selection / amplification is a general tool for directed evolution of nucleic acids and proteins , which is much more complicated for reaction promoters ( biocatalysts ) than for simple binders .
selection and identification for the former require some sort of catalyst - product pairing in an isolated compartment . in vivo selection using living cells has been typical choice for such a purpose .
meanwhile , in vitro selection , as opposed to in vivo selection , is simple and convenient to carry out , is free from cytotoxicity problems , and allows for starting with a library of great diversity .  
griffiths and tawfik proposed an in vitro compartmentalization ( ivc ) technique for the in vitro evolution of biomolecules including biocatalysts , where biocatalysts are transcribed / translated in a compartmentalized water - in - oil emulsion to allow catalyst - product pairing . in practice , the ivc technique has been applied successfully for evolving or improving biocatalysts such as ribozymes and enzymes [ 312 ] .   here
, we applied the ivc technique for evolution of trnas ( one of catalysts in protein translation systems ) [ 1316 ] and present a promising concept for in vitro selection of suppressor trnas by the combination of read - through ribosome display ( rt - rd , vide infra ) [ 17 , 18 ] .
binding of suppressor trna to an amber stop codon is in competition with that of release factor 1 ( rf1 ) to terminate the translation .
the amber codon is also often misread by the exogenous trna for gln . to maximize the suppression efficiency and minimize incorporation of gln at the amber codon
, we used a reconstituted prokaryotic cell - free translation system ( puresystem classic ) , in which rf1 , and gln and gln - trna synthetase had not been added unless otherwise stated .
balance  in the amounts of the fused mrna ( mrna - trna ) ( longer ) and the trna ( shorter ) transcribed , we put gcc immediately downstream of the first t7-promoter so as to lower the translation efficiency for the fused mrna ( it is known that a g - less sequence , immediately downstream of the t7 promoter , suppresses the transcription efficiency ) .
gel electrophoresis and blotting were carried out on a be-250 electrophoresis apparatus ( bio craft ) and a trans - blot sd system ( bio - rad ) , respectively .
the first pcr was carried out in 20  l of a reaction mixture containing 4  pmol of a forward primer containing a flag domain ( bold ) and a tag amber stop codon ( italic ) 5-d(aa gga gat ata cca atg gac tac aag gat gac gat gac aag  tag atc agt ctg att gcg gcg tta g)-3 , 4  pmol of a reverse primer with the lower t7 promoter ( underlined ) 5-d(gtt cag ccg ctc cgg cat ctc tcc tat  agt  gag  tcg  tat  tac cgg gtg act gct gag ga)-3 for the trna - fused template or 5-d(tgg cgg aga gag ggg gat ttg aac cgg gtg act gct gag ga)-3 for nonfused template , 20  ng of pdhfr , 1.25  u of pfu ultra hf dna polymerase ( stratagene ) , 4  nmol each of dntps ( toyobo ) , and 2  l of 10  pfu ultra hf reaction buffer . after the pcr reaction , the product was purified by agarose gel electrophoresis .
the second pcr was carried out in 20  l of a reaction mixture containing 4  pmol of a forward primer with the upper t7 promoter ( underlined ) and the following sequence after modification ( vide supra ) of the widely - used one 5-d(t  aat  acg  act  cac  tat  agc ccg gcc aca acg gct ggg ctc tag aaa taa ttt tgt tta act tta aga agg aga tat acc a)-3 , 4  pmol of a reverse primer with the sequence for e. coli trna for ser ( seru ) 5-d(tgg cgg aga gag ggg gat ttg aac ccc cgg tag agt tgc ccc tac
tcc ggt l1l2x yzl3 l4ac cgg tcc gtt cag ccg ctc cgg cat c)-3 ( l1~4 and xyz represent the anticodon - loop and anticodon bases , respectively ) , ~20 fmol of the purified first pcr product , 1.25  u of pfu ultra hf dna polymerase , 4  nmol each of dntps , and 2  l of 10  pfu ultra hf reaction buffer . 
