Artificial Enzymes - The Nanobiotechnology - page 61

emulsion.. Recently,. a. novel. selection. approach. based. on. this. strategy. was. developed.
to. engineer. an. RNA. polymerase. capable. of. synthesizing. RNAs. of. up. to. 95. nucleotides.
(Wochner. et. al.. 2011).. The. method. is. termed. compartmentalized. bead-tagging,. and.
consists. of. encapsulating. a. genetic. library. of. ribozymes. attached. to. magnetic. beads. in.
individual. droplets,. allowing. transcription. to. occur. within. the. droplets. to. create. the.
ribozymes,. triggering. primer. extension. by. addition. of. primer/template. duplexes. in. a.
second.emulsion,.and.detecting.the.extent.of.primer.extension.by.a.combination.of.rolling.
circle.ampliication.of.the.extended.primers.and.luorescence-activated.cell.sorting.(FACS;.
Figure.3.5)..This.method,.in.combination.with.rational.RNA.engineering,.yielded.an.RNA.
polymerase. with. greater. polymerase. activity,. idelity,. and. generality. than. the. parental.
ribozyme.. The. new. ribozyme. was. able. to. synthesize. an. enzymatically. active. ribozyme.
from.an.RNA.template.(Wochner.et.al..2011),.and.can.polymerize.sequences.half.of.its.own.
length,.bringing.us.closer.to.the.goal.of.a.completely.self-replicating.ribozyme.

1 Gene binding 2 Transcription and

ligation

3 Primer and template

binding

Primer extension

4

DNA circle annealing
and rolling circle
amplification

FACS, gene recovery,
and amplification

5 6 Fluorescent labeling 7

Figure 3.5
(See companion CD for color igure.) . Compartmentalized. bead-tagging. method. for. the. selection. of. artii-
cial. ribozymes.. (1). Hairpin. oligonucleotides. (green). and. biotinylated. genes. (red). from. a. library. are. attached.
to. streptavidin-coated. magnetic. beads. (blue. circle).. (2). Transcription. takes. place. within. a. irst. water-in-oil.
emulsion,.producing.ribozymes.that.subsequently.ligate.to.the.hairpin..(3).The.emulsion.is.broken.and.primer.
(black).and.template.(cyan).duplexes.are.attached.to.the.magnetic.beads..(4).In.a.second.emulsion,.ribozymes.
are.released.from.the.beads.and.primer.extension.can.proceed..(5).Primer.extension.ampliication.is.achieved.
by.rolling.circle.ampliication.of.a.DNA.minicircle..(6).To.facilitate.signal.detection,.luorescent-labeled.probes.
are.hybridized.to.the.DNA..(7).The.active.ribozymes.are.isolated.by.FACS.and.ampliied.by.PCR.ampliication..
(Modiied.from.Wochner,.A..et.al.,. Science ,.332,.209,.2011.)