fL
aqueous volume.
3'
transcripts
20 denaturing PAGE
Chemical 5' adenylylation (Imidazole activated
AMP)
0h 2h 4h 20h 0h 2h
4h 20h
Diagram After harvesting vesicles, there are
three fractions glass beads, mineral oil and
aqueous. These fractions were analyzed using
scintillation counting. Relative ratios are shown.
50 mM MgCl2 75 mM MgCl2
50 ?m
Asymmetric PCR
Capturing of biotin tagged DNA
Annealing DNA
Future directions
The single stranded DNA is connected via a splint
to the 5' adenylylated 3' biotin tagged DNA. The
T7 promoter is within the double stranded region.

Biotin tagged DNA is recovered by streptavidin
coated magnetic beads. In two experiments, the
specific and unspecific binding of a
phosphoylated gel purified oligo were analyzed.
For asymmetric PCR, Spacer Phosphoramidite 9
(Glen Research) was integrated into the 5' PCR
primer.

• Ligation of nicked construct using T4 DNA ligase.
• Simulation of round 1 using T4 DNA ligase.
• Recovery optimization.
• Round 1.

Taq polymerase stops at the spacer, leading to
two asymmetric strands. These are separated by
denaturing PAGE.
M C PCR
Denaturing 8 PAGE M Marker 50bp C Control PCR
140bp PCR asymmetric PCR
56.6
T7 promoter
radiolabelled gel pure primer
radiolabelled gel pure primer
N 103
5'
3'
B
5'
ppA HO
3'
p
B
p
47.3
A B C D E F
Acknowledgement Peter J. Unrau - supervisor Hani
Zaher - microscope pictures
biotin
3 native agarose gel A 50 bp ladder B
47.3 C 47.356.6 D bottom strand (N 103) E
bottom strand splint 47.356.6 F 50 bp ladder

strept- avidin
magnetic bead
strept- avidin
magnetic bead
Top strand 177 nt.
Bottom strand 152 nt.
Contact Thank you for your interest in my
project. I would be happy to answer any further
questions you might have. E.mail hebhardt_at_sfu.ca
Spacer Phosphoamidite 9 Glen Research (10-1909)
Procedure radiolabelled gel pure primers were
incubated with magnetic beads (NEB S1420S).
Beads were washed three times with 0.5xSSC. All
fractions were analyzed by scintillation
counting. W1-W3 washes 1-3 B beads.