 in vitro
coupled transcription / translation of template dnas in the reverse - phase micelle was carried out as follows .
a 50  l portion of a cell - free translation system containing 50  pm of a dna template ( 2.5  fmol ) was added gradually to 950  l of mineral oil ( sigma ) containing detergents span 85 ( nacalai tesque ) ( 4.5% v / v ) and tween 20 ( sigma ) ( 0.6% v / v ) under stirring on ice .
the diameter ( d ) of the micelles ( water droplets ) in the resulting emulsion was about 2  m , indicating that the number of micelles was n = v / v = 1.2  10 , where v is the total volume of the water phase ( 50  l ) and v is the volume of a micelle with d = 2  m .
after incubation of the mixture at 37c for 1  h , the emulsion was spun at 2000  g for 10 sec .
a 200  l portion of the supernatant was carefully taken from the upper part and mixed with 200  l of an ice - cold selection buffer ( phosphate - k , ph 7.3 ) containing 92.2  mm of k , 300  mm of na , 50  mm of mg , 0.05% of tween 20 ( wako ) , 2% of block ace ( dainippon pharmaceutical co. ) , and 1  ml of ice - cold water - saturated ether .
the mixture was inverted twenty times and centrifuged at 16100  g for 10  min at 4c , and then the organic ether phase was removed .
the water phase was washed with 1  ml of ether and mixed with 200  l of ice - cold selection buffer .
the mixture was applied on a column packed with prewashed anti - flag m2 agarose ( sigma ) and gently inverted for 2  h at 4c . after washing the gel retaining the ribosome - protein - mrna ( prm ) complex with 200  l of selection buffer five times ,
the mrnas were eluted upon collapse of the rpm complexes with 200  l of an elution buffer ( phosphate - k , ph 7.2 ) containing 92.2  mm of k , 300  mm of na , 30  mm of edta , 0.05% of tween 20 , and 2% of block ace .
the mrnas eluted were purified with an rneasy minelute cleanup kit ( qiagen ) and amplified with a qiagen one - step rt - pcr kit according to the manufacture 's protocol using a forward primer with t7 promoter ( underlined ) and a flag domain ( bold ) followed by a tag amber stop codon ( italic ) 5-d(t  aat  acg  act  cac  tat  agc ccg gcc aca acg gct ggg ctc tag aaa taa ttt tgt tta act tta aga agg aga tat acc a atg gac tac aag gat gac gat gac aag  tag)-3 and a reverse primer 5-d(tgg cgg aga gag ggg gat ttg aac ccc cgg tag agt tgc c)-3. the resulting rt - pcr products were used for the next round of selection and , after three ( for anticodon - randomized trna ) or five ( for anticodon - loop - randomized trna ) rounds , were monocloned using a pcr cloning kit ( qiagen ) and sequenced .
single - round coupled transcription / translation under normal ( noncompartmentalized ) or compartmentalized conditions ( referring to figure 1(b ) , lanes 14 or 5 , resp . ) was carried out using 400  pm of a template in 25  l of a cell - free translation system under otherwise identical conditions .
the first pcr was carried out in 20  l of a reaction mixture containing 4  pmol of a forward primer with a flag domain ( bold ) with or without a tag amber stop codon ( italic ) 5-d(aa gga gat ata cca atg gac tac aag gat gac gat gac aag [ tag ] atc agt ctg att gcg gcg tta g)-3 , 4 pmol of a reverse primer with an ochre stop codon ( italic ) 5-d(tat tca tta ccg ccg ctc cag aat ct)-3 ' , 20  ng of pdhfr , 1.25  u of pfu ultra hf dna polymerase ( stratagene ) , 4  nmol each of dntps ( toyobo ) , and 2  l of 10  pfu ultra hf reaction buffer .
the second pcr was carried out in 20  l of a reaction mixture containing 4  pmol of a forward primer with t7 promoter ( underlined ) 5-d(gaa att  aat  acg  act  cac  tat  agg gag acc aca acg gtt tcc ctc tag aaa
taa ttt tgt tta act tta aga agg aga tat acc a)-3 , 4  pmol of a reverse primer 5-d(tat tca tta ccg ccg ctc cag aat ct)-3 , ~20  fmol of the purified first pcr product , 1.25  u of pfu ultra hf dna polymerase , 4  nmol each of dntps , and 2  l of 10  pfu ultra hf reaction buffer .
template mrnas were obtained by run - off transcription of the 5-flag - tag - dhfr or 5-flag - dhfr template dna obtained using a t7-megashortscript kit ( ambion ) .
thus , a t7-transcription mixture ( 10  l ) containing 3  l of the pcr solution of template dna was incubated at 37c for 2  h. after addition of 1  u of dnase i , the mixture was incubated for additional 15  min .
mrnas were purified with an rneasy minelute cleanup kit ( qiagen ) and concentrations of the purified specimens were determined by the absorbance at 260  nm . 

template dna was prepared by pcr in 20  l of a reaction mixture containing 20  pmol of a forward primer with t7 promoter ( underlined ) 5-d(g taa  tac  gac  tca  cta  ta gga gag atg ccg gag cgg ctg aac)-3 , 20  pmol of a reverse primer 5-d(tgg cgg aga gag ggg gat ttg aac ccc cgg tag agt tgc c)-3 , 100  fmol of a template dna ( initial , rt - pcr amplified , or monocloned ) , 1.25  u of pfu ultra hf dna polymerase ( stratagene ) , 10  nmol each of dntps ( toyobo ) , and 2  l of 10  pfu ultra hf reaction buffer .
the resulting pcr solution was used for transcription using a t7-megashortscript kit ( ambion ) for 37c for 20  h. the transcribed trnas were purified by denaturing page ( 8% ) , followed by ethanol precipitation and , after dissolution in 500  l of water , further by passing successively through microcon ym-30 ( millipore ) and g-25 microspin columns ( amersham ) .
translation of a template mrna ( 5-flag - uag - dhfr or 5-flag - dhfr as a uag( ) positive control ) ( 2  g ) was carried out at 37c for 1  h in the presence of an exogenous trna ( 2  g ) in 10  l of a reconstituted cell - free translation system , in which rf1 had not been added , but gln and gln - trna synthetase had . to the reaction mixture were added 165  l of water and 175  l of a sample - loading buffer ( 125  mm tris - hcl ( ph 6.8 ) , 4% ( w / v ) sds , 20% ( w / v ) glycerol , 0.002% ( w / v ) bromophenol blue , and 10% ( v / v ) 2-mercaptoethanol ) .
the resulting solution was incubated at 95c for 5  min and applied on 15% sds - page .
western blotting was performed on a pvdf membrane ( hybond - p , amersham ) .
flag - tagged proteins were visualized with anti - flag - hrp conjugate ( sigma ) and ecl plus western blotting detection reagent ( amersham ) .
the suppression efficiencies were evaluated by comparing the band intensities of the flag - tag - dhfr protein , determined by using the image j software ( nih ) , with those of serially diluted ( 10 , 20 , 40 , 60 , 80 , 100% ) solutions of the stop - free flag - dhfr reference protein translated under otherwise identical conditions .
we previously introduced rt - rd , in which expressed protein could be fully displayed upon suppression of the stop codon(s ) downstream of the open reading frame by appropriate suppressor trnas . in this paper , we conversely apply the rt - rd technique for the selection of suppressor trnas that are coded at the 3-terminus of the very template that contains the stop codon to be suppressed .
we prepared a dsdna template containing , under the control of a t7 promoter , the rbs ( ribosome binding site ) , the atg start codon , a flag domain , the tag ( amber ) stop codon , and a spacer ( dhfr1192 ) derived from the e. coli dhfr gene covering the amino acids 164 , followed by the trna sequence together with its own t7 promoter ( figure 1(a ) ) .
there are two t7 promoters in this template , transcription of which thus generates two types of rnas ; the one that contains both an rbs and an aug serves as an mrna , and the other , which lacks them , serves only as a trna .
when the latter ( trna ) happens to suppress ( read through ) the amber ( uag ) stop codon in the transcribed mrna , a fused protein containing the flag , dhfr1192 , and trna regions would result upon translation and remain attached to the mrna with the dhfr1192-trna portion , serving as a spacer to be anchored in the ribosome tunnel to squeeze the flag - tag peptide for full display ( rt - rd ; figure 2 ) .
we first confirmed that the right amber suppressor ( trnacua ) generated in this way worked as such .
the anticodon - adjusted ( cua ) trna is known to be aminoacylated with ser by endogenous ser - trna synthetase and hence suppress ( read through ) the amber codon with concomitant incorporation of ser at that position [ 18 , 20 ] .
coupled transcription / translation for 1  h of the template fused with the gene for trnacua ( figure 1(a ) , nnn = cua ) in a reconstituted prokaryotic ( e. coli ) cell - free translation system   containing t7 rna polymerase was followed by affinity selection ( 4c and [ mg ] = 50  mm ) of the flag - tag peptide domain displayed ( rt - rd ) in the protein - ribosome - mrna ( prm ) complex .
the mrna template coding the trnacua sequence was recovered upon disruption of the ternary complex with edta , rt - pcr amplified , and identified as such ( figure 1(b ) , lane 1 ) . however , the template was by no means recovered efficiently when it was lacking ( nonfused ) the trna domain ( lane 2 ) or fused with anticodon - mismatched ( cga ) natural trna for ser ( nnn = cga ) ( lane 3 ) .
( in respect to the weak spots seen in lanes 2 and 3(figure 1(b ) ) , it is known that the stop codons can be suppressed to some extent ( up to several % ) in an rf1-minus translation system by misreading even in the absence of a suppressor trna . ) in these cases , there is no generation of the correct trna for amber suppression and hence translation stops at the amber ( uag ) codon , giving rise only to the flag peptide with no linkage to the genotype ( mrna ) .
conversely , when equal amounts of nonfused ( suppression irrelevant ) and trnacua - fused ( relevant ) templates were used , both templates were recovered ( lane 4 ) ( in respect to the apparently stronger spot for the nonfused template in lane4(figure 1(b ) ) , it is generally true that shorter templates are more easily amplified by pcr . ) this is because suppressor trnacua generated from the latter template can suppress the amber codon of the former .
to avoid such a crossover , we needed to compartmentalize the reactions of each template using a water - in - oil emulsion system [ 312 ] .
with this technique ( see below , figure 2 ) , we could selectively recover the trnacua - fused template coding the active suppressor trna ( lane 5 ) from the above mixture .
the selection cycle is shown in figure 2 . an initial pool ( a ) of fused templates ( figure 1(a ) , nnn = nnn with a diversity of 4 = 64 ) with genes for anticodon - randomized trna ( figure 3(a ) , left )
was subjected to coupled transcription / translation in a water - in - oil emulsion ( b ) [ 312 ] .
consequently , each compartment ( ~2  m ) generates and contains a pair of template - related sister rnas , a fused mrna ( shown as mrna - trna ) and a trna ( c ) .
the whole protein , susceptible to rt - rd in the form of a prm complex ( step iv in d ) , can be translated ( d ) only when the trna serves as an amber suppressor ( steps i  iii in d ) .
the ternary complex thus formed was recovered by affinity selection of the displayed flag tag after breaking the emulsion and treated with edta to afford the active trna - fused template mrnas ( e ) , which were purified and amplified by rt - pcr back to the dsdna templates ( a ) , for use in another selection cycle .
the anticodon of approximately one third of the 29 monocloned trnas was cua ( figure 3(a ) , center ) , which was the one expected based on codon - anticodon complementarity .
these results indicate that the sensitive prm complex survives the ivc and workup conditions to allow the rt - rd / ivc method to select suppressor trnas . finally , we applied the rt - rd / ivc method to the engineering of the two - base loop regions adjacent to the anticodon .
these regions are variant in various trnas but are believed to be important in stabilizing codon - anticodon interactions .
selection of fused templates with genes of anticodon - matched ( cua ) and anticodon - loop - randomized ( nn - cua - nn with a diversity of 4 = 256 ) trna ( figure 3(a ) , right ) was carried out as above .
interestingly , ~40% of the 39 monocloned trnas had the same loop sequence ( cu - cua - aa ) as the natural trna for ser ( cu - cga - aa ) even in the base modification - free conditions ( figure 3(a ) ) .
these results indicate that the anticodon - loop sequence itself has played a preserved or sophisticated role in the evolution of trnas that now have many modified bases ( since neither anticodon nor anticodon - loop region is recognized by the serenyl - trna synthetase , the selected loop sequence may play a role in stabilizing the interaction between the amber stop codon andthe suppressor anticodon . ) [ 22 , 23 ] .
suppression efficiencies were evaluated by western blotting analysis using the nonfused template lacking the trna domain in an rf1-minus translation system under noncompartmentalized conditions ( figure 3(b ) ) .
enrichment of active trna became notably pronounced after three rounds ( lanes 1113 ) ( the reason for the high suppression efficiency ( ~80% ) despite the low percentage amounts of the active trnacua ( 10% in round 3 and 38% in round 5 ) is that excess amounts of trnacua were used in the experiments for western blotting ( figure 3(b ) ) ) and the suppression efficiency of the monocloned trnacu - cua - aa showed ~100% activity ( lane9 ) compared with the suppression - free translation using a uag( ) reference template ( lane 7 ) .
interestingly , a single mutation in the anticodon - loop domain led to a dramatic loss of activity ( lanes 14 and 15 ) .
an essential aspect of it is that trna is a part of mrna ( mrna - trna fusion ) , being located downstream of the particular codon to be suppressed ( the amber stop codon in this study ) .
therefore , only active trnas that can self - suppress the codon in a water - in - oil compartment are susceptible to amplification by the read - through ribosome - display technique .  
although the concept was well demonstrated , the present method must be further optimized especially regarding the selection efficiencies .
three or five rounds of selection were required for enrichment from the small library sizes ( n = 64 and 256 ) .
one possible reason for this low selection efficiency is a misreading of stop codon in an rf1-minus translation system , which would result in the recovery of false positive trna - fused mrnas .
optimization of extraction conditions or the use of mrna display technique [ 24 , 25 ] might overcome the instability .
of course , compartmentalization conditions or mrna / trna ratios are also the factor to be checked .
furthermore , it is not easy to understand that 10 clones out of 39 ( 26% ) had the cc - cua - aa anticodon - loop sequence after 5 rounds ( figure 3(a ) ) , which turned out to be completely inactive in suppression ( figure 3(b ) , lane 14 ) . taking into account the fact that the cc - cua - aa occupies 25% of pool even in round 3 in contrast to the less percentage amounts of the active cu - cua - aa sequence ( 10% ) , it is feasible to speculate that the inactive cc - cua - aa sequence might be already abundant in the initial pool and/or has been more easily amplified regardless of the selection process .   after sufficient improvements
 here is presented a concept for in vitro selection of suppressor trnas . 
 it uses a pool of dsdna templates in compartmentalized water - in - oil micelles . 
 the template contains a transcription / translation trigger , an amber stop codon , and another transcription trigger for the anticodon- or anticodon loop - randomized gene for trnaser . upon transcription 
 are generated two types of rnas , a trna and a translatable mrna ( mrna - trna ) . 
 when the trna suppresses the stop codon ( uag ) of the mrna , the full - length protein obtained upon translation remains attached to the mrna ( read - through ribosome display ) that contains the sequence of the trna . in this way 
 , the active suppressor trnas can be selected ( amplified ) and their sequences read out . 
 the enriched anticodon ( cua ) was complementary to the uag stop codon and the enriched anticodon - loop was the same as that in the natural trnaser . 
1. Introduction
2. Materials and Methods
3. Results and Discussion